Signaling functions of L-selectin in neutrophils: alterations in the cytoskeleton and colocalization with CD18.

Ligation and clustering of L-selectin by Ab ("cross-linking") or physiologic ligands results in activation of diverse responses that favor enhanced microvascular sequestration and emigration of neutrophils. The earliest responses include a rise in intracellular calcium, enhanced tyrosine phosphorylation, and activation of extracellular signal-regulated kinases. Additionally, cross-linking of L-selectin induces sustained shape change and activation of beta2 integrins, leading to neutrophil arrest under conditions of shear flow. In this report, we examined several possible mechanisms whereby transmembrane signals from L-selectin might contribute to an increase in the microvascular retention of neutrophils and enhanced efficiency of emigration. In human peripheral blood neutrophils, cross-linking of L-selectin induced alterations in cellular biophysical properties, including a decrease in cell deformability associated with F-actin assembly and redistribution, as well as enhanced adhesion of microspheres bound to beta2 integrins. L-selectin and the beta2 integrin became spatially colocalized as determined by confocal immunofluorescence microscopy and fluorescence resonance energy transfer. We conclude that intracellular signals from L-selectin may enhance the microvascular sequestration of neutrophils at sites of inflammation through a combination of cytoskeletal alterations leading to cell stiffening and an increase in adhesiveness mediated through alterations in beta2 integrins.     

Direct injury to the bronchial vasculature in anesthetized sheep.

Injury to the bronchial vasculature may contribute to liquid and solute leakage into the lung during noncardiac pulmonary edema. The purpose of this study was to measure changes in hemodynamics, pulmonary mechanics, extravascular lung water, and lung morphometry after selectively injuring the bronchial vasculature in anesthetized sheep. In two groups of seven sheep, we injected oleic acid (0.1 ml/kg) or normal saline directly into the bronchoesophageal artery. We measured systemic and pulmonary arterial pressures, cardiac output, oxygen saturation, pulmonary resistance and compliance, and lung volumes before and 1 and 4 h after injection. The lungs were removed for measurement of extravascular water, histology, and morphometry. Four hours after injection of oleic acid, cardiac output decreased but pulmonary arterial pressure did not change. In addition, pulmonary resistance increased and dynamic compliance and vital capacity decreased. Extravascular lung water was slightly but significantly greater in the oleic acid group. Histological examination showed interstitial edema and leukocytes in airway walls and sloughing of bronchial epithelium but little or no alveolar edema. Morphometric analysis showed significant thickening of airway walls. We conclude that direct injury to the bronchial vasculature increases lung resistance, decreases dynamic compliance, and increases extravascular lung water by the accumulation of an inflammatory infiltrate in airway walls.     

The p38 mitogen-activated protein kinase mediates cytoskeletal remodeling in pulmonary microvascular endothelial cells upon intracellular adhesion molecule-1 ligation

Changes in the cytoskeleton of endothelial cells (ECs) play important roles in mediating neutrophil migration during inflammation. Previous studies demonstrated that neutrophil adherence to TNF-alpha-treated pulmonary microvascular ECs induced cytoskeletal remodeling in ECs that required ICAM-1 ligation and oxidant production and was mimicked by cross-linking ICAM-1. In this study, we examined the role of ICAM-1-induced signaling pathways in mediating actin cytoskeletal remodeling. Cross-linking ICAM-1 induced alterations in ICAM-1 distribution, as well as the filamentous actin rearrangements and stiffening of ECs shown previously. ICAM-1 cross-linking induced phosphorylation of the p38 mitogen-activated protein kinase (MAPK) that was inhibited by allopurinol and also induced an increase in the activity of the p38 MAPK that was inhibited by SB203580. However, SB203580 had no effect on oxidant production in ECs or ICAM-1 clustering. ICAM-1 cross-linking also induced phosphorylation of heat shock protein 27, an actin-binding protein that may be involved in filamentous actin polymerization. The time course of heat shock protein 27 phosphorylation paralleled that of p38 MAPK phosphorylation and was completely inhibited by SB203580. In addition, SB203580 blocked the EC stiffening response induced by either neutrophil adherence or ICAM-1 cross-linking. Moreover, pretreatment of ECs with SB203580 reduced neutrophil migration toward EC junctions. Taken together, these data demonstrate that activation of p38 MAPK, mediated by xanthine oxidase-generated oxidant production, is required for cytoskeletal remodeling in ECs induced by ICAM-1 cross-linking or neutrophil adherence. These cytoskeletal changes in ECs may in turn modulate neutrophil migration toward EC junctions.     

Early response cytokines and innate immunity: essential roles for TNF receptor 1 and type I IL-1 receptor during Escherichia coli pneumonia in mice

The early response cytokines, TNF and IL-1, have overlapping biologic effects that may function to propagate, amplify, and coordinate host responses to microbial challenges. To determine whether signaling from these early response cytokines is essential to orchestrating innate immune responses to intrapulmonary bacteria, the early inflammatory events induced by instillation of Escherichia coli into the lungs were compared in wild-type (WT) mice and mice deficient in both TNF receptor 1 (TNFR1) and the type I IL-1 receptor (IL1R1). Neutrophil emigration and edema accumulation induced by E. coli were significantly compromised by TNFR1/IL1R1 deficiency. Neutrophil numbers in the circulation and within alveolar septae did not differ between WT and TNFR1/IL1R1 mice, suggesting that decreased neutrophil emigration did not result from decreased sequestration or delivery of intravascular neutrophils. The nuclear translocation of NF-kappa B and the expression of the chemokine macrophage inflammatory protein-2 did not differ between WT and TNFR1/IL1R1 lungs. However, the concentration of the chemokine KC was significantly decreased in the bronchoalveolar lavage fluids of TNFR1/IL1R1 mice compared with that in WT mice. Thus, while many of the molecular and cellular responses to E. coli in the lungs did not require signaling by either TNFR1 or IL1R1, early response cytokine signaling was critical to KC expression in the pulmonary air spaces and neutrophil emigration from the alveolar septae.     

The accumulation of dendritic cells in the lung is impaired in CD18-/- but not in ICAM-1-/- mutant mice

Bone marrow-derived dendritic cell (DC) precursors migrate via the blood stream to peripheral tissues to adopt their sentinel function. To identify factors facilitating their emigration to the lung, mutant mice deficient in E-selectin, P-selectin, E/P-selectin, ICAM-1, or CD18 and their respective controls were examined. DCs and monocytes/macrophages were immunolabeled with M5/114 and MOMA-2 mAbs, respectively, and quantified morphometrically. Of these genotypes, the numbers of DC and MOMA-2+ cells were significantly less only in the lungs of CD18-/- mice by 68 and 35% in alveolar walls and by 28 and 26% in venous walls, respectively. DCs were reduced by 30 and 41% around large and small airways, respectively, but the number of MOMA-2+ cells in these locations was not significantly different from controls. Ablation of a single gene may be associated with augmented expression of other, related gene products. Therefore, we examined the expression of VCAM-1. Increased numbers of arteries exhibited continuous luminal VCAM-1 staining in both CD18-/- and ICAM-1-/- mutants. VCAM-1 expression was absent in pulmonary capillaries and unchanged in veins. These data suggest that under nonperturbing conditions, CD18-mediated adhesion is required for the full complement of DC precursors to accumulate in the lungs. However, the defect in CD18-/- mice is partial, suggesting that CD18-independent adhesion occurs. The alternative pathway may involve VLA-4/VCAM-1 in arteries and venules but not in capillaries. The smaller defect in ICAM-1-/- mice suggests that the CD11/CD18 complex recognizes ligands other than ICAM-1 at some sites.     

In vivo two-photon excited fluorescence microscopy reveals cardiac- and respiration-dependent pulsatile blood flow in cortical blood vessels in mice

Subtle alterations in cerebral blood flow can impact the health and function of brain cells and are linked to cognitive decline and dementia. To understand hemodynamics in the three-dimensional vascular network of the cerebral cortex, we applied two-photon excited fluorescence microscopy to measure the motion of red blood cells (RBCs) in individual microvessels throughout the vascular hierarchy in anesthetized mice. To resolve heartbeat- and respiration-dependent flow dynamics, we simultaneously recorded the electrocardiogram and respiratory waveform. We found that centerline RBC speed decreased with decreasing vessel diameter in arterioles, slowed further through the capillary bed, and then increased with increasing vessel diameter in venules. RBC flow was pulsatile in nearly all cortical vessels, including capillaries and venules. Heartbeat-induced speed modulation decreased through the vascular network, while the delay between heartbeat and the time of maximum speed increased. Capillary tube hematocrit was 0.21 and did not vary with centerline RBC speed or topological position. Spatial RBC flow profiles in surface vessels were blunted compared with a parabola and could be measured at vascular junctions. Finally, we observed a transient decrease in RBC speed in surface vessels before inspiration. In conclusion, we developed an approach to study detailed characteristics of RBC flow in the three-dimensional cortical vasculature, including quantification of fluctuations in centerline RBC speed due to cardiac and respiratory rhythms and flow profile measurements. These methods and the quantitative data on basal cerebral hemodynamics open the door to studies of the normal and diseased-state cerebral microcirculation.     

Neutrophil retention during a single transit through the pulmonary circulation.

Our laboratory has previously reported that 70-80% of polymorphonuclear cells (PMNs) are delayed with respect to erythrocytes (RBCs) in a single pass through the lungs of dogs, whereas only 5-15% of PMNs are delayed in a single pass through human lungs. Because these results were obtained using a direct blood sampling method in animals and an indirect gamma camera method in humans, the reported differences could be related to differences in measurement technique. The present study was designed to settle this question by comparing both techniques in a single species. The results show that the gamma camera technique previously used in humans underestimates the retention of PMNs with respect to RBCs during a single pass through the lung. They also show that this problem can be corrected by modifying the analysis of the data obtained using the gamma camera. We conclude that the pulmonary circulation retains PMNs with respect to RBCs to a comparable degree in animals and humans.