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.     

Direct leukocyte migration across pulmonary arterioles and venules into the perivascular interstitium of murine lungs during bleomycin injury and repair

During acute lung injury and repair, leukocytes are thought to enter the lung primarily across alveolar capillaries and postcapillary venules. We hypothesized that leukocytes also migrate across pulmonary arterioles and venules, which serve as alternative sites for leukocyte influx into the lung during acute lung injury and repair. Lung sections from C57BL/6J mice up to 14 days after intratracheal bleomycin (3.33 U/kg) or saline instillation were assessed by light, fluorescence, confocal, and transmission electron microscopy for evidence of inflammatory cell sequestration and transmigration at these sites. After bleomycin treatment, large numbers of leukocytes (including neutrophils, eosinophils, and monocytes) were present in the vascular lumina and in perivascular interstitia of pulmonary arterioles and venules, as well as within the vascular walls. Leukocytes were observed within well-defined pathways in arteriolar walls and much less structured pathways in venular walls, apparently in the process of transmigration. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were expressed at sites of leukocyte interaction with the luminal surface, especially in arterioles. Leukocytes appeared to exit from the vessels near collagen fibers into the perivascular interstitium. Results indicate that leukocytes can directly migrate across arteriolar and venular walls into the perivascular interstitium, which may represent an important but under-recognized pathway for leukocyte influx into the lung during injury and repair.     

Effect of staphylococcal toxin on the microcirculatory system]

Study of microcirculation in the mesentery of narcotized rats showed that the staphylococcal toxin causes increased migration of leukocytes, aggregation of formed elements, and disturbance of the blood flow in the small venules and arterioles. The toxin stimulates rhythmic contractile activity of the lymphatic microvessels and induces their constriction and even complete obliteration. The lymphoconstrictive effect of the toxin is weakened by verapamil and completely removed by dimethyl sulfoxide.     

Morphodynamic analysis of the role of leukocytes in the microcirculation disorder of the cerebral cortex

The aim of this work was to establish the reasons for the disturbances of microcirculation (no reflow) during various kinds of oxygen starvation of the brain. Using contact optics and video-recording, direct observations were performed and leukocyte and erythrocyte displacement was monitored in the blood vessels of rat cerebral cortex in vivo in normoxia, during the development of various kinds of oxygen deficiency up to the terminal periods of life of an organism. In normoxia leukocytes were shown to decelerate erythrocyte movement in capillaries and to cause the irregularity of the capillary blood flow. Using the color videofilm recording it was shown that at deep stages of brain oxygen deficiency the disturbances of microcirculation up to its complete cessation were explained by the occlusion of venous microvessels as a result of massive adhesion of leukocytes to the inner surface of venules and the smallest veins. The co-adhesion of leukocytes was also found to take place. The latter leads to the formation of large leukocyte aggregates in venous vessels, results in "no reflow" effect. Leukocyte aggregates in veins block the venous outflow from the brain and may become the immediate cause of organism death as a result of disruption of circulation and oxygen deficiency.     

Endotoxin stimulates monocyte-endothelial cell interactions in mouse intestinal Peyer's patches and villus mucosa

Although monocyte-endothelial cell interactions represent an initial step in controlling the recruitment of monocytes in inflamed tissues, their dynamic processes in microvessels of lymphoid (Peyer's patches) and non-lymphoid (villus) regions in gut-associated lymphoid tissue remain poorly understood. We monitored the migration of fluorescence-labelled monocytes derived from the spleen in intestinal microvessels with or without lipopolysaccharide (LPS) treatment and investigated the role of adhesion molecules, P-selectin, vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In control mice, there were few interactions between infused monocytes and the endothelium of intestinal microvessels. The monocyte-endothelial interactions (both rolling and adhesion) were significantly increased in intestinal microvessels of LPS-treated mice compared with those in controls. Anti-P-selectin monoclonal antibody (MoAb) significantly suppressed the LPS-induced increase in monocyte rolling in postcapillary venules of Peyer's patches and submucosal venules. Anti-VCAM-1 MoAbs significantly suppressed the LPS-induced increase in monocyte adhesion to postcapillary venules (PCVs) of Peyer's patches, submucosal venules, and villus capillaries. In contrast, anti-ICAM-1 MoAb significantly suppressed the number of adherent monocytes in PCV of Peyer's patches but not in submucosal venules or villus capillaries. These observations demonstrated that LPS treatment resulted in a significant increase in recruitment of monocytes both in microvessels of lymphoid and non-lymphoid regions and that P-selectin, VCAM-1 and ICAM-1 appeared to play important roles in LPS-induced interactions.     

Oxygen supply to contracting skeletal muscle at the microcirculatory level: diffusion vs. convection

An adequate supply of oxygen is essential for the normal function of all cells. Because skeletal muscle cells have the ability to vary their oxygen demand by over an order of magnitude on going from rest to vigorous contraction, it is important that mechanisms be in place to ensure that the supply of oxygen is maintained at sufficient levels. Microcirculation plays a critical role in this process, as the terminal branches of this intricate network of blood vessels determine the distribution of perfusion, as well as the structural framework for diffusion. The oxygen supply depends on proper functioning of both the convective and diffusive components of the transport system. Convection is responsible for the long-range, rapid transport of oxygen by bulk flow of the blood and diffusion is the efficient mechanism for transport over the short distances between capillaries and muscle cells. Convective transport is dominated by the movement of red blood cells, as virtually all the oxygen at normal haematocrit is carried inside them, reversibly bound to haemoglobin. Over the years, specialized techniques, many of them video-based, have been developed for use in intravital microscopy to measure the parameters needed to quantify convection and diffusion in both capillaries and the larger microvessels, arterioles and venules. Most of our knowledge of oxygen transport in the microcirculation of muscle pertains to the resting condition, because one must be able to visualize the structures of interest, such as microvessels and muscle cells, and the large tissue movements that occur during contraction preclude measurements during that time. In resting muscle it has been found that the arterioles are the primary site of the diffusion of oxygen from the circulation, where the oxygen is utilized by nearby muscle cells or diffuses directly to nearby venules or capillaries. Diffusive interactions among neighbouring capillaries have also been observed. In contracting muscles, microvessels observed immediately following the period of stimulation exhibit enhancements of both convective (increased flow of red blood cells) and diffusive (increased perfused capillary surface area) transport. The use of computational models in the interpretation of experimental studies is leading to an increased understanding of the processes that underlie the oxygen transport system in skeletal muscle.     

Pathology of microhemocirculatory vessels in congenital heart defects with left-to-right shunt

Pathology of microcirculatory vessels of greater omentum and bulbar conjunctiva in children with congenital heart disease presenting with a left-to-right shunt was studied by biomicroscopic and morphologic methods. It has been shown that in this disorder of central hemodynamics the progressive reduction of true capillaries arises both in bulbar conjunctiva, and especially in the omentum. The authors believe that such deficit of tissue blood supply leads to reduction of density in terminal vascular networks and to the simplification of the construction of microcirculatory, in which the number of main microvessels (capillaries) increases, thus combining directly arterioles and venules, resulting in blood flow centralization and decrease of the efficiency of microcirculation.     

烟碱对大鼠脑微循环中白细胞运动的影响

目的 :观察烟碱对大鼠脑微循环中白细胞运动状态的影响。方法 :采用围堤式颅窗直视大鼠大脑软脑膜微循环 ,若丹明 6G标记循环血白细胞 ,微循环仪观察软脑膜微血管 (直径 2 5～ 40 μm ,长度 10 0μm)内白细胞的运动状态。结果 :烟碱能浓度依赖性的运动大鼠脑微血管内白细胞在内皮表面的滚动和粘附 ,预先皮下注射烟碱受体拮抗剂美加明可使滚动和粘附的白细胞分别降至 14 .2± 1.1个和 1.3± 0 .7个 (对照组为 10 .2± 1.2个 )。抗E 选择素单抗能明显阻断烟碱依赖所至的白细胞滚动 ,使滚动白细胞数目从 14 .2± 1.7个减为 2 .8± 0 .8个 ,而抗αvβ3整合素抗体能使粘附的白细胞数目从 10 .6± 1.0个降至 3 .8± 1.3个。结论 :烟碱可改变大鼠脑膜微循环中白细胞的运动状态 ,使白细胞在血管内皮表面滚动或粘附于其表面 ;烟碱的作用与激活脑烟碱受体有关 ;E 选择素介导烟碱引起的白细胞滚动 ,而αvβ3整合素则与烟碱引起的白细胞与血管内皮的粘附有关。     

Alpha 2,3-sialyltransferase-IV is essential for L-selectin ligand function in inflammation

L-selectin belongs to the C-type lectin family of glycoproteins and is constitutively expressed on most leukocytes. L-selectin mediates leukocyte rolling in inflamed microvessels and high endothelial venules (HEV) via binding to specific carbohydrate structures on selectin ligands. Previous studies using sialidase treatment suggested a role of sialic acid residues in L-selectin-dependent rolling. To investigate the role of the alpha2,3-sialyltransferase (ST3Gal)-IV on L-selectin ligand activity in vivo, we studied leukocyte rolling in inflamed venules of the cremaster muscle and in Peyer's patch HEV of ST3Gal-IV-deficient mice and littermate control mice. In cremaster muscle venules with or without TNF-alpha treatment, L-selectin-dependent rolling was almost completely abolished in ST3Gal-IV(-/-) mice. In both models, L-selectin interacts with P-selectin glycoprotein ligand-1 (PSGL-1) presented by adherent leukocytes and leukocyte fragments, but not with endothelial L-selectin ligands. In contrast, L-selectin-dependent rolling in Peyer's patch HEV, which is mediated by unknown endothelial L-selectin ligands, was not impaired in the absence of ST3Gal-IV. Our in vivo data show that PSGL-1, the molecule responsible for L-selectin-mediated leukocyte interactions in inflammation, is dependent on ST3Gal-IV, while alpha2,3-sialylation by ST3Gal-IV is not necessary for L-selectin ligand activity on high endothelial cells of Peyer's patch HEV.     

Blood flow structure related to red cell flow: determinant of blood fluidity in narrow microvessels

The review article deals with phenomena of the blood flow structure (structuring) in narrow microvessels-capillaries and the adjacent arterioles and venules. It is particularly focused on the flow behavior of red blood cells (RBCs), namely, on their specific arrangements of mutual interaction while forming definite patterns of self-organized microvascular flow. The principal features of the blood flow structure in microvessels, including capillaries, include axial RBC flow and parietal plasma layer, velocity profile in larger microvessels, plug (or bolus) flow in narrow capillaries, and deformation and specific behavior of the RBCs in the flow. The actual blood flow structuring in microvessels seems to be a most significant factor in the development of pathological conditions, including arterial hypertension, brain and cardiac infarctions, inflammation, and many others. The blood flow structuring might become a basic concept in determining the blood rheological properties and disorders in the narrow microvessels. No solid theoretical (biorheological) basis of the blood flow structuring in microvessel has been found, but in the future it might become a foundation for a better understanding of the mechanisms of these properties under normal and pathological conditions in the narrowest microvessels 5 to 25 microm large. It is also a topic for further biorheological research directed to find the background of actual physiopathological phenomena in the microcirculation.     

Critical role of microvasculature basal lamina in ischemic brain injury

Cerebral vascular system can be divided into two categories: the macrovessels and microvessels. The microvessels consist of arterioles, capillaries and venules. There are three basic components in the microvasculature: endothelial cells, basal lamina and end-feet of astrocytes. The basal lamina is situated between the endothelial cells and the end-feet of astrocytes, and connects these two layers together. Damage to the basal lamina causes the dismantlement of microvascular wall structures, which in turn results in increase of microvascular permeability, hemorrhagic transformation, brain edema and compromise of the microcirculation. The present article reviews microvascular changes during ischemic brain injury, with emphasis on basal lamina damage.     

Distribution of oxygen tension on the surface of arterioles, capillaries and venules of brain cortex and in tissue in normoxia: an experimental study on rats

 The distribution of oxygen tension (PO₂) was studied in normoxia on the surface of arterioles, capillaries and venules of rat brain cortex, both longitudinally and in tissue radially from the wall of microvessels. Along the arteriolar tree, PO₂ decreased from 81.2±6.2 mmHg (mean±SD) on 1°A (first-order branch) arterioles to 61.5±12 mmHg on 5°A arterioles. Transmural flux of oxygen from blood to tissue increased markedly at the level of minute 4°A–5°A arterioles. At the arterial end of cortical capillaries, PO₂ averaged 57.9±10.6 mmHg, n=19, (or, in terms of blood oxygen saturation SO_2; 82±9%) and 258±19 μm downstream 40.9±11.5 mmHg, n=19, (SO₂ 59±18%). The averaged PO₂ drop on the capillaries studied was 17±9 mmHg, and the longitudinal PO₂ gradient was accordingly 0.07±0.04 mmHg/μm (SO₂ 0.1±0.06%/μm). The radial profiles of tissue PO₂ recorded near arterioles, capillaries and venules clearly demonstrated that all these microvessels supply oxygen to brain tissue. The PO₂ distribution on venules was characterized by pronounced heterogeneity. Received: 30 March 1998 / Received after revision: 28 July 1998 / Accepted: 25 November 1998     

The microcirculatory bed of the human myocardium in sudden cardiac death

A ventricular capillary bed was studied in 30 hearts of humans upon sudden cardiac death according to modified technique of Karnovsky-Roots. A 10-80% narrowing of coronary artery lumen was found due to formation of atherosclerotic plaques. The assessment of butyrylcholinesterase activity in myocardial sections proved helpful in providing the appearance of microcirculation from small arterioles to venules. Determination of volumetric density of microvessels suggested an inverse correlation between the degree of stenosis of major coronary arterial branches and volumetric density of ventricular capillaries in the heart.     

Receptor cleavage and P-selectin-dependent reduction of leukocyte adhesion in the spontaneously hypertensive rat

The SHR, a genetic model for hypertension and the metabolic syndrome, has attenuated leukocyte adhesion to the postcapillary endothelium by an unknown mechanism. Based on recent evidence of elevated levels of MMPs in plasma and on microvascular endothelium of the SHR with cleavage of several receptor types, we hypothesize that the reduced leukocyte-endothelial interaction is a result of enhanced proteolytic cleavage of P-selectin on the postcapillary endothelium and PSGL-1 on leukocytes. The attenuated rolling interactions of SHR leukocytes with the endothelium were restored by chronic treatment with a broad-spectrum MMP inhibitor (CGS) for 24 weeks. The SHR MMP levels, in plasma and mesentery, as well as the systolic blood pressure, decreased significantly with treatment. In the SHR mesentery, labeling of P-selectin in the postcapillary venules by immunohistochemistry demonstrated, on average, a 31% lower extracellular P-selectin density compared with the normotensive WKY. A significantly lower extracellular PSGL-1 density on the membranes of SHR neutrophils compared with the WKY also supported our hypothesis. In vivo stimulation of the mesenteric postcapillary venules with histamine demonstrated that the SHR had an attenuated response, as measured by leukocyte rolling velocity on the endothelium. The reduced P-selectin and PSGL-1 density, on SHR postcapillary endothelium and on SHR leukocytes, respectively, was restored significantly by chronic MMP inhibition. The impaired ability of SHR leukocytes to reduce rolling velocity upon inflammatory stimulation led to fewer firmly adhered leukocytes to the endothelium as a contributor to immune suppression.     

Leukocyte behaviour and permeability in the rat mesenteric microcirculation following induction of ovulation

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS), a highly dangerous and incompletely understood complication of ovulation induction with exogenous gonadotrophins, can include haemoconcentration, hypovolaemia, hypotension, acute renal insufficiency, thromboembolism and ultimately death. Using intravital microscopy, we examined microvascular permeability and leukocyte-endothelial cell interactions in the rat mesenteric microcirculation associated with induction of ovulation. METHODS: In female rats treated with hMG and hCG, mesenteric venules were observed by intravital microscopy assisted by a video imager. Erythrocyte velocity was monitored, and rolling and adhesion of leukocytes were studied by transmission video images. Transvascular leakage of fluorescein isothiocyanate-labelled albumin was assessed by epi-illumination. RESULTS: Administration of hMG and hCG significantly increased vascular protein leakage within a few hours, and also reduced rolling velocities of leukocytes in venules and increased numbers of leukocytes adherent to endothelium at 16 h following hCG injection. The administration of antibodies against intracellular adhesion molecule (ICAM)-1 inhibited these reactions. CONCLUSION: By induction of ovulation, vascular permeability is increased not only at the surface of the ovary but also in the mesentery. Alteration of leukocyte behaviour in the microcirculation through mechanisms involving ICAM-1 is one likely cause of the protein leakage.     

Involvement of CD44 in leukocyte trafficking at the blood-retinal barrier

In the present study, we investigated the involvement of CD44 in leukocyte trafficking in vivo at the blood-retinal barrier using experimental autoimmune uveoretinitis (EAU) as a model system. Leukocyte trafficking was evaluated using adoptive transfer of calcein-AM (C-AM)-labeled spleen cells harvested from syngeneic mice at prepeak severity of EAU to mice at a similar stage of disease. CD44 and its ligand hyaluronan were up-regulated in the eye during EAU. CD44-positive leukocytes were found sticking in the retinal venules and postcapillary venules but not in the retinal arterioles nor in mesenteric vessels. Preincubation of in vitro C-AM-labeled leukocytes with anti-CD44 monoclonal antibodies (mAb; IM7) or high molecular weight hyaluronic acid (HA) before transfer significantly suppressed leukocyte rolling but not sticking in retinal venules and also reduced cell infiltration in the retinal parenchyma. Administration of the HA-specific enzyme hyaluronidase to mice before cell transfer also reduced leukocyte infiltration, suggesting that CD44-HA interactions are involved in leukocyte recruitment in EAU. This was further supported by the observation that disease severity was reduced by administration of anti-CD44 mAb (IM7) at the early leukocyte-infiltration stage. Further studies also indicated that CD44 activation was associated with increased levels of apoptosis, and this may also be in part responsible for the reduction in disease severity. These findings demonstrate that CD44 is directly involved in leukocyte-endothelial interaction in vivo and influence the trafficking of primed leukocytes to the retina and their overall survival.     

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.     

Recurrent obstructive apneas trigger early systemic inflammation in a rat model of sleep apnea

Obstructive sleep apnea (OSA) is associated with vascular disorders possibly due to systemic inflammation. To determine whether repeated episodes of OSA in a rat model lead to endothelial cell activation and systemic leukocyte recruitment in the microcirculation. Three experimental groups (apnea, sham and naive) were studied. The apnea group was instrumented and subjected to repeated obstruction for 3h (rate 60/h, length 5s) using a special device. The sham group was only instrumented and the naive group was used as a control. Leukocyte-endothelial cell interactions (intravital microscopy) and expression of P-selectin (immunohistochemistry) were determined in colonic venules. The apnea group induced a significant increase in the flux of leukocytes rolling, number of rolling leukocytes and number of adherent leukocytes when compared with the sham or naive groups. P-selectin was up-regulated only in the apnea group. This experimental model of recurrent obstruction demonstrates rapid endothelial cell activation, suggesting the onset of an inflammatory response.     

Spontaneous leukocyte rolling in rat and mouse microvessels is independent of mast cell activity

OBJECTIVE AND DESIGN: The role of mast cells in spontaneous leukocyte rolling in venules of the mouse cremaster muscle and rat mesentery was investigated. MATERIALS: The experiments were carried out using mast cell-deficient rats (Ws/Ws), WBB6F1 mice (W/Wv), and their congenic littermates (wild type). Treatment: Administration of compound 48/80 intraperitoneally (50 microg) in rats and intrascrotally (5 microg) in mice, 4 h prior to the experiments. METHODS: Intravital microscopy of the terminal vascular beds in mouse cremaster muscle and rat mesentery. RESULTS: The level of spontaneous leukocyte rolling and the rolling velocity in venules of mast cell-deficient animals exactly matched that seen in wild-type animals. Challenge with compound 48/80 markedly increased leukocyte adhesion and emigration in venules of wild-type animals. In contrast, the number of adherent and extravascular leukocytes was very low in compound 48/80-challenged animals lacking mast cells and did not differ from that seen in control animals treated with phosphate-buffered saline. CONCLUSIONS: The presence or activation of mast cells has no bearing on spontaneous leukocyte rolling, at least not in rat and mouse microvessels.     

Electrical resistance of arterioles and venules in the hamster cheek pouch

The electrical resistance of the vascular endothelium was determined on single microvessels in the hamster cheek pouch in order to obtain information about this variable in a mammalian preparation. So far, the technique has only been applied to frog microvessels. The technique consists of injection of current into the vascular lumen via a microelectrode and recording of the ensuing intravascular potential distribution by a second microelectrode. Cable theory was used for the analysis. The average diameter of the vessels under study was 41 micron for arterioles and 28 micron for venules. The average resistance of the vessel wall at 37 degrees C was 19 omega cm2 and 3.3 omega cm2, respectively. For the venules this is somewhat lower than what has been recorded on muscle capillaries (Olesen & Crone 1983) in the frog at room temperature, whilst the values on arterioles are rather similar. The calculated sodium permeabilities, PNa+, were for arterioles 4 X 10(-5) cm X s-1 and for venules 23 X 10(-5) cm X s-1. The high permeability values for arterioles and venules indicate that the vascular exchange function may not be limited to capillaries only.