The role of endothelial cell apoptosis in inflammatory and immune diseases.

The integrity of the endothelial lining of the vasculature is essential for vascular homeostasis and normal organ function. Endothelial injury or dysfunction has been implicated in the pathogenesis of diverse vascular diseases. Studies in vitro have demonstrated that a wide variety of stimuli can induce programmed cell death (apoptosis) of endothelial cells, and have suggested that apoptosis could be an important mechanism of vascular injury, resulting in vascular leak, inflammation, and coagulation. In this review, we focus on the potential role of endothelial apoptosis in the initiation and progression of inflammatory and immune disorders, reviewing human diseases and in vivo models in which endothelial cell apoptosis has been demonstrated. Although endothelial cell apoptosis is observed in many inflammatory and immune disorders, we find that there is, as yet, only limited experimental evidence demonstrating that it is critical to the pathogenesis of disease.

C1-inhibitor reduces the ischaemia-reperfusion injury of skeletal muscles in mice after aortic cross-clamping.

BACKGROUND: Both C1-inhibitor (C1-INH) and antibodies against the CD18 adhesion molecule have been shown to reduce ischaemia-reperfusion injuries. The objective of this study was to investigate the effect of increased ischaemia times and to determine whether inhibiting C1 or blocking the CD18 function was protective in skeletal muscle ischaemia-reperfusion injury after aortic cross-clamping. MATERIALS AND METHODS: BALB/c mice were subjected to aortic cross-clamping below the renal artery for 60, 75 or 105 min, followed by 3 h of reperfusion. Two-thirds of a total dose of anti-CD18 antibody (40 mg/kg) or human C1-INH (1,000 IU/kg) was given by intraperitoneal injection before ischaemia and one-third immediately after the clamping. Creatine kinase (CK) in the plasma was used as an indicator of muscle injury severity. RESULTS: There was a consistent rise in the plasma CK concentration proportional to the length of ischaemia (P < 0.0005). C1-INH treatment significantly (P = 0.012) reduced the plasma CK for the ischaemia times of 75 and 105 min. The anti-CD18 antibody did not have any effect, as demonstrated by the CK values that were similar to controls (P = 0.836). CONCLUSION: The data support a beneficial role for C1-INH in the treatment of ischaemia-reperfusion injuries of skeletal muscles.

A constitutive cytoprotective pathway protects endothelial cells from lipopolysaccharide-induced apoptosis.

Lipopolysaccharide (LPS) has been implicated as the bacterial component responsible for much of the endothelial cell injury/dysfunction associated with Gram-negative bacterial infections. Protein synthesis inhibition is required to sensitize the endothelium to lipopolysaccharide-induced apoptosis, suggesting that a constitutive or inducible cytoprotective protein(s) is required for endothelial survival. We have identified two known endothelial anti-apoptotic proteins, c-FLIP and Mcl-1, the expression of which is decreased markedly in the presence of cycloheximide. Decreased expression of both proteins preceded apoptosis evoked by lipopolysaccharide + cycloheximide. Caspase inhibition protected against apoptosis, but not the decreased expression of c-FLIP and Mcl-1, suggesting that they exert protection upstream of caspase activation. Inhibition of the degradation of these two proteins with the proteasome inhibitor, lactacystin, prevented lipopolysaccharide + cycloheximide-induced apoptosis. Similarly, lactacystin protected against endothelial apoptosis induced by either tumor necrosis factor-alpha or interleukin-1beta in the presence of cycloheximide. That apoptosis could be blocked in the absence of new protein synthesis by inhibition of the proteasome degradative pathway implicates the requisite involvement of a constitutively expressed protein(s) in the endothelial cytoprotective pathway. Finally, reduction of FLIP expression with antisense oligonucleotides sensitized endothelial cells to LPS killing, demonstrating a definitive role for FLIP in the protection of endothelial cells from LPS-induced apoptosis.

Fas (CD95) induces alveolar epithelial cell apoptosis in vivo: implications for acute pulmonary inflammation.

Activation of the Fas/FasL system induces apoptosis of susceptible cells, but may also lead to nuclear factor kappaB activation. Our goal was to determine whether local Fas activation produces acute lung injury by inducing alveolar epithelial cell apoptosis and by generating local inflammatory responses. Normal mice (C57BL/6) and mice deficient in Fas (lpr) were treated by intranasal instillation of the Fas-activating monoclonal antibody (mAb) Jo2 or an irrelevant control mAb, and studied 6 or 24 hours later using bronchoalveolar lavage (BAL), histopathology, DNA nick-end-labeling assays, and electron microscopy. Normal mice treated with mAb Jo2 had significant increases in BAL protein at 6 hours, and BAL neutrophils at 24 hours, as compared to lpr mice and to mice treated with the irrelevant mAb. Neutrophil recruitment was preceded by increased mRNA expression for tumor necrosis factor-alpha, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-2, macrophage chemotactic protein-1, and interleukin-6, but not interferon-gamma, transforming growth factor-ss, RANTES, eotaxin, or IP-10. Lung sections from Jo2-treated normal mice showed neutrophilic infiltrates, alveolar septal thickening, hemorrhage, and terminal dUTP nick-end-labeling-positive cells in the alveolar septae and airspaces. Type II pneumocyte apoptosis was confirmed by electron microscopy. Fas activation in vivo results in acute alveolar epithelial injury and lung inflammation, and may be important in the pathogenesis of acute lung injury.

NF-kappaB activation is required for human endothelial survival during exposure to tumor necrosis factor-alpha but not to interleukin-1beta or lipopolysaccharide.

In the presence of a protein synthesis inhibitor, cycloheximide, tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), or lipopolysaccharide (LPS) induces human umbilical vein endothelial cells (HUVECs) to undergo apoptosis, suggesting that constitutive or inducible cytoprotective pathways are required for cell survival. We studied the correlation between nuclear factor-kappaB (NF-kappaB) activation and cell death induced by TNF-alpha, IL-1beta, or LPS. Adenovirus-mediated overexpression of a dominant-negative IkappaBalpha (inhibitor of kappaB) mutant blocked NF-kappaB activation by gel shift assay and blocked induction of vascular cell adhesion molecule-1 protein by TNF-alpha, IL-1beta, and LPS, a NF-kappaB-dependent response. In cells overexpressing the IkappaBalpha mutant, TNF-alpha induced cell death, whereas IL-1beta or LPS did not. We conclude that cell survival following TNF-alpha stimulation is NF-kappaB-dependent but that a constitutive or inducible NF-kappaB-independent pathway(s) protects IL-1beta- or LPS-treated HUVECs from cell death.

The role of leukocyte emigration and IL-8 on the development of lipopolysaccharide-induced lung injury in rabbits.

Leukocyte emigration and alveolar macrophage-derived cytokines may contribute to lung microvascular injury associated with adult respiratory distress syndrome. We have used mAbs against cell adhesion molecules on leukocytes (anti-CD18 and anti-CD49d) or against IL-8 to investigate these contributions. Intratracheal (i.t.) instillation of LPS (50 microg/kg) caused a significant increase in bronchoalveolar lavage polymorphonuclear leukocytes (PMNs) without an increase in mononuclear cells (MNCs) or an increase in lung permeability. Injection of LPS (10 microg/kg) i.v. at 24 h after i.t. LPS caused significant increases in bronchoalveolar lavage PMNs, MNCs, IL-8, and monocyte chemotactic protein-1, as well as increases in lung permeability. Rabbits that were administered i.t. LPS followed by i.v. LPS and treated with anti-CD18 mAb had a significantly lower lung permeability index and emigration of fewer PMNs but no change in MNC emigration compared with saline treatment. Anti-IL-8 mAb treatment resulted in a significantly lower lung permeability index with no change in PMN emigration compared with no treatment. These results suggest that PMN emigration is necessary but not sufficient for the development of LPS-induced lung injury, and that IL-8 plays a significant role in PMN-dependent lung injury, independent of PMN emigration.

Outcome after hemorrhagic shock in trauma patients.

BACKGROUND: It is essential to identify patients at high risk of death and complications for future studies of interventions to decrease reperfusion injury. METHODS: We conducted an inception cohort study at a Level I trauma center to determine the rates and predictors of death, organ failure, and infection in trauma patients with systolic blood pressure < or = 90 mm Hg in the field or in the emergency department. RESULTS: Among the 208 patients with hemorrhagic shock (blood pressure < or = 90 mm Hg), 31% died within 2 hours of emergency department arrival, 12% died between 2 and 24 hours, 11% died after 24 hours, and 46% survived. Among those who survived > or = 24 hours, 39% developed infection and 24% developed organ failure. Increasing volume of crystalloid in the first 24 hours was strongly associated with increased mortality (p = 0.00001). CONCLUSION: Hemorrhage-induced hypotension in trauma patients is predictive of high mortality (54%) and morbidity. The requirement for large volumes of crystalloid was associated with increased mortality.

A1 is a constitutive and inducible Bcl-2 homologue in mature human neutrophils.

During the process of terminal differentiation toward mature neutrophils, the anti-apoptotic proteins Bcl-2 and Bcl-x become down-regulated and eventually cease to be expressed, whereas the death-promoting Bcl-2 homologue, Bax, persists. Thus, the disappearance of anti-apoptotic homologues was thought to account for the early demise of mature neutrophils. However, although the survival of mature human neutrophils can be prolonged by a variety of factors, no anti-apoptotic Bcl-2 homologues have previously been identified. Human A1 is a Bcl-2 homologue previously shown to be present in endothelial cells and to convey anti-apoptotic function in vitro. We describe here that human A1 mRNA is constitutively expressed in mature neutrophils and is up-regulated by G-CSF and LPS, agonists that promote neutrophil survival. In addition, we show progressive A1 mRNA accumulation in HL-60 cells during all-trans retinoic acid-driven neutrophilic differentiation. Our findings suggest that A1 may have an important role in neutrophilic development and in modulating mature neutrophil survival.

Antibiotic therapy determines subcutaneous Escherichia coli abscess formation after CD18 inhibition in rabbits.

Monoclonal antibodies (MAbs) that interrupt polymorphonuclear neutrophil (PMN)-endothelial cell adhesion can ameliorate PMN-mediated injury, including burn-induced inflammatory injury, but can also impair PMN-mediated defense against bacterial infection. We report the effects of combined anti-adhesion and antibiotic therapy on local infectious sequelae after subcutaneous Escherichia coli inoculation in rabbits treated with anti-CD18 (60.3) or anti-P-selectin (PB1.3) MAb. Ampicillin or ceftriaxone were administered for 72 hours. PMN emigration was assessed at 24 hours and local infectious sequelae at 7 days. In ampicillin/60.3-treated rabbits, E. coli inoculation resulted in impaired PMN emigration and increased infectious complications, with abscesses forming at a 10,000-fold lower inoculation concentration compared with other MAb-antibiotic treatment groups. We conclude that (1) CD18, but not P-selectin blockade interferes with PMN emigration and host defense to subcutaneous E. coli, and (2) appropriate antibiotic therapy can prevent the local infectious events caused by CD18 inhibition.

Leukocyte Adhesion Deficiency Type II is a generalized defect of de novo GDP-fucose biosynthesis. Endothelial cell fucosylation is not required for neutrophil rolling on human nonlymphoid endothelium.

Leukocyte Adhesion Deficiency Type II (LAD II) is a recently described syndrome and the two patients with this defect lack fucosylated glycoconjugates. These glycoconjugates include the selectin ligand, sialyl LewisX, and various fucosylated blood group antigens. To date, the molecular anomaly in these patients has not been identified. We localized the defect in LAD II to the de novo pathway of GDP-fucose biosynthesis, by inducing cell-surface expression of fucosylated glycoconjugates after exposure of lymphoblastoid cell lines from the LAD II patients to exogenous fucose. This defect is not restricted to hematopoietic cells, since similar findings were elicited in both human umbilical vein endothelial cells (HUVEC) and fibroblasts derived from an affected abortus. We have used these LAD II endothelial cells to examine the consequence of fucosylation of endothelial cells on the rolling of normal neutrophils in an in vitro assay. Neutrophil rolling on LPS-treated normal and LAD II HUVEC was inhibited by an E-selectin monoclonal antibody at both high and low shear rates. LAD II HUVEC lacking fucosylated glycoproteins supported leukocyte rolling to a similar degree as normal HUVEC or LAD II cells that were fucose-fed. At low shear rates, an L-selectin antibody inhibited neutrophil rolling to a similar degree whether the LAD II cells had been fucose-fed or not. These findings suggest that fucosylation of nonlymphoid endothelial cells does not play a major role in neutrophil rolling and that fucose is not a critical moiety on the L-selectin ligand(s) on endothelial cells of the systemic vasculature.

Mild hypothermia during reperfusion reduces injury following ischemia of the rabbit ear.

Ischemia and reperfusion causes tissue injury that can be partially prevented by mild hypothermia. In this study we postulated that hypothermic protection could occur if imposed only during reperfusion. Rabbit ears were partially amputated, the central artery occluded for 6 h followed by reperfusion at an ambient temperature of either 20 or 24 degrees C resulting in ischemic ear temperatures of 22.5 vs. 24.7 degrees C. Ear temperature of rabbits remaining in the 24 degrees C room increased with reperfusion to 32.4 degrees C whereas those moved to the 20 degrees C room increased to 30.0 degrees C by 2 h of reperfusion. Ear volume was used as a measure of tissue edema and was measured for 7 days after the ears were allowed to reperfuse. Normalized myeloperoxidase content (polymorphonuclear cell accumulation) was significantly greater in the 24 degrees C ischemia-24 degrees C reperfusion group compared with the other groups. Ear edema was significantly less in the two groups exposed to 20 degrees C reperfusion compared with the 24 degrees C ischemia-24 degrees C reperfusion group. Peak ear volume was 5.0 times baseline for the 24 degrees C ischemia-24 degrees C reperfusion, 4.0 times baseline for the 20 degrees C ischemia-24 degrees C reperfusion, 3.4 times baseline for the 24 degrees C ischemia-20 degrees C reperfusion, and 3.3 times baseline for the 20 degrees C ischemia-20 degrees C group. We conclude that mild hypothermia reduces PMN accumulation and is more effective in preventing tissue injury when imposed during reperfusion compared with during ischemia.

Cytoplasmic domain of E-selectin contains a non-tyrosine endocytosis signal.

E-selectin is an activation-dependent, endothelial cell-restricted adhesion molecule that is internalized and degraded rapidly once expressed on the cell surface. Tyrosine-containing structural motifs play an important role in the internalization of a number of integral proteins, and the membrane-proximal E-selectin cytoplasmic tyrosine residue (Tyr582) conforms to the endocytosis motif proposed previously. To determine the endocytosis motif in E-selectin, we selectively introduced truncation, substitution, and deletion mutations to the cytoplasmic tail of E-selectin. We analyzed the internalization kinetics of surface-expressed wild-type and mutant E-selectin constructs in transiently transfected Chinese hamster ovary cells using 125I-labeled E-selectin monoclonal antibody (125I-P6E2) in an acid elution assay. Interestingly, truncation immediately membrane proximal to Tyr582 (DeltaDGS construct) did not alter internalization kinetics significantly (DeltaDGS versus wild-type, mean surface half-life = 42 versus 45 min, respectively). Thus, it appears that the tyrosine residues are not required for internalization of E-selectin. Additional analyses indicated that Ser581 was necessary but alone was insufficient for surface E-selectin endocytosis. Thus, we conclude that there exists a novel non-tyrosine-containing endocytosis signal in the cytoplasmic tail which involves Ser581 and residues membrane-proximal to it.

Binding of human peripheral blood polymorphonuclear leukocytes to E-selectin (CD62E) does not promote their activation.

E-selectin (CD62E) is a cytokine-inducible endothelial cell adhesion molecule that tethers polymorphonuclear leukocytes (PMNs) and supports PMN rolling under conditions of flow. We examined whether interaction of PMNs with E-selectin also leads to activation of CD11b/CD18 (Mac-1, alphaMbeta2), an event that can promote firm adhesion. PMNs were added to monolayers of IL-1beta-activated HUVECs and Chinese hamster ovary (CHO) cells transfected with E-selectin cDNA. PMN activation was assessed by 1) increased CD11b/CD18 surface expression, 2) appearance of activation epitope on CD11b/CD18 (CD11b*) detected by mAb CBRM1/5, and 3) decreased L-selectin (CD62L) expression, as determined by flow cytometry. Both adherent and nonadherent supernatant PMNs became activated on IL-1beta-pretreated HUVECs. This activation was not affected by CD62E-blocking mAb P6E2. The activation state of PMNs adhered to CHO cells transfected with E-selectin cDNA was not increased over background and was similar to that of PMNs exposed to parent CHO cells. The findings were confirmed using confocal microscopy, which allowed staining of the cells for CD11b* in situ. In concert, the results suggest that PMN binding to E-selectin does not elicit inter-receptor signaling that could result in strengthening of PMN adhesion to endothelium.

The role of leukocyte and endothelial adhesion molecules in ischemia-reperfusion injury.

Reperfusion of ischemic tissue is associated with an acute inflammatory response that may further exacerbate vascular and tissue damage. Compelling evidence from a variety of animal models indicates that neutrophils are the principle effector cells of the reperfusion injury and that blockade of neutrophil adhesion to endothelium attenuates ischemia-reperfusion injury. "Anti-adhesion" therapy may represent a new approach to treatment of the many diverse clinical disorders in which ischemia-reperfusion occurs.

CD18 adhesion blockade decreases bacterial clearance and neutrophil recruitment after intrapulmonary E. coli, but not after S. aureus.

Leukocyte emigration in the lung occurs by both CD18-dependent and -independent mechanisms that are stimulus specific. We examined the effect of CD18 blockade (mAb 60.3) on neutrophil (PMN) emigration into, and bacterial clearance from, the lung. After intravenous treatment with either mAb 60.3 or saline, rabbits were given an intralobar inoculation with 10(9) colony-forming units of either Staphylococcus aureus or Escherichia coli. Four hours after inoculation, lungs were lavaged to assess PMN emigration. CD18 blockade reduced PMN emigration to E. coli by 76% but only 45% to S. aureus. Experiments to determine bacterial recovery from the lungs at 4, 8, and 24 h after inoculation showed that CD18 blockade impaired the early (4 h) clearance of E. coli but not S. aureus. These findings suggest that PMN emigration to intrapulmonary S. aureus is largely CD18-independent. In contrast, intrapulmonary E. coli elicits CD18-mediated PMN emigration. CD18 blockade results in impaired clearance of E. coli but not S. aureus from the lung.

Neutrophil adhesion molecule expression is comparable in perinatal rabbits and humans.

BACKGROUND: Human newborns, particularly those born before full term, are more susceptible to bacterial infections as a result of impaired host defense mechanisms. Compared with adults, circulating leukocytes from human newborns (preterm and full-term gestations) and newborn rabbits (full-term gestation) have low resting levels of CD62L (L-selectin) and do not significantly increase surface expression of CD18 after inflammatory stimulation. To determine the potential utility of preterm rabbits in investigations of perinatal human conditions, the authors compared the surface expression of the beta 2-integrin CD18 and CD62L (L-selectin) on polymorphonuclear leukocytes (PMNs) from perinatal rabbits and perinatal humans, both under resting conditions and after in vitro activation with inflammatory stimulants. METHODS: After erythrocyte lysis of whole-blood samples, leukocytes from 7-day-old, full-term (31-day gestation), and preterm (24-day gestation) rabbits, as well as full-term (37-42 week gestation) and preterm (27-36 week gestation) human newborns were prepared and stimulated in vitro at 37 degrees C with either C5a or phorbol myristate acetate. After fluorescence labeling of CD18 and CD62L with monoclonal antibodies, PMN adhesion molecule expression was assessed by flow cytometry. RESULTS: Constitutive CD18 expression was not significantly different between perinatal and adult humans but was reduced in all perinatal rabbits compared with adults. Inflammatory stimulation caused significant increases in CD18 expression in adult human PMNs but not in full-term and preterm newborns. Changes in CD18 expression in adult and preterm rabbits after stimulation, although in the same direction as humans, were more variable. In both species, constitutive CD62L expression on PMNs from all perinates was significantly lower than in adults. However, CD62L was shed to similar degrees after inflammatory stimulation in all groups. CONCLUSIONS: Preterm rabbits may provide a potentially useful experimental model to study PMN adhesion and host defense in the perinatal period, particularly preterm gestations. Specific advantages and limitations of rabbits in such studies are discussed.

Blocking L-selectin function attenuates reperfusion injury following hemorrhagic shock in rabbits.

Leukocyte adhesion molecule (LAM) blockade reduces ischemia-reperfusion injury. We tested the hypothesis that a monoclonal antibody (MAb) that recognizes a functional epitope of L-selectin would decrease hemorrhagic shock-induced reperfusion injury. Anesthetized rabbits were subjected to 2 h of hemorrhagic shock (cardiac output reduced to 30% of baseline), then given one of the following treatments: MAbs that recognize functional domains of L-selectin (LAM1-3), CD18 (60.3), MAbs that recognize a nonfunctional domain on L-selectin (LAM1-14), or saline, immediately before resuscitation with shed blood. Additional fluids were administered as needed to maintain cardiac output at baseline levels for 6 h. The cumulative fluid resuscitation after MAb LAM1-3 (58 +/- 34 ml/kg) was not significantly different from after MAb 60.3 (21 +/- 24 ml/kg) or MAb LAM1-14 (66 +/- 51 ml/kg), but it was significantly less than saline-treated controls (142 +/- 142 ml/kg). However, two animals treated with MAb LAM1-14 died before 6 h. If their resuscitation volumes are projected to 6 h by linear regression, then the LAM1-14-treated group required significantly greater volume (101 +/- 99 ml/kg) than the MAb LAM1-3-treated group. We conclude that MAbs to a functional domain on L-selectin are protective against reperfusion-injury following hemorrhagic shock.

Antibodies against CD14 protect primates from endotoxin-induced shock.

Lipopolysaccharide (LPS), residing in the outer membrane of all gram-negative bacteria, is considered a major initiating factor of the gram-negative septic shock syndrome in humans. LPS forms a complex with the LPS binding protein (LBP) in plasma, and LPS-LBP complexes engage a specific receptor, CD14, on the surface of myeloid cells, leading to the production of potent proinflammatory cytokines. The major goal of this study was to test the importance of the CD14 pathway in vivo in a primate model that is similar to human septic shock. Primates were pretreated with one of two different inhibitory anti-CD14 mAbs, then challenged with intravenous endotoxin (375 microg/kg/h) for 8 h. The anti-CD14 treatment regimens were successful in preventing profound hypotension, reducing plasma cytokine levels (TNF-alpha, IL-1beta, IL-6, and IL-8), and inhibiting the alteration in lung epithelial permeability that occurred in animals treated with LPS and an isotype-matched control antibody. These results demonstrate for the first time the importance of the CD14 pathway in a primate model that is similar to human septic shock. Inhibition of the CD14 pathway represents a novel therapeutic approach to treating this life-threatening condition.