Leukocyte kinetics in the microcirculation

The transport of leukocytes in the microcirculation is specific for the type, size, and the rheological and adhesive properties, the microanatomy of the host organ, and the hemodynamics. The adhesion to the endothelium is determined largely by the degree of activation via chemotactic factors. Leukocyte motion differs from that of red cells or platelets in several respects. When granulocytes enter into capillaries, they are deformed just like red cells. Under normal flow conditions, the time to deform at the entry to capillaries is typically 1,000 times larger than for the red cell, leading to temporary obstruction of the capillaries. After entry, granulocytes move with lower velocity than red cells which causes a cell train formation inside the capillary. At the venular side, the granulocyte is displaced from the center stream toward the endothelium by faster moving red cells. This process leads to systematic attachment of the granulocytes to the endothelium. At a reduced perfusion pressure or in the presence of locally elevated levels of chemotactic factors, the granulocytes may not be able to pass through the capillary network, which leads to microvascular obstruction. Organs with a narrow capillary network may thereby become filters for circulating granulocytes. This event is accompanied in many situations with damage to the host organ.     

内毒素血症大鼠肠系膜微循环中血管内皮细胞与白细胞的粘附性变化

本实验采用中文吖啶橙荧光标记技术,结合微循环观察用显微超高速摄录像装置,观察了内毒素对微血管内白细胞与微静脉血管内皮细胞的粘附性的影响。结果表明,内毒素对大鼠的血压、微血管口径和微动脉血流速度影响不大,微静脉血流速度在滴注内毒素后４５和６０ｍｉｎ下降了１６．６７％和１７．９５％（Ｐ＜０．０５）;但内毒素能迅速改变微静脉内的白细胞流态,明显增加附壁滚动的白细胞数和粘附白细胞密度指数,经测量同一微静脉内的白细胞和红细胞流速,求得白细胞与微静脉内皮细胞之间的破裂力在５ｍｉｎ和１５ｍｉｎ时下降了２５．９６％和４２．８８％（Ｐ＜０．０１）,下降趋势持续整个实验过程;说明内毒素能明显地增加白细胞与微静脉血管内皮细胞之间的粘附力。由此提示,研究白细胞与微静脉血管内皮细胞之间粘附力增强机制及寻找其抑制因素对改善微循环紊乱、抢救休克具有重要的临床意义。     

Substance P induces degranulation of mast cells and leukocyte adhesion to venular endothelium

Substance P (SP), one of the established neurotransmitters, evokes an immunoinflammatory response involving leukocyte adhesion to venular endothelium and the degranulation of mast cells. The pathogenetic relationship between these responses, however, remains unresolved. In this study, we propose to examine the changes associated with the activation of mast cells, as well as leukocyte adhesion to venular endothelium by in vivo observation of the rat mesentery. The use of an in vitro assay for intracellular Ca²⁺ mobilization and the degranulation of mast cells demonstrated the significant upper shift of concentration response to SP (10⁻⁴–10⁻⁵ M). In vivo experiments on the mesenteric microcirculation also showed that SP induced a significant increase in the number of degranulated mast cells as well as in the number of leukocytes adherent to the venular wall. Tranilast, a mast cell stabilizer, as well as SP antagonist (CP-96,345) significantly attenuated the extent of mast cell degranulation and leukocyte adhesion elicited by SP. Although an immunoneutralization against CD18 by WT-3 significantly attenuated the leukocyte adhesion, it had no influence on the mast cell degranulation after SP superfusion. These separate in vivo observations show that SP induces leukocyte adhesion to the venular endothelium, possibly through the degranulation of mast cells.     

ATP-mediated release of arachidonic acid metabolites from venular endothelium causes arteriolar dilation

This study was designed to test the hypothesis that venular administration of ATP resulted in endothelium-dependent dilation of adjacent arterioles through a mechanism involving cyclooxygenase products. Forty-three male golden hamsters were anesthetized with pentobarbital sodium (60 mg/kg ip), and the cremaster muscle was prepared for in vivo microscopy. ATP (100 microM) injected into venules dilated adjacent arterioles from a mean diameter of 51 +/- 4 to 76 +/- 6 microm (P < 0.05, n = 6). To remove the source of endothelial-derived relaxing factors, the venules were then perfused with air bubbles to disrupt the endothelium. Resting arteriolar diameter was not altered after disruption of the venular endothelium (51 +/- 5 microm), and the responses to venular ATP infusions were significantly attenuated (59 +/- 4 microm, P < 0.05). To determine whether the relaxing factor was a cyclooxygenase product, ATP infusion studies were repeated in the absence and presence of indomethacin (28 microM). Under control conditions, ATP (100 microM) infusion into the venule caused an increase in mean arteriolar diameter from 55 +/- 4 to 78 +/- 3 microm (P < 0.05, n = 6). In the presence of indomethacin, mean resting arteriolar tone was not significantly altered (49 +/- 4 microm), and the response to ATP was significantly attenuated (54 +/- 4 microm, P < 0.05, n = 6). These studies show that increases in venular ATP concentrations stimulate the release of cyclooxygenase products, possibly from the venular endothelium, to vasodilate the adjacent arteriole.     

Luminol-dependent photoemission from single neutrophil stimulated by phorbol ester and calcium ionophore--role of degranulation and myeloperoxidase

Luminol-dependent photonic burst from phorbol ester-treated single neutrophil was visually investigated by using an ultrasensitive photonic image intensifier microscope. Neutrophils stimulated by phorbol myristate acetate (0.1 microgram/ml) alone produced a negligible level of photonic activities in the presence of luminol (10 micrograms/ml). The additional application of 0.1 microM Ca2+ ionophore A23187 induced explosive changes of photonic burst corresponding to the distribution of neutrophils, and these photonic activities were gradually spread to extracellular space. Sodium azide, which prevents myeloperoxidase activity, inhibited Ca2+ ionophore-induced photonic burst from phorbol ester-treated neutrophil. These findings suggest a prerequisite role of degranulation and myeloperoxidase release in luminol-dependent photoemission from stimulated neutrophils.     

几种不同刺激对血管内白细胞粘附的影响

目的：研究几种刺激引起白细胞与内皮细胞粘附的变化。方法：本实验采用脉冲电刺激、缺血再灌、内毒素和白介素-8等物理或药物的作用,观察大鼠肠系膜细静脉内白细胞粘附及白细胞和血管内皮粘附之间的差别。结果：缺血再灌、内毒素、内毒素、脉冲电刺激和白介纱-8（IL-8）作用后肠系膜细静脉白细胞粘附数量比正常组明显增多,IL-8用药后30min细静脉内白细胞粘附数量最多、缺血再灌、内毒素、脉冲电刺激后白细胞粘附数量大致相同。结论：缺血再灌、内毒素、脉冲电刺激能诱导白细胞的粘附作用。造成内皮损伤,IL-8诱导白细胞的粘附作用最强。     

Differentiated microdomains of the luminal plasmalemma of murine muscle capillaries: segmental variations in young and old animals

We investigated the luminal surface of the continuous endothelium of the microvasculature of the murine heart and diaphragm to find out whether it has differentiated microdomains. The probes were ferritin molecules, cationized to pI's 6.8, 7.15, 7.6, 8.0 and 8.4, which were introduced by retrograde or anterograde perfusion through the aorta or vena cava after the blood was removed from the vasculature. The pattern of labeling was analyzed by electron microscopy and assessed quantitatively by morphometry in arterioles, capillaries, and venules identified in bipolar microvascular fields in the diaphragm. The results showed that the plasmalemma proper was heavily but discontinuously labeled by all cationized ferritins (CF) used, the labeling being less extensive on the venular endothelium. CF had access as individual molecules to a fraction of the vesicular population opened on the luminal front of the endothelium. Plasmalemmal vesicle labeling increased from approximately 10 to approximately 25% as the pI decreased from 8.4 to 6.8. Vesicle labeling also increased with CF concentration in the perfusate. All CF binding sites were removed by pronase and papain. Heparinase and heparitinase caused only a slight reduction in CF labeling. Neuraminidase decreased the extent and density of labeling, especially on the plasmalemma proper of the venular endothelium; this decrease was particularly pronounced in old animals.     

Signal transduction in monocytes and granulocytes measured by multiparameter flow cytometry.

The novel calcium indicator fura red and the oxidative burst indicator dihydrorhodamine (both excited at 488 nm) were used in combination with multiparameter flow cytometry to allow simultaneous kinetic measurements of calcium fluxes and oxidative bursts in monocytes and granulocytes. Using this method it was possible to obtain direct evidence for the following cell type- and stimulus-specific differences in signal transduction pathways: 1) n-formyl-methionyl-leucyl-phenylalanine (FMLP)/cytochalasin B-induced oxidative burst is several-fold higher in granulocytes than in monocytes although the calcium fluxes have similar amplitudes in the two cell types; 2) stimulus-induced calcium fluxes in granulocytes are mainly due to release from intracellular stores, whereas monocytes mobilize calcium mainly by influx from the medium; 3) the FMLP/cytochalasin B-induced calcium flux in monocytes is less sensitive to the G-protein inhibitor pertussis toxin than the flux in granulocytes; 4) in contrast to FMLP/cytochalasin B, the protein kinase C activator phorbol myristate acetate (PMA) induces an oxidative burst that is not preceded by a cytoplasmic calcium flux; 5) the PMA-induced oxidative burst can be triggered in monocytes and granulocytes that are depleted of intracellular calcium ions, whereas that induced by FMLP/cytochalasin B can not; 6) the G-protein inhibitor pertussis toxin blocks an early event in the signal transduction pathway of FMLP/cytochalasin B, as shown by inhibition of both calcium fluxes and oxidative burst; and 7) 100 nM of the protein kinase inhibitor staurosporine blocks the FMLP/cytochalasin B-induced respiratory burst by interfering with a step downstream to cytoplasmic calcium fluxes, whereas only 10-20 nM is necessary to block PMA-induced oxidative burst.     

Preferential activity of Tie2 promoter in arteriolar endothelium

The tyrosine kinase Tie2/Tek (the receptor for angiopoietins) is considered one of the most reliable markers of the endothelial phenotype, across organisms, organs, and developmental stages. However, endothelium is intrinsically heterogeneous in origin, composition and function, presenting an arteriolar/venular asymmetry. In this regard, the expression of Tie2 along the vascular tree, although thought to be homogenous, has not been systematically investigated. Therefore we questioned whether the activity of Tie2 promoter is uniform in the microvascular endothelium. To this end, we analyzed in situ the expression of the markers beta-galactosidase [LacZ(Tie2)] and green fluorescent protein (GFP) [GFP(Tie2)], placed under the Tie2 promoter in transgenic mice, in whole mount tissue samples, which allow the simultaneous evaluation of its relative distribution in various microvascular compartments. In the mesenteries of LacZ(Tie2) and GFP(Tie2) mice, we found that the activity of Tie2 promoter is asymmetrically distributed, being much stronger in arteries and arterioles than on the venular side of the vascular tree. This observation was replicated in the diaphragm of LacZ(Tie2) mice. The capillaries presented a mosaic pattern of Tie2 promoter activity. Stimulation of angiogenesis either by wounding, or by intraperitoneal injection of Vascular Endothelial Growth Factor (VEGF), revealed that the arteriolar/venular asymmetry is established at endothelial cellular level early during new capillary formation, even before the starting of the microvascular blood flow. In conclusion, a strong Tie2 promoter activity qualifies as a novel marker of the arteriolar phenotype in microvascular endothelium.     

Capillary plugging by granulocytes and the no-reflow phenomenon in the microcirculation

Granulocytes are large, stiff viscoelastic cells that adhere naturally to the vascular endothelium. On their passage through the capillary network they have to be deformed, and recent evidence indicates that they may impose a significant hemodynamic resistance. The entry time of granulocytes into capillaries is about three orders of magnitude longer than that for red cells. Inside the capillary the granulocytes move with a lower velocity than red cells. Under conditions when the capillary perfusion pressure is reduced and/or elevated levels of inflammatory products are present that increase the adhesion stress to the endothelium, granulocytes may become stuck in the capillary. In such a situation, the granulocytes form a large contact area with the capillary endothelium, they obstruct the lumen, and they may initiate tissue injury. After the restoration of the perfusion pressure the granulocytes may not be removed from the capillary owing to the adhesion to the endothelium. Capillary plugging by granulocytes appears to be the mechanism responsible for the no-reflow phenomenon, and together with oxygen free radical formation and lysosomal enzyme activity may constitute the origin for ischemic injury as well as other microvascular occlusive diseases.     

Five steps in leukocyte extravasation in the microcirculation by chemoattractants

For in vivo study of the phenomena observed in vitro, PMN (polymorphonuclear leukocyte) extravasation was analysed quantitatively in the microcirculation of the hamster cheek pouch using a video system. Topical application of leukotriene B(4) or N-formyl-methionylleucyl- phenylalanine increased dose dependently the number of PMNs adhering to the venules. Eighty to 90% of the adhering PMNs disappeared from the vascular lumen into the venular wall within 10-12 rain after the adhesion. After PMNs had passed through the endothelial cell layer, they remained in the venular wall for more than 30 min after application of the chemoattractants and appeared in the extravascular space. Thus, the process could be divided into five steps: (1) rolling and (2) adhesion to the endothelium, (3) passage through the endothelial layer (4) remaining in the venular wall, and (5) passage through the basement membrane.     

Open junctions in the endothelium of the postcapillary venules of the diaphragm

We have previously established that approximately 30% of the endothelial junctions in the pericytic venules of the mouse diaphragm are open to a gap of approximately 30--60 A, and are fully permeated by hemeundecapeptide (H11P) (mol diam approximately 20 A). To estimate the size limit for molecules that can permeate these junctions, we have administered graded tracers intravenously and studied their behavior at the level of pericytic venules in bipolar microvascular fields (BMFs) in the mouse diaphragm. Horseradish peroxidase (HRP) (mol diam approximately 50 A) permeated only approximately 50% of the open junctions of the venular endothelium. Outflow through venular junctions appeared to be modest since the tracer remained restricted to the perivenular spaces. Hemoglobin (Hb, mol diam 64 x 55 x 50 A) permeated only a few (less than 5%), and ferritin (mol diam 110 A), practically none, of the endothelial junctions of the pericytic venules. The findings suggest that under normal conditions the size limit for permeant molecules for open venular junctions is approximately 60 A. Replicas of freeze-fracture preparations from appropriate regions in BMF showed that the intercellular junctions of the venular endothelium have the same organization as previously described for the corresponding segments of the microvasculature in the omentum and mesentery: discontinuous creases or grooves either free of or marked by few intramembrane particles only. Administration of histamine (topically or systemically) and 5-hydroxytryptamine (5-HT) (topically) resulted in typical focal separations of the endothelial junctions and intramural deposits of large tracer particles (carbon black) in the postcapillary venules.     

Evaluation as a function of granulocytes in hyperimmunoglobulinemia E syndrome with recurrent infections

The hyper-IgE syndrome with recurrent infections (HIESRI) is characterized by skin and respiratory infections due to Staphylococcus aureus and several fungi infections which are frequently associated with tissue damage. A deficiency in the chemotaxis of phagocytic cells has been documented to explain these findings; however, the expression of adhesion molecules, the secretion of cytokines that activate granulocytes and the production of oxygen reactive molecules have not been evaluated in HIESRI. Six HIESRI patients were evaluated for the following parameters: (1) secretion of GM-CSF and IL-5 by mitogen and antigen-activated mononuclear cells, (2) the chemotactic response of FMLP-activated granulocytes, (3) the respiratory burst of PMA-activated granulocytes, and (4) the expression of L-selectin and CD11b in PMA-activated granulocytes. Human recombinant GM-CSF and culture supernatants were evaluated for capacity to modulate granulocytic function. Compared to controls, HIESRI patients showed a normal production of GM-CSF and an increase in the basal secretion of IL-5. No significant differences were observed for chemotaxis, respiratory burst or L-selectin and CD11b expression. The GM-CSF did not modulate these functions in granulocytes from HIESRI patients, but culture supernatants applied to granulocytes inhibited chemotaxis, increased respiratory burst and caused the shedding of L-selectin from the granulocyte surface. The 6 HIESRI patients were nonsymptomatic during the time of this research due to a program of continued treatment; findings suggest that granulocytes are activated more easily in response to proinflammatory factors and that production of these factors is higher in HIESRI.     

Activation of human phagocytes through carbohydrate antigens (CD15, sialyl-CD15, CDw17, and CDw65).

The leukocyte carbohydrate (CHO) Ag CD15, sialyl-CD15, and CDw65 have recently been found to function as ligands for CD62 and ELAM-1 cell adhesion molecules on platelets and endothelium, respectively. Cell adhesion ligands also may act as receptors capable of signal transduction. We therefore investigated the possibility that these CHO Ag and CDw17, a glycolipid Ag whose expression is regulated by leukocyte activation, may have receptor-like characteristics. The effects of antibody cross-linking of CHO Ag on phagocyte activation were measured by using flow cytometry and fluorescent indicators for cytoplasmic calcium ions, oxidative burst, and the granule-associated proteins CD11b and CD67. Cross-linking of CD15, sialyl-CD15, CDw65, or CDw17 induced a moderate release of calcium ions into the cytoplasm of granulocytes, a strong activation of oxidative burst, and a low up-regulation of CD11b and CD67 compared to the effects of treatment with 4 microM FMLP. The results suggest a role for CHO Ag in leukocyte signal transduction and support the view that these molecules are involved in phagocyte activation.     

Changes in the venular epithelium of the mesentery in rats after application of hydrogen peroxide

The change of the venular permeability and endothelial structure was studied with the aid of fluorometry and electron microscopy after application of H2O2. Transmural transfer rate of FITC-albumin++ and permeable fraction of the vessel walls were increased. There were formed the "leaks" and transendothelial canals in the venular endothelium. Numerous local membrane injuries of the endotheliocytes and pericytes, degranulation of the mast cells and destruction of thrombocytes into the vessel lumen were detected.     

Effects of serine protease inhibitors on oxyradical burst from phagocytizing neutrophils—analysis by chemiluminescence counting and its microscopic imaging

Reaction difference of oxyradical generation and luminol-dependent photoemission of zymosan- and phorbol ester-treated neutrophils were investigated using a conventional photomultiplier and ultrasensitive photonic imaging technique. Zymosan-treated cells released a concentrated photonic burst corresponding to the cellular distribution. In contrast, phorbol ester-treated cells produced a negligible level of photoemission, and the additional application of Ca²⁺ ionophore enhanced the photonic burst, which was gradually spread out into extracellular space. Serine protease inhibitors did not attenuate PMA-induced chemiluminescence but did attenuate zymosan-induced chemiluminescence. This suggests the involvement of serine protease in the respiratory burst of phagocytizing neutrophils.     

Inhaled NO reduces leukocyte-endothelial cell interactions and myocardial dysfunction in endotoxemic rats

Inhaled nitric oxide (NO) has been shown to have some protective effect in the peripheral distal inflamed vasculature. The objective of the study was to determine whether inhaled NO would reduce endotoxin-induced leukocyte activation and myocardial contractile dysfunction. Rats were treated with either saline or endotoxin (10 mg/kg iv) and then allowed to breathe (4 h) either air or air plus NO (10 ppm). In endotoxemic rats, mesenteric venular endothelium leukocyte firm adhesion increased compared with control rats (1.15 +/- 0.32 vs. 4.08 +/- 0.96 leukocytes/100 microm; P < 0.05). Inhaled NO significantly attenuated endotoxin-induced venular endothelium leukocyte adhesion (4.08 +/- 0.96 vs. 1.86 +/- 0.76 leukocytes/100 microm; P < 0.05) and FITC-conjugated anti-intercellular adhesion molecule-1 fluorescence intensity. Endotoxin-induced myocardial dysfunction and leukocyte content increases were reduced in inhaled NO-treated rats. These observations suggest that inhaled NO reduces the degree of cardiovascular dysfunction and inflammation in endotoxemic rats.     

Dual mechanisms in priming of the chemoattractant-induced respiratory burst in human granulocytes. A Ca2+-dependent and a Ca2+-independent route

After interaction with so-called priming agents, the respiratory burst in human granulocytes does not become activated, but is enhanced upon subsequent stimulation with the chemoattractant FMLP. Investigating the mechanism of the priming reaction, we found that a transient rise in the cytosolic free calcium concentration [( Ca2+]i) suffices to irreversibly prime human granulocytes. Thus, platelet-activating factor (PAF) induced a transient increase in [Ca2+]i and primed the cells to an enhanced respiratory burst upon subsequent interaction with FMLP. Artificially, the transient rise in [Ca2+]i was mimicked by addition and subsequent removal of the Ca2+ ionophore ionomycin; this treatment too, primed the respiratory burst of the granulocytes. The priming induced by ionomycin was completely abolished when [Ca2+]i changes were buffered during exposure of the cells to the ionophore. The priming induced by PAF was only partially inhibited under [Ca2+]i-buffering conditions during priming, indicating that multiple pathways exist in the priming of granulocytes by PAF.     

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body’s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).     

Microparticle adhesive dynamics and rolling mediated by selectin-specific antibodies under flow

In vitro studies were performed to characterize the relative performance of candidate receptors to target microparticles to inflammatory markers on vascular endothelium. To model the interactions of drug-bearing microparticles or imaging contrast agents with the vasculature, 6 micron polystyrene particles bearing antibodies, peptides, or carbohydrates were perfused over immobilized E- or P-selectin in a flow chamber. Microparticles conjugated with HuEP5C7.g2 (HuEP), a monoclonal antibody (mAb) specific to E- and P-selectin, supported leukocyte-like rolling and transient adhesion at venular shear rates. In contrast, microparticles conjugated with a higher affinity mAb specific for P-selectin (G1) were unable to form bonds at venular flow rates. When both HuEP and G1 were conjugated to the microparticle, HuEP supported binding to P-selectin in flow which allowed G1 to form bonds leading to stable adhesion. While the microparticle attachment and rolling performance was not as stable as that mediated by the natural ligands P-selectin Glycoprotein Ligand-1 or sialyl Lewis(x), HuEP performed significantly better than any previously characterized mAb in terms of mediating microparticle binding under flow conditions. HuEP may be a viable alternative to natural ligands to selectins for targeting particles to inflamed endothelium.