Leukocytes in chemotactic-fragment-induced lung inflammation. Vascular emigration and alveolar surface migration.

Lung inflammation was induced in rabbits by intratracheal injections of chemotactic fragments obtained from zymosan-activated serum (CF-ZAS), and the route of vascular emigration and alveolar surface interaction of polymorphonuclear leukocytes (PMNs) and monocytes migrating into the lung was characterized by transmission (TEM) and scanning (SEM) electron-microscopic examination. Leukocytes migrated from capillaries and venules into the alveolar wall interstitium by adherence to the vascular endothelium and migration through the endothelial intracellular junction to attain a position between a reapposed endothelial cell junction and the vascular basement membrane. The cells then migrated into the interstitium through a narrow opening in the basement membrane. Leukocyte entrance into the alveolar space from the interstitium appeared to occur through small openings in the epithelial basement membrane at or near the Type I epithelial intercellular junction. Once in the alveolus, PMNs and macrophages demonstrated surface adherence and spreading along with evidence of migration, pseudopod extension, interalveolar pore transit, and retraction fiber formation. This study indicates the leukocyte influx into the alveolus in acute chemotactic-factor-induced inflammation is via a continuum of migrational activity, beginning at the pulmonary capillary endothelial surface and persisting on the alveolar epithelial surface.     

Direct in vivo observations of P-selectin glycoprotein ligand-1-mediated leukocyte–endothelial cell interactions in the pulmonary microvasculature in abdominal sepsis in mice

Objective

P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to play a significant role in septic lung injury. However, the detailed role of PSGL-1 in the pulmonary leukocyte recruitment remains elusive. We have developed a method based on intravital fluorescence microscopy of the lung microcirculation to examine the role of PSGL-1 in the extravasation process of leukocytes in septic lung damage.

Methods

Male C57BL/6 mice were treated with a control antibody or an anti-PSGL-1 antibody prior to cecal ligation and puncture (CLP). Leukocyte–endothelium interactions and microvascular hemodynamics were studied in pulmonary arterioles, capillaries and venules 4 h after CLP.

Results

Immunoneutralization of PSGL-1 decreased CLP-induced leukocyte rolling in pulmonary arterioles and venules significantly. Inhibition of PSGL-1 had no effect on leukocyte adhesion in venules, whereas the number of adherent leukocytes in lung arterioles and the number of trapped leukocytes in capillaries were markedly decreased. Moreover, immunoneutralization of PSGL-1 improved microvascular perfusion in the lung of septic animals.

Conclusions

Taken together, these results document that PSGL-1 mediates leukocyte rolling in arterioles and venules. However, inhibition of PSGL-1 only decreases leukocyte adhesion in arterioles, suggesting that leukocyte rolling is not a prerequisite for pulmonary venular adhesion of leukocytes in sepsis. In addition, our data show that capillary trapping of leukocytes is dependent on PSGL-1 function.     

Morphologic assessment of leukocyte-endothelial cell interactions in mesenteric venules subjected to ischemia and reperfusion

Intravital microscopic studies of the mesenteric microcirculation have demonstrated that leukocyte adherence and emigration in postcapillary venules are a characteristic feature of tissues exposed to ischemia-reperfusion. The objectives of this study were to determine whether: (1) neutrophils are the predominant leukocytes that adhere and emigrate in postischemic mesenteric venules, and (2) leukocyte adherence and/or emigration are a prerequisite for reperfusion-induced increases in venular permeability. Leukocyte kinetics in cat mesenteric venules (25–35m diameter) were evaluated using both intravital microscopy and quantitative morphometry. The intestine and mesentery were exposed to 60 min of ischemia, followed by 60 min reperfusion. Some animals were pretreated with a monoclonal antibody (MoAb IB₄) against the leukocyte adhesion glycoprotein, CD11/CD18. Vessels observed by intravital microscopy and adjacent venules of similar diameter were excised and processed for light (LM) and electron microscopy (EM). Horseradish peroxidase (HRP), administered intravenously, was used to assess vascular permeability by EM. By LM, the control (nonischemic) mesentery is sparsely populated by plasma cells, mast cells, and leukocytes; 30–50% of the resident population is neutrophils. Ischemia-reperfusion led to a significant increase in the number of extravascular cells, with neutrophils accounting for >80% of the total cell population. Control and ischemic venules demonstrated no leakage of HRP into the interstitium. However, venules exposed to ischemia and reperfusion demonstrated HRP leakage between endothelial cells and into the surrounding interstitium; neutrophils were adherent to the luminal surface of the endothelium, transmigrating the vessel wall, and in the surrounding interstitium. Animals pretreated with MoAb IB₄ presented the same cell profile as nonischemic controls, with no adherent or transmigrating neutrophils. However, some HRP leakage was noted following reperfusion in venules treated with MoAb IB₄. The results of this study indicate that: (1) neutrophils are the predominate leukocytes that adhere and emigrate in postischemic venules, and (2) inhibition of leukocyte adhesion does not completely prevent the venular dysfunction associated with ischemia-reperfusion.Supported by a grant from the National Institutes of Diabetes and Digestive and Kidney Diseases (DK 33594).     

A murine model to study leukocyte rolling and intravascular trafficking in lung microvessels

The cascade of leukocyte interactions under conditions of blood flow is well established in the systemic microcirculation, but not in lung microcirculation. We have developed a murine model to study lung microcirculation by transplanting lung tissue into dorsal skin-fold window chambers in nude mice and examining the ability of leukocytes to traffic within revascularized lung microvessels by intravital microscopy. The revascularized lung allograft demonstrated a network of arterioles, capillaries, and postcapillary venules with continuous blood flow. Stimulation of the lung allograft with TNF-alpha induced leukocyte rolling and adhesion in both arterioles and venules. Treatment with function-blocking anti-selectin mAb revealed that P- and L-selectin are the predominant rolling receptors in the lung microvessels, with E-selectin strengthening P-selectin-dependent interactions. Intravital microscopic studies also demonstrated that during their transit in capillaries, some leukocytes undergo shape change and continue to roll as elongated cells in postcapillary venules. Furthermore, the revascularized microvessels demonstrated the ability to undergo vasoconstriction in response to superfusion with endothelin-1. Overall, these studies demonstrate that the revascularized lung allograft is responsive to various external stimuli such as cytokines and vaso-active mediators and serves as a model to evaluate the interaction of leukocytes with the vascular endothelium in the lung microcirculation under acute as well as chronic experimental conditions.     

Leukocyte emigration and migration in the vagina following mating in the rabbit

Within 45 minutes after mating in the rabbit, numerous heterophil leukocytes adhere to the endothelium of venules in the vagina. Initial association appears to occur via small protuberances from the leukocyte which fit into small indentions in the endothelial cell. Following adherence, leukocytes flatten and pass between endothelial cells. A regular intercellular space separates the leukocyte from the endothelial cells. Leukocytes subsequently migrate through the connective tissue to the epithelium. By three hours postcoitus, the region beneath the basement lamina of the vaginal epithelium is crowded with numerous juxtaposed leukocytes. Leukocytes subsequently migrate between epithelial cells into the vaginal lumen where they actively engulf spermatozoa. Spermatozoa appear to be ingested head first. Numerous small filaments are observed in the leukocyte cytoplasm in the region adjacent to the sperm head. Degranulation of azurophyl granules follows sperm uptake. The leukocyte response can be elicited either by spermatozoa (from the epididymis) or by semen (from vasectomized bucks), but is not elicited by ovulation (with human chorionic gonadotropin). It is suggested that the response may be initiated because the vagina does not distinguish between semen, spermatozoa and bacterial infection.     

Periarteriolar localization of mast cells promotes oriented interstitial migration of leukocytes in the hamster cheek pouch.

As studied by intravital microscopy, mast cell-dependent inflammatory reactions evoked by antigen or compound 48/80 in the hamster cheek pouch involved leakage of plasma and emigration of leukocytes exclusively from the venules. The leukocyte diapedesis and subsequent tissue migration induced by antigen or compound 48/80 were oriented from the venules towards adjacent arterioles. In contrast, leukocyte emigration induced by a mast cell-independent stimulus, leukotriene B4, did not show preferential orientation towards arterioles. Moreover, mast cells were abundant in the hamster cheek pouch, and they were localized predominantly along arterioles, rather than along venules. Because mast cells are considered to be the source of the chemotactic mediators causing the leukocyte emigration, the periarteriolar mast cell localization may be of functional significance by creating chemotactic gradients between arterioles and venules, thereby promoting oriented and effective interstitial migration of leukocytes. Whether or not a similar mechanism is operative in other species and tissues remains to be established, however, arteriolar predominance of mast cells was observed also in rat calvarial periosteum and in mouse skin.     

Tubulointerstitial inflammation, cast formation, and renal parenchymal damage in experimental pyelonephritis.

Some basic changes in experimental pyelonephritis were studied by transmission and scanning electron microscope. Initially, bacteria settled and multiplied in capillaries and venules. Leukocytes first marginated and then escaped from the capillaries, particularly to the wide peritubular interstitium. After opening the tubular basement membrane, the infiltrating leukocytes were immediately localized in the tubular wall between epithelial cells but were never seen between the epithelial cells and the underlying basement membrane. The inflammatory cells seemed not to be able to pass through the tight junctions of the nonnecrotic tubular epithelium. As a consequence of severe inflammatory injury, the tight junctions exhibited alterations of intermediate junction type. Where circumscribed necrosis of the tubular walls occurred, leukocytes appeared in the lumen. Thus, pus casts originated from these sites, apparently as drainage of interstitial abscesses. The secondary/regressive and regenerative/tubular changes were similar to those occurring after various tubular lesions.     

Active participation of endothelial cells in inflammation

Leukocyte migration from the blood into tissues is vital for immune surveillance and inflammation. During this diapedesis of leukocytes, the leukocytes bind to endothelial cell adhesion molecules and then migrate across the vascular endothelium. Endothelial cell adhesion molecules and their counter-receptors on leukocytes generate intracellular signals. This review focuses on the active function of endothelial cells during leukocyte-endothelial cell interactions. We include a discussion of the "outside-in" signals in endothelial cells, which are stimulated by antibody cross-linking or leukocyte binding to platelet-endothelial cell adhesion molecule-1, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Some of these signals in endothelial cells have been demonstrated to actively participate in leukocyte migration. We suggest that some of the adhesion molecule signals, which have not been assigned a function, are consistent with signals that stimulate retraction of lateral junctions, stimulate endothelial cell basal surface adhesion, or induce gene expression.     

Responses of microvasculature in the paired cheek pouches of hamsters to blood loss and blood substitution

Comparison of arteriolar and venular responses in the left and right cheek pouches of hamsters to blood loss and blood substitution showed that blood loss led to a greater constriction of arterioles and venules in the right pouch than in the left and that blood substitution decreased arteriolar constriction and restored venular size in the right pouch, but resulted in a more marked constriction of both arterioles and venules in the left pouch. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 1, pp. 30–32, January, 1996 Presented by V. V. Kupriyanov, Member of the Russian Academy of Medical Sciences     

Effect of blood gases and pH on thromboembolic reactions in rabbit mesenteric microvessels

The influence of changes in systemic blood gas and pH values on the thromboembolic reaction following wall puncture was studied in rabbit mesenteric arterioles and venules (diameter 20–40 m), using intravital videomicroscopy. Under normal circumstances the number of emboli produced was higher in arterioles than in venules (6 and 1, respectively). The initial thrombus growth, the number of emboli produced per vessel and the total duration of the embolisation period were not significantly influenced by changes in blood gas and pH values in both arterioles and venules. Therefore, the observed difference in thromboembolic reaction between arterioles and venules cannot be explained by differences in blood gas and pH values in these microvessels. Since reduced velocity, as a measure of wall shear rate, did not correlate with the thromboembolic reaction in arterioles or venules, fluid dynamics can also not explain the difference, indicating that the thrombogenic or anuthrombogenic activity of arteriolar and venular walls differs following injury. A combination of hypercapnia and hypoxia was found to result in a prolongation of the average time period needed to produce a new embolus in both vessel types. This prolongation in embolus production time was largely due to the occurrence of periods, in which the thrombus did not grow, reflecting hampering of the adhesion and aggregation of blood platelets to a growing thrombus under hypereapnic/hypoxic conditions.     

Endothelial gaps and adherent leukocytes in allergen-induced early- and late-phase plasma leakage in rat airways.

Exposure of sensitized individuals to antigen can induce allergic responses in the respiratory tract, manifested by early and late phases of vasodilatation, plasma leakage, leukocyte influx, and bronchoconstriction. Similar responses can occur in the skin, eye, and gastrointestinal tract. The early-phase response involves mast cell mediators and the late-phase response is leukocyte dependent, but the mechanism of leakage is not understood. We sought to identify the leaky blood vessels, to determine whether these vessels contained endothelial gaps, and to analyze the relationship of the gaps to adherent leukocytes, using biotinylated lectins or silver nitrate to stain the cells in situ and Monastral blue as a tracer to quantify plasma leakage. Most of the leakage occurred in postcapillary venules (< 40-microns diameter), whereas most of the leukocyte migration (predominantly neutrophils) occurred in collecting venules. Capillaries and arterioles did not leak. Endothelial gaps were found in the leaky venules, both by silver nitrate staining and by scanning electron microscopy, and 94% of the gaps were distinct from sites of leukocyte adhesion or migration. We conclude that endothelial gaps contribute to both early and late phases of plasma leakage induced by antigen, but most leakage occurs upstream to sites of leukocyte adhesion.     

Leukocyte-endothelial interactions in pial arterioles and venules on development of cerebral ischemia in rats

In vivo microscopy was used to study the interaction between leukocytes and the pial venular and arteriolar endothelium in rats during cerebral ischemia evoked by bilateral ligation occlusion of the carotid arteries. Specimens were obtained from 40 arterioles and 30 venules (diameter up to 40 μm) of the pia mater from Wistar rats (n = 7) subjected to ischemia for 5 h to respiratory arrest. The experimental data demonstrated significant differences in changes in the adhesion of leukocytes to the endothelium of arterial and venous microvessels during the development of hypoxia. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 94, No. 1, pp. 45–52, January, 2008.     

Inflammatory cell migration into the central nervous system: a few new twists on an old tale

Understanding the mechanisms of leukocyte trafficking into the brain might provide insights into how to modulate pathologic immune responses or enhance host protective mechanisms in neuroinflammatory diseases such as multiple sclerosis. This review summarized our knowledge about the sites for leukocyte entry into the central nervous system, highlighting the routes from blood into the perivascular space and brain parenchyma through the blood-brain barrier. We further discussed the multistep paradigm of leukocyte-endothelial interactions at the blood-brain barrier, focusing on the adhesion molecules and chemokines involved in leukocyte transmigration. Luminal chemokines, which are immobilized on endothelial surfaces, initiate leukocyte integrin clustering and conformational change, leading to leukocyte arrest. Some leukocytes undergo post-arrest locomotion across the endothelial surface until interendothelial junctions are identified. Leukocytes then extend protrusions through the interendothelial junctions, in search of abluminal chemokines, which will serve as guidance cues for transmigration. Extravasating cells first accumulate in the perivascular space between the endothelial basement membrane and the basement membrane of the glia limitans. Matrix metalloproteases may be involved in leukocyte transverse across glia limitans into the brain parenchyma. The adhesion molecules and chemokine receptors provide attractive targets for neuroinflammatory diseases because of their important role in mediating central nervous system inflammation.     

Leukocyte fluid shear response in the presence of glucocorticoid

Leukocytes respond to physiological fluid shear stress ( approximately 1.5 dyn/cm(2)) by cytoplasmic reorganization. The cytoplasm is also influenced, however, by glucocorticoids. In this study, we explore how glucocorticoids may affect the leukocyte fluid shear response. Normal leukocytes, exposed to fluid shear in vitro during active migration, retract pseudopods accompanied by modestly decreasing intracellular calcium ions. In contrast, dexamethasone (DX)-treated leukocytes project pseudopods after shear exposure with a significant rise in intracellular calcium ions, an effect that can be blocked by voltage-dependent calcium channel blockers. Although a cyclic adenine monophosphate analog blocks calcium influx and pseudopod projection by DX, inhibition of A-kinase induces reversal of the shear response, as seen with DX treatment. DX also reverses the leukocyte shear response in vivo in the rat circulation. Leukocytes that adhere to the endothelium in postcapillary venules of control rats return into the circulation only after pseudopod retraction, and in DX-treated rats, adherent leukocytes return into the circulation still with projecting pseudopods. The fraction of circulating leukocytes with pseudopods in DX-treated rats is higher than in controls. Thus, the reversal of leukocyte shear response by glucocorticoids may contribute to an enhanced incidence of circulating leukocytes with pseudopods, a process that affects the kinetics of these cells in the microcirculation.     

婴儿和足月胎儿下颌下腺的微血管构筑

用血管铸型扫描电镜法，观察了婴儿和足月胎儿下颌下腺的微血管构筑，腺泡微动脉自小叶内部行至腺泡周围分成毛细胞管网，或穿出小叶至小叶的基底面，沿小叶表面延伸并分成毛细血管网，腺泡毛细血管网排列稀疏。足月胎儿的纹状管周围为毛细血管后微静脉和微静脉交织吻合成的网状微静脉丛。婴儿的纹状管周围为排列密集的毛细血管网。足月胎儿的腺泡毛细血管汇入纹状管周围微静脉丛，婴儿的与纹状管周围毛细血管网相连。小叶间导管周围     

Reduction in shear stress, activation of the endothelium, and leukocyte priming are all required for leukocyte passage across the blood--retina barrier

The passage of leukocytes across the blood-retina barrier at the early stages of an inflammatory reaction is influenced by a complex series of interactions about which little is known. In particular, the relationship between hydrodynamic factors, such as shear stress and leukocyte velocity, to the adherence and subsequent extravasation of leukocytes into the retina is unclear. We have used a physiological method, scanning laser ophthalmoscopy, to track labeled leukocytes circulating in the retina, followed by confocal microscopy of retinal flatmounts to detect infiltrating cells at the early stage of experimental autoimmune uveitis. This has shown that retinal vessels are subjected to high shear stress under normal circumstances. During the inflammatory reaction, shear stress in retinal veins is reduced 24 h before leukocyte infiltration. This reduction is negatively correlated with leukocyte rolling and sticking in veins and postcapillary venules, the sites of leukocyte extravasation. Activation of vascular endothelial cells is also a prerequisite for leukocyte rolling and infiltration. In addition, antigen priming of leukocytes is influential at the early stage of inflammation, and this is seen clearly in the reduction in rolling velocity and adherence of the primed leukocytes in activated retinal venules, 9 days postimmunization.     

Cocaine-induced cerebral vascular damage can be ameliorated by Mg2+ in rat brain

Cocaine HCl (10 micrograms/ml) delivered perivascularly to the surface of the rat brain resulted in rapid contraction of pial arterioles, which reduced the diameters by 26% compared to controls. This was followed by venular vasospasm and rupture of postcapillary venules and micro-hemorrhages at postcapillary sites. Administration of Mg aspartate HCl, by intraarterial or intravenous infusion (1, 10 and 20 mumol/min), before or after the cocaine, produced dose-dependent inhibition (20-85%) of the cocaine-induced arteriolar spasms and prevention and attenuation of the venular vasculotoxicity and hemorrhaging. These data suggest that magnesium salts might be useful agents in the treatment of cocaine-induced intoxication and prevention of brain damage.     

哮喘大鼠气管微循环中白细胞粘附的活体观察

哮喘大鼠气管微循环中白细胞粘附的活体观察苗会１胡清华１薛全福２新见英幸３庄逢源１支气管哮喘（简称哮喘）是当今世界常见的慢性呼吸道疾病，近年的报道表明其患病率及死亡率有上升趋势［１］。哮喘的发病机制至今尚未完全阐明。自１９８０年代提出气道慢性炎症学说以...     

Neurogenic inflammation in the rat trachea. II. Identity and distribution of nerves mediating the increase in vascular permeability

Summary This study addresses the question of whether increased vascular permeability, which is a prominent feature of neurogenic inflammation in the respiratory tract, is mediated by sensory axons that end near venules in the airway mucosa. In these experiments, neurogenic inflammation was produced in the tracheal and bronchial mucosa of atropine-treated Long-Evans rats by electrical stimulation of the left or right superior laryngeal nerve and/or cervical vagus nerve. The particulate tracer Monastral blue was injected intravenously to localize the sites of increased vascular permeability, and microspectrophotometry was used to measure the amount of extravasated Monastral blue in the trachea and thereby quantify the increase in vascular permeability. In some rats, selective denervations were made to locate the cell bodies of neurons that mediate the increase in vascular permeability; in others, fluorescence immunohistochemistry and quantitative electron microscopic methods were used to determine which structures in the tracheal mucosa are innervated by these neurons. The study revealed that the vagally mediated increase in vascular permeability was sudden, transient (half-life=2.4 min) and restricted to venules. Stimulation of the left or right superior laryngeal nerve increased the permeability of venules in the extrathoracic trachea, whereas stimulation of either vagus nerve increased vascular permeability in the intrathoracic trachea and bronchi. All nerves had bilateral effects in the trachea, but the vagus nerves had largely unilateral effects in the bronchi. Neurons that mediated the increase in venular permeability had their cell bodies in the jugular (superior sensory) ganglion of the vagus nerve or rostral portion of the nodose (inferior sensory) ganglion. Preganglionic autonomic vagal neurons in the brain stem were not essential for this increase in venular permeability. Few nerves identifiable by substance P-immunohistochemistry or electron microscopy were located near the affected venules, and no nerves were within 1 m of the walls of venules. However, the epithelium and arterioles of the airway mucosa were densely innervated. All intraepithelial nerves were within 0.1 m of epithelial cells, and at least two-thirds of nerves near arterioles were within 1 m of the vessel walls. We conclude that the increase in venular permeability associated with neurogenic inflammation in the trachea and bronchi of rats is mediated by sensory axons that travel in the vagus nerves and superior laryngeal nerves. We question whether tachykinins from the sensory nerves mediate the increase in vascular permeability through a direct action on venules, and raise the possibility that these nerves evoke the release from epithelial cells of mediators that contribute to the increase in vascular permeability.     

Mitochondrial biogenesis in the pulmonary vasculature during inhalational lung injury and fibrosis

Cell survival and injury repair is facilitated by mitochondrial biogenesis; however, the role of this process in lung repair is unknown. We evaluated mitochondrial biogenesis in the mouse lung in two injuries that cause acute inflammation and in two that cause chronic inflammation and pulmonary fibrosis. By using reporter mice that express green fluorescent protein (GFP) exclusively in mitochondria, we tracked mitochondrial biogenesis and correlated it with histologic lung injury, proliferation, and fibrosis. At 72 hours after acute LPS or continuous exposure to hyperoxia (Fio2, 1.0), the lungs showed diffuse infiltration by inflammatory cells in the alveolar region. In reporter mice, patchy new mitochondrial fluorescence was found in the alveolar region but was most prominent and unexpected in perivascular regions. At 14 days after instillation of asbestos or bleomycin, diffuse chronic inflammation had developed, and green fluorescence appeared in inflammatory cells in the expanded interstitium and was most intense in smooth muscle cells of pulmonary vessels. In all four lung injuries, mitochondrial fluorescence colocalized with mitochondrial superoxide dismutase, but not with proliferating cell nuclear antigen. These data indicate that vascular mitochondrial biogenesis is activated in diverse inhalational lung injuries along with oxidative stress. This finding indicates a unique and unexpected mechanism of metabolic adaptation to pulmonary fibrotic injuries.