VAP-1 and CD73, endothelial cell surface enzymes in leukocyte extravasation

Leukocyte extravasation from the blood into tissues is crucial for normal immune surveillance and in inflammation. Traditionally molecules belonging to selectin, chemokine, integrin, and immunoglobulin super families are thought to mediate the multiple adhesive and activation events needed for a successful emigration cascade. Recently, emerging evidence suggests that enzymes expressed on the surface of endothelial cells and leukocytes also contribute to the leukocyte extravasation cascade. Here we briefly review the role of vascular adhesion protein-1 (VAP-1) and CD73, 2 cell surface enzymes, in leukocyte migration form the blood into the tissues. Importantly, specific enzyme inhibitors, gene-deficient mice, and recombinant enzymes have recently unambiguously shown that the catalytic activity of these enzymes regulates the leukocyte traffic. The concept of enzymatic regulation of leukocyte extravasation provides new insight into the multi-step adhesion cascade and opens new possibilities for inhibiting inappropriate inflammatory reaction through the use of small molecule enzyme inhibitors.     

Cellular softening mediates leukocyte demargination and trafficking,thereby increasing clinical blood counts

Leukocytes normally marginate toward the vascular wall in large vessels and within the microvasculature. Reversal of this process, leukocyte demargination, leads to substantial increases in the clinical white blood cell and granulocyte count and is a well-documented effect of glucocorticoid and catecholamine hormones, although the underlying mechanisms remain unclear. Here we show that alterations in granulocyte mechanical properties are the driving force behind glucocorticoid- and catecholamine-induced demargination. First, we found that the proportions of granulocytes from healthy human subjects that traversed and demarginated from microfluidic models of capillary beds and veins, respectively, increased after the subjects ingested glucocorticoids. Also, we show that glucocorticoid and catecholamine exposure reorganizes cellular cortical actin, significantly reducing granulocyte stiffness, as measured with atomic force microscopy. Furthermore, using simple kinetic theory computational modeling, we found that this reduction in stiffness alone is sufficient to cause granulocyte demargination. Taken together, our findings reveal a biomechanical answer to an old hematologic question regarding how glucocorticoids and catecholamines cause leukocyte demargination. In addition, in a broader sense, we have discovered a temporally and energetically efficient mechanism in which the innate immune system can simply alter leukocyte stiffness to fine tune margination/demargination and therefore leukocyte trafficking in general. These observations have broad clinically relevant implications for the inflammatory process overall as well as hematopoietic stem cell mobilization and homing.Leukocyte margination within the microvasculature and in larger blood vessels is an integral part of the inflammatory process and innate immune system (1, 2). This margination phenomenon is twofold, involving sequestration of leukocytes in the capillary bed (3, 4) as well as movement of leukocytes toward the blood vessel wall (Fig. 1A) (5, 6). Recent experimental and computational data, including our own, indicate that the mechanical properties of leukocytes play a major role in margination and are sufficient to drive leukocytes in whole blood toward the vessel wall (7–12).Open in a separate windowFig. 1.A reductionist microfluidic approach to investigate whether alterations in the mechanical properties of leukocytes can cause demargination in the microvasculature and larger blood vessels. (A) Under homeostatic conditions, leukocytes marginate via two main mechanisms: sequestration within the microvascular capillary beds and movement toward the vessel wall in veins. (B) Transit times of leukocytes through a microfluidic capillary bed model were measured to determine the degree of margination within the microvasculature. (C) CBC performed on blood samples obtained from one healthy human subject before and after ingestion of dexamethasone (Dex) showed an expected increase in the WBC and granulocyte (Gran) counts. (D) Leukocytes isolated from the same subject after dexamethasone ingestion have shorter transit times than pretreatment control leukocytes obtained from the same subject, with less obstructed leukocytes within the microfluidic device (P < 0.05 via Mann−Whitney test). (E) Using confocal videomicroscopy and a microfluidic large veins model, the distances of leukocytes from the wall of the microchannel were measured to determine degree of margination. (F) CBCs performed on blood samples obtained from a second healthy human subject also showed the expected WBC and Gran increase after dexamethasone ingestion. (G) Leukocytes within whole-blood samples collected after dexamethasone ingestion demarginated away from the vessel wall in higher proportions compared with pretreatment controls (N_control = 4,894 cells, N_dex6hour = 4,398 cells, P < 0.01 via chi-square test), which correlates to the dexamethasone-induced increases in WBC and granulocyte counts.What is not known is whether leukocyte softening can cause the reversal of leukocyte margination, which would indicate that leukocyte stiffness may be modulated by the immune system as an additional biophysical means to mediate leukocyte trafficking. To that end, we explored whether leukocyte stiffness alterations play a role in leukocyte demargination induced by glucocorticoid and catecholamine hormones. Although this phenomenon, which causes significant increases in the white blood cell (WBC) count within the clinical complete blood count (CBC) and specifically involves the granulocyte subpopulation of leukocytes, has been well documented from a clinical perspective for decades, the underlying mechanisms remain unclear (13–15). This leukocyte demargination effect can be induced via in vivo ingestion of an exogenous glucocorticoid, such as dexamethasone, or catecholamine, such as epinephrine, both of which are used clinically as an antiinflammatory agent and a vasopressor, respectively. Canonically, glucocorticoid- and catecholamine-induced demargination is attributed to down-regulation of adhesion molecules such as L- and P-selectin (16). However, in humans, although glucocorticoid infusion is associated with decreased leukocyte L-selectin expression, this does not occur until several hours after leukocyte demargination and concurrent increase in leukocyte count has already transpired (17). Furthermore, L- and P-selectin-deficient mice exhibit no abnormalities in leukocyte margination compared with wild type, suggesting additional mechanisms are likely involved (18, 19). Finally, leukocyte margination toward the vessel wall occurs in vitro in the absence of intact endothelium, questioning the need for specific interactions between these ligands and their adhesion molecules during this process (5, 6).Mechanistically, computational models have determined that cell−cell collisions between leukocytes and softer erythrocytes enhance leukocyte margination (7, 8, 20, 21). Computational research from our own group and others indicate that modulating only the cell mechanical properties, such as stiffness, alters these physical interactions and thus changes in margination, but this has not been validated experimentally (9–12, 20–22). Furthermore, decreases in cell stiffness may also reduce leukocyte sequestration within the capillary bed, as softer cells could deform to release into the circulation. Here we demonstrate that granulocyte softening is the driving force behind glucorticoid- and catecholamine-induced demargination, including leukocyte release both out of the capillary bed and away from the vascular wall of larger vessels, and provides a cellular mechanical mechanism in which the mechanical properties of leukocytes directly contribute to increases in the WBC and granulocyte counts observed clinically. In addition, our findings reveal a biophysical answer to an old hematologic question regarding how glucocorticoids and catecholamines cause leukocyte demargination.     

白细胞黏附分子CD54、CD62L、CD44在慢性肺源性心脏病患者外周血白细胞的表达

目的探讨白细胞黏附分子CD54、CD62L、CD44在慢性肺源性心脏病（chronic cor pulmonale,CCP）外周血白细胞（PBC）上的表达。方法采用流式细胞术,选用单克隆抗体检测CCP患者40例（B组：B1组急性加重期20例,B2组经治疗后进入缓解期20例）外周血中性粒细胞（N）、淋巴细胞（L）、单核细胞（M）表面CD54、CD62L及CD44的表达,同时设立慢性阻塞性肺疾病（chronic obstructive pulmonary disease,COPD）患者40例（A组）及健康者20例（C组）作为对照组。结果B1组、B2组N和LCD54,MCD54和CD62L阳性表达率较C组显著升高（P〈0.05）,但B1组与B2组间无显著性差异（P〉0.05）;B1组、B2组N和LCD62L,及N、L和MCD44阳性表达率与C组间均无显著性差异（P〉0.05）。A组LCD54、CD62L和CD44,MCD54阳性表达率较C组显著升高（P〈0.05）;A组NCD62L阳性表达率较C组降低（P〈0.05）;A组NCD54、MCD62L、N和MCD44阳性表达率与C组间均无显著性差异（P〉0.05）。B组N和LCD54,MCD54和CD62L阳性表达率较C组显著升高（P〈0.01）;B组N和LCD62L,N、L和MCD44阳性表达率与C组间均无显著性差异（P〉0.05）。B组NCD54、MCD62L阳性表达率较A组显著升高（P〈0.05）;A组LCD44阳性表达率较B组显著升高（P〈0.05）;A组L和MCD54、N和LCD62L、N和LCD44阳性表达率与B组间均无显著性差异（P〉0.05）。结论CCP患者通过外周血N表面CD54、L表面CD54、M表面CD54和CD62L表达上调,参与自身的病理生理过程。在从COPD发展成为CCP的过程中,L和M表面CD54,尤其是N表面CD54和M表面CD62L表达上调可能在慢性肺动脉高压的形成中起重要作用。而白细胞黏附分子CD44可能不起重要作用。     

Changes in gastrointestinal lymph and blood vessels in patients with cirrhotic portal hypertension

Background. The aim of this study was to characterize the lymph vessels in different parts of the gastrointestinal tract and also to evaluate morphometric changes in these vessels during cirrhotic portal hypertension. Methods. Sixteen patients with cirrhotic portal hypertension and 18 control subjects without portal hypertension were enrolled in the study. Tissue specimens were collected at autopsy or surgery, and were stained enzyme histochemically, using 5′-nucleotidase and alkaline phosphatase to distinguish lymph vessels and blood vessels, respectively. The numbers of vessels and their luminal areas were estimated using computer graphics software (National Institutes of Health [NIH] image program). Results. The numbers and luminal areas of the lymph vessels varied considerably among the different organs of the gastrointestinal tract, both in controls and in the patients with cirrhotic portal hypertension. There was no significant difference in the numbers of lymph vessels between controls and patients with cirrhotic portal hypertension. However, the luminal area of the lymph vessels in the esophagus and stomach was significantly greater in the patients with cirrhotic portal hypertension than in the controls. These differences in lymph vessels were not seen in the small intestine and colon. Conclusions. These data indicate that dilatation of lymph vessels may be related to the absorption of excess interstitial fluid, resulting from congestion, in cirrhotic portal hypertension. Received: November 30, 2000 / Accepted: April 13, 2001     

Critical but divergent roles for CD62L and CD44 in directing blood monocyte trafficking in vivo during inflammation

Using noninvasive in vivo imaging and experimental autoimmune uveoretinitis as a model, we show for the first time that the mechanisms controlling blood monocyte recirculation through peripheral and lymphoid tissues alter during inflammation. The recirculation of monocytes in mice with ocular inflammation but not controls was found to depend on the selectin CD62-ligand (CD62L) and on CD44. Not only was rolling efficiency ablated or markedly reduced in antibody-treated mice, but most of the labeled monocytes also disappeared from the circulation within seconds, anti-CD44-treated monocytes homing to the lymph nodes and anti-CD62L-treated monocytes homing to the spleen. Our data indicate that, although PSGL-1 has a partial role in the transmigration of monocytes into the inflamed retina, CD62L has a key role in regulating recruitment of monocytes to lymphoid tissue from the blood during inflammation and that CD44 is required to maintain CD62L(+) inflammatory monocytes within the circulation during inflammation. This effect was systemic, because sequestered monocytes accumulated in mesenteric as well as draining cervical lymph nodes, and inflammation dependent, because depletion of circulating blood monocytes was much reduced or absent in normal mice and accumulations of adoptively transferred monocytes in the lymphoid tissues did not occur.     

The biological role of blood vessels during endochondral bone formation

Endothelial cells play an important role in endochondral bone formation. In the chondro-osseous junction, endothelial cells appear to invade into cartilage by the cellular mechanism of angiogenesis evidenced by cell duplication, disappearance of basement membranes and activated migration. The endothelial cells penetrate the unmineralized transverse partition of the cartilage columns.     

Gefitinib affects functions of platelets and blood vessels via changes in prostanoids balance.

Prostaglandins (PGs) and thromboxane (TX) produced by cyclooxygenase (COX) have a great influence on vascular systems and platelet functions. The serum levels of epidermal growth factor (EGF) and PGs were measured in patients with lung cancer treated with gefitinib, and the influence of EGF on platelet aggregation was investigated. Twenty patients were investigated. The serum level of TXB(2) increased significantly in all patients who received gefitinib for 2 weeks (before vs. after = 94.1 +/- 47.3 vs. 190.9 +/- 54.3, p<0.01). TXB(2) also increased significantly in responders without concurrent chemotherapy (before vs. after = 79.3 +/- 35.5 vs. 194.5 +/- 58.1, p<0.05), but not in non-responders (before vs. after = 106. 5 +/- 65.8 vs. 162.2 +/- 52.8, N.S.). PG 6-keto F1alpha and PGE(2) did not exhibit significant changes. Furthermore, EGF showed no significant change (after vs. before = 234 +/- 35 vs. 276 +/- 72, N.S.). Although there was no correlation between the levels of EGF and TXB(2) (N.S.), the PG 6-keto F2alpha/TXB(2) ratio decreased significantly (before vs. after = 0.054 +/- 0.018 vs 0.033 +/- 0.015, p<0.05). The secondary platelet aggregation observed after high-dose adenosine diphosphate stimulation was inhibited after a 1-minute preincubation with EGF. Platelet aggregation in patients after gefitinib administration tended to accelerate and secondary aggregation was observed after low-dose adenosine diphosphate stimulation. We conclude that careful observation is needed for patients with chronic obstructive pulmonary disease, pulmonary fibrosis, and thromboembolic diseases receiving gefitinib. Furthermore, measurement of prostanoids may be a good predictor of the beneficial and adverse effects. Moreover, the combination of gefitinib with a COX inhibitor that regulates TXA(2)/PGI(2) balance should be evaluated.     

Vasa vasorum of subcutaneous veins and lymph vessels

After intraarterial ink-injection the vasa vasorum of veins and lymphatic vessels in adults, dead born children and fetuses were demonstrated micropreparatorily. In their walls the larger veins of the saphena system possess a capillary network corresponding to the connective tissue and muscle structures/patterns in the vessel wall. No vasa vasorum could be found in the subcutaneous veins less than 1 mm in diameter. The lymphatic vessels of the inner prefascial bundle show a one-dimensional capillary network in the outer layers of the wall. Like the vasa vasorum of the saphena system it develops in the second half of fetal life.     

Morphological analysis of lymph vessels and capillaries in gastric carcinoma

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Microfluidic chambers for monitoring leukocyte trafficking and humanized nano-proresolving medicines interactions

Leukocyte trafficking plays a critical role in determining the progress and resolution of inflammation. Although significant progress has been made in understanding the role of leukocyte activation in inflammation, dissecting the interactions between different leukocyte subpopulations during trafficking is hampered by the complexity of in vivo conditions and the lack of detail of current in vitro assays. To measure the effects of the interactions between neutrophils and monocytes migrating in response to various chemoattractants, at single-cell resolution, we developed a microfluidic platform that replicates critical features of focal inflammation sites. We integrated an elastase assay into the focal chemotactic chambers (FCCs) of our device that enabled us to distinguish between phlogistic and nonphlogistic cell recruitment. We found that lipoxin A₄ and resolvin D1, in solution or incorporated into nano–proresolving medicines, reduced neutrophil and monocyte trafficking toward leukotriene B₄. Lipoxin A₄ also reduced the elastase release from homogenous and heterogenous mixtures of neutrophils and monocytes. Surprisingly, the effect of resolvin D1 on heterogenous mixtures was antisynergistic, resulting in a transient spike in elastase activity, which was quickly terminated, and the degraded elastin removed by the leukocytes inside the FCCs. Therefore, the microfluidic assay provides a robust platform for measuring the effect of leukocyte interactions during trafficking and for characterizing the effects of inflammation mediators.     

The role of phagocytic cells in human blood leukocyte suspensions for in vitro colony-forming cells

The iron ingestion method was used to separate colony stimulating cells from in vitro colony forming cells (CFU-C) in human white blood cell (WBC) suspensions. The depletion of phagocytic cells (granulocytes and monocytes) from WBC eliminated spontaneous colony growth, without influencing the total number of CFU-C. Thus, phagocyte-free human WBC can be used as a target for measuring human colony stimulating activity (CSA). Such leukocytes can be cryopreserved, thus making possible the use of standardized target cell preparations for longitudinal studies. Hemolysate added to phagocyte-free blood leukocytes did not induce colony formation but could enhance it in the presence of a suitable CSA. Spontaneous colony formation of unseparated WBC was significantly reduced by selective destruction of granulocytes by freezing in 10% DMSO, thus indicating that both granulocytes and monocytes are involved in the production of endogenous CSA in WBC suspensions.     

The CD nomenclature of leukocyte antigens

The author gives the basic information about human leucocyte antigens of haematopoietic cells. At present, the membrane markers (antigens) included into CD nomenclature are gradually detected, classified and ranged, many of them being well defined as to genetic determination, chemical structure and function.     

Human leukocyte differentiation antigens and CD classification

The autor summarizes the information about human leukocyte antigens. Expression of membrane markers is included in accordance with CD (Cluster of Differentiation) classification. The CD molecules are used ubiquitously in human medical research, immunodiagnosis and treatment and also CD profiles are intended to be used by biologists, pathologists and clinicians generally. Profiles nearly 400 known leukocyte cell surface molecules are summarized in CD system (CD1-CD350). Most CD molecules are integral membrane proteins that have one or more pass through the plasma cell membranes. Knowing which regions of a protein are intracellular or extracellular is important in the selection of peptides for immunization, for the expression of domains of the protein, and to understand the interaction of the protein with other proteins. CD markers are great targets for diagnostic and research of different types of diseases as a potential treatment for a variety of tumors.     

The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during murine development

The processes of angiogenesis and hematopoiesis require a high degree of coordination during embryogenesis. Whereas much is understood about the development of the vascular system in avian embryos, little information has been attained in mammals, predominantly because there are no specific markers for either blood vessels or hematopoietic cells in any developing mammalian system. We have recently shown that murine CD34 (mCD34) is expressed on the vascular endothelium in all organs and tissues of the adult mouse as well as on a small percentage of presumably hematopoietic stem cells in the bone marrow and fetal liver. Here we show that mCD34 is also expressed on the endothelium of blood vessels and on a subset of hematopoietic-like cells throughout murine development. mCD34 is first observed on the yolk sac endothelium of day 7.5 embryos and on a subset of hematopoietic cells within these yolk sacs. mCD34 expression is maintained on vessels and hematopoietic cells in all organs and tissues throughout embryogenesis. In addition, mCD34 is localized on growth conelike filopodial processes that appear at the budding edge of newly sprouted capillaries. Double staining of capillaries for mCD34 and laminin shows that these growth conelike processes seem to be free of laminin, whereas the formed capillaries seem to be coated with this extracellular matrix protein. Analysis of vessels in developing brain shows that these filopodial processes seem to be directed toward the ventricular epithelium, a previously described site of vascular endothelial growth factor synthesis. Finally, we show that the vascular structures of developing murine embryoid bodies also express mCD34. These data suggest that mCD34 is a useful marker for the analysis of the development of the blood vascular system in murine embryos.     

Endoscopic photoconversion reveals unexpectedly broad leukocyte trafficking to and from the gut

Given mounting evidence of the importance of gut-microbiota/immune-cell interactions in immune homeostasis and responsiveness, surprisingly little is known about leukocyte movements to, and especially from, the gut. We address this topic in a minimally perturbant manner using Kaede transgenic mice, which universally express a photoconvertible fluorescent reporter. Transcutaneous exposure of the cervical lymph nodes to violet light permitted punctual tagging of immune cells specifically therein, and subsequent monitoring of their immigration to the intestine; endoscopic flashing of the descending colon allowed specific labeling of intestinal leukocytes and tracking of their emigration. Our data reveal an unexpectedly broad movement of leukocyte subsets to and from the gut at steady state, encompassing all lymphoid and myeloid populations examined. Nonetheless, different subsets showed different trafficking proclivities (e.g., regulatory T cells were more restrained than conventional T cells in their exodus from the cervical lymph nodes). The novel endoscopic approach enabled us to evidence gut-derived Th17 cells in the spleens of K/BxN mice at the onset of their genetically determined arthritis, thereby furnishing a critical mechanistic link between the intestinal microbiota, namely segmented filamentous bacteria, and an extraintestinal autoinflammatory disease.Interactions between the gastrointestinal microbiota and the immune system, particularly their impact on the initiation and progression of autoimmune and inflammatory diseases, are garnering much attention of late (1). Aberrant accumulation of leukocytes in the intestine is a cardinal feature of inflammatory bowel disease, a build-up that reflects, at least in part, an influx of immune cells (2). In addition, gut-microbiota/leukocyte cross-talk influences systemic immune responses, in particular extraintestinal autoimmune diseases. For example, it has been reported that one bacterial species, segmented filamentous bacteria, can protect female nonobese diabetic (NOD) mice from development of autoimmune diabetes (3) yet drive inflammatory arthritis in the K/BxN mouse model (4).The intestinal tract is a site of frequent antigenic challenge, whether dietary or microbial, and therefore needs to be continuously replenished with circulating cells to optimize immune responses to would-be pathogens. Indeed, a few recent studies have demonstrated that blood-circulating Ly6C^hi monocytes continually enter the healthy colon and subsequently differentiate in situ into tissue macrophage or dendritic cell populations (5–7). T cells are “imprinted” for gut homing primarily in the mesenteric lymph nodes (MLNs), entering the intestine via recirculation from the blood, which is facilitated by mucosal addressin cell-adhesion molecule-1 and C-C motif chemokine ligand 25 (8).Concerning the other direction, exit from the gut, much less is known, although it is widely considered that there is little movement of leukocytes beyond the MLNs. It was recently shown that, at steady state, the intestinal microbiota controls transport of both pathogenic and commensal bacterial antigens from the gut lumen to the MLNs, which relies on ferrying by mononuclear phagocytes (9). More generally, most investigators adhere to the concept that both cells and antigens drain from the gut directly into the MLNs. Many years ago, however, dye-tracking experiments established that the gastrointestinal tract is serviced by a series of LNs—at least five discrete entities—and specific regions of the gut are drained by particular nodes (10, interpreted as per ref. 11).Given ample indications of a role for leukocyte migration in enteric diseases, surprisingly little is known about gut-associated immune-cell trafficking. Additionally, the interpretation of much of the existing data has been complicated by the systems used: notably, transfer models. To our knowledge, no one has yet reported specific, in vivo labeling of physiological frequencies of gut-resident leukocytes and subsequent monitoring of their migratory capabilities. Here, we report optimization of a means to follow immune-cell migration to and from the gut in a minimally perturbant manner. Upon exposure to violet light, the photoconvertible fluorescent protein, Kaede, irreversibly changes in color from green to red, so that cells are punctually tagged at the site of photoconversion. A line of mice expressing Kaede in all cell-types was engineered, and has already been used to monitor immune-cell migration from the lymphoid organs or the skin (12–15). Some of the advantages of this system are that it permits precise delineation of the origin of migratory populations, is amenable to the study of cell efflux as well as influx, allows simultaneous analysis of a multitude of migratory cell subsets as they traffic to numerous locations, and is very sensitive.Using the standard Kaede transgenic (tg) mouse system, as well as a novel endoscopic adaptation of it, we have performed a series of experiments tracking movement of leukocytes to and from the intestine. The following major questions were addressed: To what extent do immune cells circulate into and out of the gut under physiological conditions? Do migration patterns vary between different immune-cell lineages or lymphoid-cell functional subsets? Is it possible to visualize the arrival of colon-derived immune cells at a pathogenetically important site in a gut-distal autoinflammatory disease?     

Intravital 2-photon imaging,leukocyte trafficking,and the beating heart

Intravital two-photon microscopy allows for the analysis of single-cell dynamics within intact tissues. As it is well recognized that molecular cues that regulate leukocyte trafficking into inflammatory sites differ between various tissues, it is important to study organ-specific responses. Recently, intravital two-photon microscopy has been expanded to moving organs in the mouse such as beating hearts. Unlike previous experimental approaches to image cardiac tissue explants or isolated perfused heart preparations by two-photon microscopy, intravital imaging accounts for the mechanical force transmitted to vessels by the heartbeat and accurately assesses dynamic leukocyte behavior in the coronary vessels and myocardial tissue. Intravital two-photon imaging of beating hearts is a promising experimental tool that will help elucidate cellular and molecular immune processes that contribute to a variety of cardiovascular diseases.     

Liver ischemia/reperfusion induces an increase of microvascular leukocyte flux,but not heterogeneity of leukocyte trafficking

Abstract: Leukocytic response plays a major role in the manifestation of hepatic ischemia/reperfusion (I/R) injury. To clarify whether post-ischemic hepatic leukocyte accumulation is based on increased leukocyte flux to the hepatic tissue due to systemic inflammation or chemoattractant activities or whether it represents solely a local tissue response without changing overall leukocyte flux and trafficking characteristics through the microvasculature, we studied acinar and sinusoidal leukocyte flux and distribution in rat livers in vivo both under normal (sham, n=8) and post-ischemic (60′ ischemia/75′ reperfusion) conditions (I/R, n=8), using fluorescence epi-illumination microscopy (rhodamine-6G). Hepatic ischemia/reperfusion significantly (p<0.05) increased acinar leukocyte flux (58.4±20.9 cells/min vs 36.4±12.8 cells/min in sham controls); however, it did not exhibit increased heterogeneity of acinar leukocyte distribution, as indicated by the unchanged coefficient of variance (CV) of 0.36±0.16 (sham controls: 0.31 ±0.14). In parallel, analysis of individual sinusoidal leukocyte flux demonstrated significantly (p<0.05) higher values (8.9±3.7 cells/min) after ischemia/reperfusion when compared with sham controls (5.7±1.9 cells/min), which, however, was not associated with increased heterogeneity of sinusoidal leukocyte trafficking (CV: 0.85±0.15 vs 0.85±0.16 in sham controls) and manifestation of preferential pathways. Analysis of blood cell count did not demonstrate an overall increase of total blood leukoycte count; however, an increased (p<0.01) fraction of polymorphonuclear leukocytes (65.2±11.2%) and stab cells (9.5±7.9%) during post-ischemic reperfusion when compared with sham controls (8.8±3.5% and 0.2±0.4%) was demonstrated. Thus, the increase of hepatic leukocyte flux after ischemia/reperfusion may be the result of both the manifestation of a systemic inflammatory response and the increase of local chemoattractant activities, such as the production and release of the cytokine-induced neutrophil chemoattractant of the IL-8 family.