Leukocyte-vessel Wall Interactions Research Articles

Between Rho(k) and a hard place: the relation between vessel wall stiffness, endothelial contractility, and cardiovascular disease.

Vascular stiffness is a mechanical property of the vessel wall that affects blood pressure, permeability, and inflammation. As a result, vascular stiffness is a key driver of (chronic) human disorders, including pulmonary arterial hypertension, kidney disease, and atherosclerosis. Responses of the endothelium to stiffening involve integration of mechanical cues from various sources, including the extracellular matrix, smooth muscle cells, and the forces that derive from shear stress of blood. This response in turn affects endothelial cell contractility, which is an important property that regulates endothelial stiffness, permeability, and leukocyte-vessel wall interactions. Moreover, endothelial stiffening reduces nitric oxide production, which promotes smooth muscle cell contraction and vasoconstriction. In fact, vessel wall stiffening, and microcirculatory endothelial dysfunction, precedes hypertension and thus underlies the development of vascular disease. Here, we review the cross talk among vessel wall stiffening, endothelial contractility, and vascular disease, which is controlled by Rho-driven actomyosin contractility and cellular mechanotransduction. In addition to discussing the various inputs and relevant molecular events in the endothelium, we address which actomyosin-regulated changes at cell adhesion complexes are genetically associated with human cardiovascular disease. Finally, we discuss recent findings that broaden therapeutic options for targeting this important mechanical signaling pathway in vascular pathogenesis.

Ways to Enhance Lymphocyte Trafficking into Tumors and Fitness of Tumor Infiltrating Lymphocytes

The tumor is a hostile microenvironment for T lymphocytes. Indeed, irregular blood flow, and endothelial cell (EC) anergy that characterize most solid tumors hamper leukocyte adhesion, extravasation, and infiltration. In addition, hypoxia and reprograming of energy metabolism within cancer cells transform the tumor mass in a harsh environment that limits survival and effector functions of T cells, regardless of being induced in vivo by vaccination or adoptively transferred. In this review, we will summarize on recent advances in our understanding of the characteristics of tumor-associated neo-angiogenic vessels as well as of the tumor metabolism that may impact on T cell trafficking and fitness of tumor infiltrating lymphocytes. In particular, we will focus on how advances in knowledge of the characteristics of tumor ECs have enabled identifying strategies to normalize the tumor-vasculature and/or overcome EC anergy, thus increasing leukocyte-vessel wall interactions and lymphocyte infiltration in tumors. We will also focus on drugs acting on cells and their released molecules to transiently render the tumor microenvironment more suitable for tumor infiltrating T lymphocytes, thus increasing the therapeutic effectiveness of both active and adoptive immunotherapies.

Recent advances in vascular biology: Selected highlights from IVBM 2008

Thromb Haemost 2009; 101: 997–998 The contributions encapsulated in this themed issue of Thrombosis and Haemostasis derive from invited lectures at the 15th International Vascular Biology Meeting (IVBM) held in June, 2008 in Sydney, Australia. IVBMs, which are held every two years, have become the most prestigious international biomedical conference in blood vessel biology and disease. The scientific programme featured 130 talks couched in seven plenary sessions (14 keynote speakers), 17 workshops (2–3 invited speakers and 2–3 speakers selected from abstracts), two sponsored symposia, and almost 400 posters. The programme also featured three memorial orations honoring true giants in vascular biology that had passed away since the last IVBM in Amsterdam in 2006, namely Judah Folkman, Tucker Collins and Rudi Busse. The following provides a brief synopsis of six areas covered at IVBM 2008. Angiogenesis underpins normal physiological processes such as reproduction and wound healing, as well as a wide range of vascular disorders such as atherogenesis, and invasive tumour growth and metastasis. Angiogenesis involves the suppression of adhesion receptors, such as ICAM-1, VCAM-1, E-selectin and CD34 in tumour endothelium (1). Angiogenesis inhibitors are able to normalise endothelial adhesion molecule expression in tumour blood vessels, restoring leukocyte vessel wall interactions and enhancing inflammatory infiltration in the tumours, thus making tumours more vulnerable to attack by the immune system. Arjan Griffioen (VU University Medical Center, Amsterdam, The Netherlands) and Florry Vyth-Dreese (Netherlands Cancer Institute, Amsterdam, The Netherlands) have reviewed (2) the potential of angiostasis therapy to stimulate immune-mediated tumour inhibitory activity and speculate on new therapeutic approaches involving angiostasis in immunotherapy. The various mechanisms with which angiogenesis inhibitors might improve immuno-targeted approaches include tumour cell lysis stimulating tumour antigen presentation, reduced inhibitory molecule load that improves immune reactivity, limited release of immunosuppressive cytokines into the tumour microenvironment, and the creation of a pro-inflammatory microenvironment. Blood vessels adjust their calibre size in order to adapt to the demands of tissues or organs for nutrients and oxygen. The mechanisms involved in calibre size determination in blood vessels are not well understood. Nobuyuki Takakura and Hiroyasu Kidoya (Osaka University, Osaka, Japan) recently discovered that apelin, a factor secreted from endothelial cells following activation of Tie2 receptor tyrosine kinase, regulates the calibre size of blood vessels through its cognate endothelial G proteincoupled receptor APJ (3). Takakura and Kidoya have reviewed (4) the cooperative effect of apelin and VEGF on endothelial cell proliferation and capillary tube formation, and apelin’s regulation of cell-to-cell aggregation and cord hollowing. Integrins are heterodimeric cell-surface molecules that mediate molecular interactions between cells and their extracellular matrix (ECM) environment (5). These signalling receptors transmit information in both “inside-out” and “outside-in” directions across the cell membrane. A necessary and sufficient final step in integrin activation is the binding of the cytoskeletal protein talin to the integrin β-subunit. Brian Petrich (University of California, San Diego, USA) has reviewed (6) our current understanding of the mechanisms regulating the interaction of talin and that of numerous other proteins (such as filamin and kindlin) with the β-integrin subunit and modulation of integrin activation. Our growing appreciation of the functional significance of integrin signalling in haemostasis and thrombosis has been facilitated by mutational analysis in recombinant cell lines and very recently by the use of genetically modified mice. These and other advances focusing mainly on integrin αIIbβ3 (GPIIb-IIIa) are reviewed. Antibodies represent the fastest growing class of human therapeutics and the second largest class of drugs after vaccines, and these molecules are widely used as therapeutic agents in a diverse range of clinical settings, including cancer, inflammatory disease and cardiovascular disease. Single-chain antibodies (7) are one of the smallest possible formats for a recombinant antibody. Karlheinz Peter and colleagues (Baker IDI Heart and Diabetes Institute, Melbourne, Australia) have described (8) the © 2009 Schattauer GmbH, Stuttgart

Epigenetic Regulation of Tumor Endothelial Cell Anergy: Silencing of Intercellular Adhesion Molecule-1 by Histone Modifications

Tumors can escape from immunity by repressing leukocyte adhesion molecule expression on tumor endothelial cells and by rendering endothelial cells unresponsive to inflammatory activation. This endothelial cell anergy is induced by angiogenic growth factors and results in reduced leukocyte-vessel wall interactions, thereby attenuating infiltration of leukocytes into the tumor. This report describes a novel mechanism of endothelial cell anergy regulation. We recently reported that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors have angiostatic activity. Here, we studied whether epigenetic mechanisms regulate this angiogenesis-mediated escape from immunity. We found that DNMT inhibitors 5-aza-2'-deoxycytidine and zebularine, as well as HDAC inhibitor trichostatin A, reexpressed intercellular adhesion molecule-1 (ICAM-1) on tumor-conditioned endothelial cells in vitro, resulting in restored leukocyte-endothelial cell adhesion. In addition, treatment with DNMT or HDAC inhibitors in vivo also restored ICAM-1 expression on tumor endothelial cells from two different mouse tumor models. Furthermore, leukocyte-vessel wall interactions in mouse tumors were increased by these compounds, as measured by intravital microscopy, resulting in enhanced leukocyte infiltration. We show that ICAM-1 down-regulation in tumor endothelial cells is associated with ICAM-1 promoter histone H3 deacetylation and loss of histone H3 Lys(4) methylation but not with DNA hypermethylation. In conclusion, our data show that ICAM-1 is epigenetically silenced in tumor endothelial cells by promoter histone modifications, which can be overcome by DNMT and HDAC inhibitors, suggesting a new molecular mechanism based on which novel therapeutic approaches for cancer can be pursued.

Anti‐angiogenesis therapy can overcome endothelial cell anergy and promote leukocyte‐endothelium interactions and infiltration in tumors

Tumor escape from immunity, as well as the failure of several anti-cancer vaccination and cellular immunotherapy approaches, is suggested to be due to the angiogenesis-mediated suppression of endothelial cell (EC) adhesion molecules involved in leukocyte-vessel wall interactions. We hypothesized that inhibition of angiogenesis would overcome this escape from immunity. We investigated this in vivo by means of intravital microscopy and ex vivo by immunohistochemistry in two mouse tumor models. Angiogenesis inhibitors anginex, endostatin, and angiostatin, and the chemotherapeutic agent paclitaxel were found to significantly stimulate leukocyte-vessel wall interactions by circumvention of EC anergy in vivo, i.e., by the up-regulation of endothelial adhesion molecules in tumor vessels. This was confirmed by in vitro studies of cultured EC at the protein and mRNA levels. The new angiostatic designer peptide anginex was most potent at overcoming EC anergy; the enhanced leukocyte-vessel interactions led to an increase in the numbers of tumor infiltrating leukocytes. While anginex inhibited tumor growth and microvessel density significantly, the amount of infiltrated leukocytes (CD45), as well as the number of CD8+ cytotoxic T lymphocytes, was enhanced markedly. The current results suggest that immunotherapy strategies can be improved by combination with anti-angiogenesis.

Basic Fibroblast Growth Factor (bFGF, FGF-2) Potentiates Leukocyte Recruitment to Inflammation by Enhancing Endothelial Adhesion Molecule Expression

Basic fibroblast growth factor (bFGF, FGF-2) is a potent angiogenic factor and endothelial cell mitogen. Although bFGF levels are increased in chronically inflamed tissue, its role in inflammation is unclear. We investigated the effect of bFGF on acute dermal inflammation and the recruitment of monocytes, T cells, and neutrophils. Leukocyte recruitment to inflamed sites was quantified with radiolabeled leukocytes. Intradermal injection of bFGF in rats did not induce leukocyte recruitment or inflammation. However, the recruitment of leukocytes to inflammation induced by tumor necrosis factor-alpha, interferon-gamma, C5a, or a delayed hypersensitivity reaction was enhanced by bFGF by 55 to 132% (P < 0.05). Either acute or prolonged bFGF treatment of dermal sites had this effect. The potentiating effect of bFGF on leukocyte recruitment was also seen in joints. There was no associated modulation of vascular permeability, blood flow, or angiogenesis in the sites by bFGF. However, the expression of the endothelial cell adhesion molecules (CAMs) for leukocytes, P-selectin, E-selectin, and ICAM-1, was significantly up-regulated in the inflamed tissue by bFGF, as quantified by radiolabeled anti-CAM antibody binding in vivo. Thus, although not directly proinflammatory, bFGF synergistically potentiates inflammatory mediator-induced leukocyte recruitment, at least in part, by enhancing CAM up-regulation on endothelium.

Detection of leukocytes in contact with the vessel wall from in vivo microscope recordings using a neural network.

Leukocytes play an important role in the host defense as they may travel from the blood stream into the tissue in reacting to inflammatory stimuli. The leukocyte-vessel wall interactions are studied in post capillary vessels by intravital video microscopy during in vivo animal experiments. Sequences of video images are obtained and digitized with a frame grabber. A method for automatic detection and characterization of leukocytes in the video images is developed. Individual leukocytes are detected using a neural network that is trained with synthetic leukocyte images generated using a novel stochastic model. This model makes it feasible to generate images of leukocytes with different shapes and sizes under various lighting conditions. Experiments indicate that neural networks trained with the synthetic leukocyte images perform better than networks trained with images of manually detected leukocytes. The best performing neural network trained with synthetic leukocyte images resulted in an 18% larger area under the ROC curve than the best performing neural network trained with manually detected leukocytes.

Effect of repetitive asphyxia on leukocyte-vessel wall interactions in the developing chick intestine

Background/Purpose: Information on leukocyte-vessel wall interactions (LVWI) during development of the immature intestine is scarce. The authors designed an experimental model for studying the microcirculation in the developing intestine of chick fetuses at days 13 (n = 12), 15 (n = 17), and 17 (n = 19) of incubation (0.6, 0.7, and 0.8 of the incubation time, respectively) using intravital microscopy. Methods: The authors investigated whether episodes of asphyxia increase LVWI and induce tissue damage in the developing intestine. Asphyxia was induced by clamping of the chorioallantoic vein for 6 periods of 5 minutes each, with 5-minute intervals, whereas in sham groups a sham procedure was performed. Video recordings were made before as well as 10, 20, and 30 minutes after the end of the asphyxia or sham protocol. Results: Baseline number of rolling leukocytes per minute significantly increased (P <.001) from 0 at 0.6 incubation to 1.5 and to 4 at 0.7 and 0.8 incubation time, respectively. At 0.6 and 0.7 incubation no adherent leukocytes were observed under baseline conditions, whereas at 0.8 incubation single leukocytes adhered to the venular wall. LVWI variably increased during the course of the experiments. Asphyxia neither enhanced LVWI nor induced histological damage in the intestine. Conclusions: These findings indicate that (1) leukocyte-vessel wall interactions mature during fetal development, and (2) repetitive episodes of asphyxia induce neither an inflammatory response nor histological tissue injury in the developing intestine from 0.6 to 0.8 incubation. The authors hypothesize that immaturity of leukocyte-vessel wall interactions, as part of the nonspecific host defense to invading bacteria, might play a role in the development of necrotizing enterocolitis in premature neonates. J Pediatr Surg 35:49-55. Copyright © 2000 by W.B. Saunders Company.

Effect of repetitive asphyxia on leukocyte-vessel wall interactions in the developing chick intestine

Information on leukocyte-vessel wall interactions (LVWI) during development of the immature intestine is scarce. The authors designed an experimental model for studying the microcirculation in the developing intestine of chick fetuses at days 13 (n = 12), 15 (n = 17), and 17 (n = 19) of incubation (0.6, 0.7, and 0.8 of the incubation time, respectively) using intravital microscopy. The authors investigated whether episodes of asphyxia increase LVWI and induce tissue damage in the developing intestine. Asphyxia was induced by clamping of the chorioallantoic vein for 6 periods of 5 minutes each, with 5-minute intervals, whereas in sham groups a sham procedure was performed. Video recordings were made before as well as 10, 20, and 30 minutes after the end of the asphyxia or sham protocol. Baseline number of rolling leukocytes per minute significantly increased (P < .001) from 0 at 0.6 incubation to 1.5 and to 4 at 0.7 and 0.8 incubation time, respectively. At 0.6 and 0.7 incubation no adherent leukocytes were observed under baseline conditions, whereas at 0.8 incubation single leukocytes adhered to the venular wall. LVWI variably increased during the course of the experiments. Asphyxia neither enhanced LVWI nor induced histological damage in the intestine. These findings indicate that (1) leukocyte-vessel wall interactions mature during fetal development, and (2) repetitive episodes of asphyxia induce neither an inflammatory response nor histological tissue injury in the developing intestine from 0.6 to 0.8 incubation. The authors hypothesize that immaturity of leukocyte-vessel wall interactions, as part of the nonspecific host defense to invading bacteria, might play a role in the development of necrotizing enterocolitis in premature neonates.

Total warm ischemia and reperfusion impairs flow in all rat gut layers but increases leukocyte-vessel wall interactions in the submucosa only.

To study the effect of warm ischemia and reperfusion (I/R) on local perfusion and leukocyte-vessel wall interactions in vivo in all small bowel layers, and to quantify small bowel tissue injury histologically and by measuring intestinal fatty acid binding protein (I-FABP) release from the enterocytes. Gut injury as a result of I/R plays a pivotal role in a variety of clinical conditions, such as small bowel transplantation, heart or aortic surgery, and (septic) shock. The precise mechanism behind I/R injury and the role of microvascular changes remain unclear. The influence of warm I/R of the gut on microvascular parameters in the different gut layers has not been studied before. Anesthetized Lewis rats were either subjected to 30 minutes of ischemia and 1 hour of reperfusion or sham-treated as controls. After ligating the inferior mesenteric artery, total warm ischemia was induced by clamping the superior mesenteric artery. Intravital video microscopic measurements were obtained at intervals. Tissue injury of the small bowel and other organs was histologically evaluated afterward. In addition, plasma levels of I-FABP were determined to measure enterocyte damage. After ischemia, mean red blood cell velocity decreased significantly in all layers of the small bowel, but no diameter changes were observed. Leukocyte-vessel wall interactions increased in the submucosa but not in the muscle layers. Plasma levels of I-FABP significantly increased from 30 minutes of reperfusion onward. The intestinal mucosa was severely injured; no histologic damage was detected in other tissues. This is the first in vivo study showing that total warm ischemia of the rat gut impairs perfusion in the whole small bowel, whereas leukocyte-vessel wall interactions increase in the submucosal layer only. Therefore, the early inflammatory response to I/R seems to be limited to the submucosa. Both microvascular effects may have contributed to the severe morphologic and functional mucosal injury observed after I/R.

Repetitive asphyxia does not enhance leukocyte-vessel wall interactions in the immature fetal intestine

Department of Surgery1, University Hospital Maastricht, Departments of Physiology and Biophysics, Maastricht University, the Netherlands, Departments of Surgery and Pediatrics, University of Auckland, New Zealand

Serial In Vivo Observations of Cerebral Vasculature after Treatment with a Large Single Fraction of Radiation

To test whether single high doses of radiation, similar to those used with radiosurgery, given to normal cerebral vasculature can cause changes in leukocyte-vessel wall interactions and tissue perfusion, a rat pial window model was used to view the cerebral vasculature, facilitating repeated in vivo observations of microcirculatory function. An attachment for a 4 MV linear accelerator was designed to deliver a well-collimated 2.2-mm beam of radiation to a selected region of rat brain. Sequential measurements of leukocyte-endothelial cell interactions, relative change in blood flow with laser Doppler flowmetry and vessel length density were performed prior to and at 24 h and 3 weeks after treatment with 15, 22.5 or 30 Gy, given in a single fraction. Significant increases in leukocyte-endothelial cell interactions were seen 24 h and 3 weeks after irradiation that were dependent on dose, particularly in arteries. Changes were apparent in both arteries and veins at 24 h, but by 3 weeks the effects in arteries predominated. Decreases in vessel length density and blood flow were observed and became greater with time after treatment. A variety of morphological changes were observed in irradiated arteries, including formation of aneurysmal structures, endothelial denudation and thrombus formation. These results suggest that: (1) An increase in leukocyte-vessel wall interactions occurs after irradiation; (2) cerebral arterioles are more sensitive than veins to radiation administered in this fashion; and (3) the increase in leukocyte-vessel wall interactions likely contributes to reduction of or loss of arteriolar flow, with resultant loss of flow to dependent microvascular vessels.

Effects of Different Durations of Total Warm Ischemia of the Gut on Rat Mesenteric Microcirculation

Background: Gut injury due to ischemia and reperfusion (I/R) plays a pivotal role in many clinical conditions, such as small bowel transplantation, heart or aortic surgery in adults, and necrotizing enterocolitis in neonates. The influence of ischemic events on microcirculatory mechanisms is not well understood. Therefore, we studied,in vivo,local perfusion and leukocyte–vessel wall interactions before and after different periods of total warm ischemia of the whole gut and subsequent reperfusion in mesenteric microvessels. Materials and Methods: Groups of pentobarbital-anaesthetized Lewis rats were subjected to 15 (n= 9), 30 (n= 12), or 60 min (n= 5) of total warm gut ischemia and 2 h reperfusion. As control a sham group (n= 10) was included. After ligating the inferior mesenteric artery, total warm ischemia was induced by clamping the superior mesenteric artery. Before and at different time periods after start of reperfusion intravital video microscopic measurements were performed. Results: Rats subjected to 60 min ischemia died during the early reperfusion phase. Fifteen, 30, and 60 min ischemia induced in venules a significant decrease in blood flow, while diameter changes were not observed. This flow decrease was severe in the 15- and 30-min ischemia groups, dropping to 40 and 25% of control, respectively. Following 60 min ischemia blood flow did not exceed 10% of control. The total number of interacting leukocytes, a parameter which includes both leukocyte rolling and adhesion in venules, increased up to 5 or 10 times its control value following 15 or 30 min ischemia, respectively. Leukocyte–vessel wall interactions could not be studied in the 60-min ischemia group, due to the low blood flow. Conclusions: Even short periods of total warm ischemia of the whole gut induce severe attenuation of venular blood flow with an increase in leukocyte–vessel wall interactions. These changes increase with prolongation of the ischemic period. A 60-min period of total warm ischemia is fatal during the early reperfusion phase.

Ischemia/reperfusion injury in rat mesenteric venules: Red blood cell velocity and leukocyte rolling

The authors determined the effects of 15 (n = 9) and 30 (n = 12) minutes of warm ischemia on the rat mesentery and compared the results with those of a sham-operated group (n = 10). Red blood cell velocity and number of rolling leukocytes were assessed before ischemia as well as 10, 20, 30, 60, 90, and 120 minutes after the start of reperfusion. Leukocyte rolling is considered to be an early step of the inflammatory process. Leukocytes roll along the vessel wall at a velocity that is clearly lower than that of the other blood cells. The preischemic values of red blood cell velocity and number of rolling leukocytes in the 15- and 30-minute ischemia groups did not differ from those of the sham group. In the sham group, no significant changes in red blood cell velocity and number of rolling leukocytes were observed over time. Compared with the sham group, the red blood cell velocity of the 15-minute ischemia group was significantly lower at 30, 60, 90, and 120 minutes after the start of reperfusion the number of rolling leukocytes did not differ significantly. For the 30-minute ischemia group, red blood cell velocity also was significantly lower at 20, 30, 60, 90, and 120 minutes after the start of reperfusion, and the number of rolling leukocytes was higher at 10, 20, and 30 minutes after the start of reperfusion. The results of this study indicate that short periods of total warm ischemia of the rat small bowel and subsequent reperfusion result in a significantly impaired microcirculatory blood flow in the mesentery. However, a prolonged period of ischemia is required to increase leukocyte-vessel wall interactions. In the future, this model will enable us to study the effect of pharmacological interventions during an early stage of the inflammatory response to ischemia/reperfusion in the gut.

Intermittence of blood flow in liver sinusoids, studied by high-resolution in vivo microscopy

Kupffer cell migration and leukocyte-vessel wall interactions cause temporary slowing and/or stoppage of blood flow through individual liver sinusoids. Such temporal heterogeneity of flow was quantified in anesthetized mice and rats. Video recordings of red blood cell flow in 44 networks containing 8-16 sinusoids each were analyzed for 5- to 10-min periods. Flow was graded "fast," "slow," "stopped," or "reversed" based on red blood cell velocity. The mean numbers of flow changes (between grades) per minute in zone 1 vs. zone 3 were 1.39 vs. 0.78 (mouse) and 1.25 vs. 0.09 (rat). The mean percentage of time for each flow grade differed significantly between zones 1 and 3 and between species. For example, fast flow was present in zone 1 sinusoids for 51% of the time in mice and for 74% in rats; in zone 3 the corresponding numbers were 76 and 95%. Flow stasis was present in zone 1 sinusoids for 19% of the time in mice and for 7% in rats; in zone 3 the corresponding numbers were 2 and 0%. Thus considerable intermittence of perfusion exists, and the flow conditions create very different microenvironments for hepatocytes in zone 1 vs. zone 3.

Polymorphonuclear leukocytes enhance release of growth factors by cultured endothelial cells.

Porcine aortic endothelial cells (PAECs) in culture constitutively secrete polypeptide (endothelium-derived) growth factors (EDGFs) into the surrounding medium. Incubation of PAECs with human peripheral blood polymorphonuclear leukocytes (PMNs) caused a significant increase in EDGF release as assessed by [3H]thymidine incorporation into BALB/c 3T3 mouse fibroblasts and cell proliferation assay. The effect was time dependent and correlated with the number of PMNs, reaching a maximum with a 1:1 PAEC to PMN ratio. Generation of mitogenic activity was prevented by cycloheximide, indicating a requirement for de novo protein synthesis. Antibody-mediated inhibition assays suggested that mitogenic activity was due to platelet-derived growth factor and basic fibroblast growth factor. When supernatant from N-formyl-methionyl-leucyl-phenylalanine-stimulated PMNs was substituted for PMNs during incubation with PAECs, powerful mitogenic activity was generated, indicating the involvement of soluble mediators. A role for free oxygen radicals was ruled out by experiments in which superoxide dismutase and catalase did not prevent the increase in mitogenic activity. By contrast, serine protease inhibitors such as soybean trypsin inhibitor, alpha 1-antitrypsin, and eglin C reduced the PMN-stimulating activity by 70%, 80%, and 100%, respectively. The possible involvement of cathepsin G and elastase was investigated. Cathepsin G and elastase, when substituted for PMNs, increased the release of EDGFs in a dose-dependent fashion, mimicking the effect of PMNs. These findings suggest a new role for leukocyte-vessel wall interactions in the proliferative feature of atherosclerosis.

Increased biosynthesis of vasoactive prostanoids in Schönlein-Henoch purpura.

Schönlein-Henoch purpura (SHP) is an acute immune-mediated vasculitis characterized by infiltration of polymorphonuclear leukocytes into the vessel wall causing damage to the vascular endothelium by the release of proteolytic enzymes. The local inflammatory and thrombotic process may be regulated by increased biosynthesis of vasoactive prostanoids. We investigated the biosynthesis of thromboxane A2 (TxA2), a potent vasoconstrictor and platelet agonist, prostacyclin (PGI2), a vasodilator and platelet antagonist, and prostaglandin E2, a mediator of inflammation, in 14 children with SHP by physicochemical analysis of index metabolites in plasma and urine. TxA2 and PGI2 biosynthesis in the systemic circulation was significantly elevated in the acute phase of the disease and correlated with the degree of clinical symptoms. Recurrent episodes of the disease were associated with phasic increases of plasma and urinary TxA2 and PGI2 metabolites. Renal TxA2 formation was highest in two patients presenting with the nephrotic syndrome and extracapillary glomerulonephritis. Prostaglandin E2 biosynthesis in the systemic circulation was increased in the acute phase of the disease. The enhanced TxA2 formation is consistent with phasic platelet activation in SHP. The increased PGI2 biosynthesis reflects endothelial cell damage and may be a response of vascular endothelium to modulate platelet-vessel wall and leukocyte-vessel wall interactions. Increased prostaglandin E2 formation, which probably derives from activated polymorphonuclear leukocytes and macrophages, is thought to be related to the inflammatory process in SHP.

Inducible endothelial functions in inflammation and coagulation.

The studies discussed have established that inflammatory or immune cytokines, such as IL-1, TNF, and LT, as well as bacterial endotoxin, can act directly on vascular endothelial cells to modulate two important functional properties. The first of these, the inducible expression of E-LAMs, provides a mechanism for the local regulation of leukocyte-vessel wall interactions. This endothelial-dependent mechanism may be relevant to a broad spectrum of pathologic processes, including inflammation, delayed hypersensitivity reactions, and atherogenesis. The second, modulation of endothelial tissue factor PCA and fibrinolytic components, has important implications for the local balance of prothrombotic and antithrombotic influences at the blood-vessel wall interface. Thus, under the influence of inflammatory stimuli, vascular endothelial cells may actively contribute to the development and maintenance of intravascular or perivascular fibrin. Although the endothelial effector mechanisms of these functional alterations are distinct, their induction by similar stimuli points to important interrelationships of leukocyte-vessel wall adhesion and thrombosis. Further understanding of the regulation of endothelial expression of E-LAMs and coagulant properties should contribute to our understanding of vascular disease.

Consequences of leukocyte-vessel wall interactions in inflammatory and immune reactions.

Consequences of leukocyte-vessel wall interactions in inflammatory and immune reactions.

Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines.

Increased leukocyte adhesion to the endothelial lining of blood vessels is an essential event in inflammation and the pathogenesis of certain vascular diseases. We have studied the effect of interleukin 1 (IL-1), an inflammatory/immune mediator, on endothelial-leukocyte adhesion using quantitative in vitro assays. Selective pretreatment of cultured human umbilical vein endothelial monolayers with IL-1 (5 U/ml, 4 h) resulted in an 18.3 +/- 2.6-fold increase in human peripheral blood polymorphonuclear leukocyte (PMN) adhesion (mean +/- SEM, n = 16) and a 2.6 +/- 0.3-fold increase in monocyte adhesion (n = 7) over basal levels. IL-1-treated endothelial monolayers also supported increased adhesion of the promyelocytic cell line HL-60 and the monocytelike cell line U937 (33.0 +/- 6.0-fold, n = 6 and 4.9 +/- 0.5-fold, n = 15, respectively). In contrast, selective IL-1 pretreatment of leukocytes, or the addition of IL-1 during the adhesion assay, did not alter endothelial-leukocyte adhesion. Conditioned medium from IL-1-treated endothelial cultures also did not promote leukocyte adhesion to untreated monolayers. IL-1 induction of endothelial adhesivity was concentration dependent (maximum, 10 U/ml), time dependent (peak, 4-6 h), and reversible, was blocked by cycloheximide (10 micrograms/ml) or actinomycin D (5 micrograms/ml) but not by acetylsalicylic acid (100 microM), and occurred without detectable endothelial cell damage. IL-1 treatment of SV40-transformed human endothelial cells and dermal fibroblasts did not increase their adhesivity for leukocytes. These data suggest that IL-1 can act selectively on human vascular endothelium to increase its adhesivity for circulating blood leukocytes, and thus to localize leukocyte-vessel wall interactions at sites of inflammation in vivo.