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Incidences of cardiovascular diseases (CVD) are high among virologically suppressed HIV-infected individuals. Monocyte activation and trafficking are key mechanisms in the evolution of CVD. We studied the ability of cenicriviroc (CVC), a dual C-C chemokine receptor type 2 (CCR2) and CCR5 antagonist, to influence the migration of monocytes from HIV-infected individuals on antiretroviral therapy (ART). Monocytes were derived from 23 ART-suppressed HIV-infected and 16 HIV-uninfected donors. In a trans-endothelial migration model, monocytes, and human aortic endothelial cells (HAoECs) were exposed to cenicriviroc and migrated monocytes, quantified. Expression of CCR2 and CCR5 on monocytes and adhesion molecules (E-selectin, ICAM-1, VCAM-1, PECAM-1, and CD99) on HAoECs were measured. The single antagonists, BMS-22 (CCR2), and maraviroc (CCR5), served as controls. When both HAoECs and monocytes together were exposed to the antagonists, cenicriviroc led to a greater decrease in monocyte migration compared to BMS-22 or vehicle in both HIV-infected and HIV-uninfected groups (P < 0.05), with maraviroc having no inhibitory effect. Cenicriviroc treatment of HAoECs alone decreased monocyte migration in the HIV-infected group when compared to vehicle (P < 0.01). Inhibition of migration was not evident when monocytes alone were exposed to cenicriviroc, BMS-22 or maraviroc. Incubation of HAoECs with cenicriviroc decreased E-selectin expression (P = 0.045) but had limited effects on the other adhesion molecules. Cenicriviroc inhibits monocyte trans-endothelial migration more effectively than single chemokine receptor blockade, which may be mediated via disruption of monocyte-endothelial tethering through reduced E-selectin expression. Cenicriviroc should be considered as a therapeutic intervention to reduce detrimental monocyte trafficking. Cenicriviroc, a dual CCR2 and CCR5 antagonist, inhibits monocyte trafficking in an in vitro trans-endothelial migration assay more effectively than a single chemokine receptor blockade.

Human and mouse neutrophils are known to form tethers when rolling on selectins in vitro. Tethers are ∼0.2 μm thin, ∼5–10 μm-long structures behind rolling cells that can swing around to form slings that serve as self-adhesive substrates. Here, we developed a mouse intravital imaging method, where the neutrophil surface is labeled by injecting fluorescently labeled mAb to Ly-6G. Venules in the cremaster muscle of live mice were imaged at a high frame rate using a confocal microscope equipped with a fast resonant scanner. We observed 270 tethers (median length 3.5 μm) and 31 slings (median length 6.9 µm) on 186 neutrophils of 15 mice. Out of 199 tether break events, 123 were followed by immediate acceleration of the rolling cell, which shows that tethers are load-bearing structures in vivo. In venules with a high wall shear stress (WSS; > 12 dyn/cm2), median rolling velocity was higher (19 μm/s), and 43% of rolling neutrophils had visible tethers. In venules with WSS < 12 dyn/cm2, only 26% of rolling neutrophils had visible tethers. We conclude that neutrophil tethers are commonly present and stabilize rolling in vivo.

Selectin-ligand interactions are important for leukocyte homing and functionality. The roles of selectin-ligand interactions in modulating immunity to intracellular infections are not completely understood. Mice lacking the expression of fucosyltransferase-IV and -VII (Fucosyltransferase-IV and -VII double knockout, FtDKO) exhibit deficient functionality of selectin-ligand interactions. We addressed the kinetics of infection and immunity to Listeria monocytogenes (LM), an intracellular pathogen, in FtDKO mice. These mice exhibited enhanced ability to clear infection and increased survival to a lethal dose of LM infection relative to wild-type (WT) C57BL/6J controls. This was associated with increased levels of neutrophils, monocytes, and dendritic cells (DCs) in the blood and/or infected organs. Adoptive transfer of bone marrow (BM) cells from FtDKO mice to WT mice resulted in enhanced neutrophil numbers and improved clearance of LM bacteria in recipients. In vivo depletion of myeloid innate immune cells, particularly neutrophils, monocytes, macrophages, and DCs, using anti-Ly-6G (RB6-8C5) monoclonal antibody, reduced the ability of FtDKO mice to curtail LM infection. Nevertheless, depletion using anti-Ly-6G (1A8) known to exclusively deplete neutrophils did not abrogate increased resistance of FtDKO mice to LM infection, suggesting a role for other myeloid innate immune cells in this model. Examination of BM hematopoietic progenitors through flow cytometry and cell culture colony-forming unit assay showed increased frequencies of granulocyte-macrophage progenitors in FtDKO relative to WT mice, Overall, our results indicate that functional selectin ligand deficiency enhances innate immune-mediated resistance to systemic LM infection despite defective leukocyte migration and lymphocyte homing.

Neutrophils are the first cells arriving at sites of tissue injury or infection to combat invading pathogens. Successful neutrophil recruitment to sites of inflammation highly depends on specific molecular mechanisms, fine-tuning the received information into signaling pathways and converting them into well-described recruitment steps. This review highlights the impact of vascular flow conditions on neutrophil recruitment and the multitude of mechanisms developed to enable this sophisticated process under wall shear stress conditions. The recruitment process underlies a complex interplay between adhesion and signaling molecules, as well as chemokines, in which neutrophils developed specific mechanisms to travel to sites of lesion in low and high shear stress conditions. Rolling, as the first step in the recruitment process, highly depends on endothelial selectins and their ligands on neutrophils, inducting of intracellular signaling and subsequently activating β2 integrins, enabling adhesion and postadhesion events. In addition, subcellular structures, such as microvilli, tethers, and slings allow the cell to arrest, even under high wall shear stress. Thereby, microvilli that are pulled out from the cell body form tethers that develop into slings upon their detachment from the substrate. In addition to the above-described primary capture, secondary capture of neutrophils via neutrophil–neutrophil or neutrophil–platelet interaction promotes the process of neutrophil recruitment to sites of lesion. Thus, precise mechanisms based on a complex molecular interplay, subcellular structures, and cell–cell interactions turn the delicate process of neutrophil trafficking during flow into a robust response allowing effective neutrophil accumulation at sites of injury.

H2S donor attenuates inflammatory responses through the L-selectin involved pathway, in the cutaneous reverse passive Arthus reaction. H2S has been highlighted recently as an endogenous, gaseous signaling molecule, especially in inflammations. The deposition of IC induces an acute inflammatory response with tissue injury. To assess the roles of H2S in the IC-induced diseases, the cutaneous, reverse passive Arthus reaction was conducted using NaHS as a H2S donor. Furthermore, we conducted similar experiments using selectin−/− mice to determine the involvement of selectin molecules in the H2S-mediated pathway. Exogenous application of NaHS dramatically attenuated inflammatory reactions in WT mice associated with Arthus reaction. Namely, mRNA expressions of TNF-α, IFN-γ, and neutrophil numbers were reduced significantly in the lesional skins of NaHS-treated WT mice relative to untreated ones. NaHS treatment significantly reduced these three parameters in the lesional skins of E- and P-selectin−/− mice but not in those of L-selectin−/− mice. Quite similar results were obtained in the blocking study using WT mice injected with mAb to E-, P-, and L-selectin. Our results indicated that the exogenous application of NaHS attenuates inflammatory responses in reverse passive Arthus reaction through a L-selectin-involved pathway but not through E- or P-selectin pathways.

Leukocyte trafficking from the blood into the tissues represents a key process during inflammation and requires multiple steps mediated by adhesion molecules and chemoattractants. Inflammation has a detrimental role in several diseases, and in such cases, the molecular mechanisms controlling leukocyte migration are potential therapeutic targets. Over the past 20 years, leukocyte migration in the CNS has been investigated almost exclusively in the context of stroke and MS. Experimental models of ischemic stroke have led to the characterization of adhesion molecules controlling leukocyte migration during acute inflammation, whereas EAE, the animal model of MS, has provided similar data for chronic inflammation. Such experiments have led to clinical trials of antileukocyte adhesion therapy, with consistently positive outcomes in human subjects with MS, showing that interference with leukocyte adhesion can ameliorate chronic inflammatory CNS diseases. This review summarizes our current understanding of the roles of adhesion molecules controlling leukocyte–endothelial interactions in stroke and MS, focusing on recently discovered, novel migration mechanisms. We also discuss the growing evidence suggesting a role for vascular inflammation and leukocyte trafficking in neurodegenerative diseases such as AD. Moreover, we highlight recent findings suggesting a role for leukocyte–endothelial interactions in the pathogenesis of seizures and epilepsy, thus linking endothelial activation and leukocyte trafficking to neuronal electrical hyperactivity. These emerging roles for leukocytes and leukocyte adhesion mechanisms in CNS diseases provide insight into the mechanisms of brain damage and may contribute to the development of novel therapeutic strategies.

Platelets, monocytes, and endothelial cells are instrumental in the development and progression of cardiovascular diseases. Inflammation, a key process underlying cardiovascular disorders, is accompanied and amplified by activation of platelets and consequent binding of such platelets to the endothelium. There, platelet-derived chemokines, in conjunction with increased expression of adhesion molecules, promote the recruitment of circulating monocytes that will eventually migrate across the endothelial lining of the vessel into the tissues. Additionally, platelets may already become activated in the circulation and may form platelet-monocyte complexes, which show increased adhesive and migratory capacities themselves but also facilitate recruitment of noncomplexed leukocytes. They should therefore be considered as important mediators of inflammation. In molecular terms, these events are additionally governed by chemokines released and presented by the endothelium as well as the different classes of endothelial adhesion molecules that regulate the interactions among the various cell types. Most important in this respect are the selectins and their ligands, such as P-selectin glycoprotein (GP) ligand 1, and the integrins binding to Ig-like cell adhesion molecules as well as to GP, such as von Willebrand factor, present in the extracellular matrix or on activated endothelium. This review aims to provide an overview of these complex interactions and of their functional implications for inflammation and development of cardiovascular disease.

After birth, the clinical course of neonates is frequently complicated by infections leading to sepsis and septic shock due to infections triggered by pathogens [2]. These inflammatory processes, which are commonly caused by gram-negative and -positive bacteria, are the major causes for morbidity and mortality in neonates [3]. It is estimated that neonatal infections cause the death of 1 million neonates/year worldwide [3]. Neonates surviving sepsis display retarded growth and poor neurodevelopmental outcomes [2, 4]. Preterm infants are especially prone to infections as a result of their immature immune system, and 20% of all preterm infants develop a bacterial sepsis during the first 3 days postpartum, rising to as much as 46% in preterm infants, 25 weeks of gestational age [5]. The innate immune system is the first line of defense against exogenous pathogens. As a part of the innate immune system, neutrophils are actively recruited to sites of inflammatory processes, where they combat exogenous pathogens by phagocytosis, release of proteolytic enzymes, and production of cell-toxic ROS. A defect in leukocyte recruitment, such as seen in the “leukocyte adhesion deficiency” syndrome, is accompanied by recurrent infections and a shortened life expectancy. Aside from combating pathogens, the innate immune system, including neutrophils, also participates in the development of an adaptive immune system in neonates. Neutrophil recruitment into peripheral tissues in response to inflammatory stimuli occurs in a cascade-like fashion [6]. The initial contact (capturing) of circulating neutrophils with the endothelium is mediated by Pand E-selectin, expressed on inflamed endothelial cells, binding to their respective receptors on neutrophils. Following the initial contact of neutrophils with the endothelium, neutrophils roll on the inflamed vascular endothelium and are activated by different stimuli. The binding of selectins to their respective receptors on neutrophils induces an intracellular signaling cascade in neutrophils, leading to the activation of 2-integrins. E-selectin binding to PSGL-1 on neutrophils forces the 2-integrin LFA-1 into the extended conformation, which can subsequently bind to its ligand ICAM-1 and induce slow leukocyte rolling. In addition to selectins, chemokines are presented by the inflamed vascular endothelium and bind to GPCRs on neutrophils, leading to firm arrest. Following arrest, neutrophils crawl on the endothelial cells before they transmigrate into the inflamed tissue. Recent research indicates that the function of the innate immune systems differs among preterm infants, term neonates, and adults. Although it is a well-known fact that preterm infants are especially vulnerable to infections as a result of immaturity of their immune system, the underlying mechanisms responsible for this decreased pathogen clearance are only poorly understood. Possible explanations for the immaturity of neutrophils could be a yet-underdeveloped expression pattern of receptors and adhesion molecules on the cell surface or an altered intracellular signaling that limits neutrophil activation during early stages of fetal development. In this issue of the Journal of Leukocyte Biology, Nussbaum and colleagues [1] demonstrated that neutrophils from extremely preterm infants ( 30 weeks of gestation) display severe rolling and adhesion defects. The ability of neutrophils to roll and adhere is closely associated with the gestational age of the preterm infant. This observation is in line with previously published reports showing reduced adhesion of human term neonatal neutrophils under flow conditions compared with adult neutrophils [7]. Mechanistically, this may be explained by different expression levels of surface adhesion receptors in term infants and a diminished response of neonatal neutrophils to the stimulation with chemokines (Fig. 1) [7, 8]. Similarly, endothelial cells from preterm infants also demonstrate a gestational age-related, decreased expression pattern of adhesion molecules, resulting in diminished interaction with neutrophils (Fig. 1) [9]. Notably, the observed correlation between neutrophil rolling and adhesive properties with gestational age fits well with the clinical observation that the prevalence of neonatal sepsis is inversely correlated to gestational age [5]. In addition, whereas the expression of TLRs, in general, is not different between term neonates and adults, it has

Human milk is thought by many authorities to be preferable to formula as a source of nutrients for infants. Some of the benefits may stem from its high concentration of unbound oligosaccharides (5-10 g/L). These sugars have structural similarities to selectin ligands, known to mediate important cell–cell interactions in the immune system. Platelet-neutrophil complexes (PNC) exist in healthy individuals but have been implicated in disease states. Formation of these complexes requires selectins and as such, could be influenced by human milk oligosaccharides (HMO). Here, we investigate this possibility by examining the effect of HMO on the formation of PNC and activation of associated neutrophils. We collected blood from 10 healthy volunteers, activated platelets with adenosine 5′-diphosphate, and added HMO, oligosaccharide standards, or phosphate-buffered saline as a control. We determined the influence of HMO on PNC formation and adjacent neutrophil activation with fluorescein-activated cell sorter analysis after labeling with antibodies for the platelet marker CD42a and the neutrophil activation marker CD11b. Within physiologically achievable concentrations (6.25-125 μg/mL), an acidic HMO fraction reduced PNC formation up to 20%, which was similar to the effect seen with high concentrations of sialyl-Lewis x. Associated neutrophils showed a dose-dependent decrease in β 2 integrin expression, up to 30%, at high but physiological concentrations. The neutral HMO fraction had no effect. These results support the hypothesis that acidic HMO serve as anti-inflammatory components of human milk and thus, contribute to the lower incidence of inflammatory diseases such as necrotizing enterocolitis in breast-fed versus formula-fed infants.

P-selectin glycoprotein ligand-1 (PSGL-1) mediates rolling of leukocytes on P-selectin-expressing endothelial cells under shear flow. Function-blocking monoclonal antibodies (mAbs) against mouse and human PSGL-1 recognize an anionic segment at the N-terminus of PSGL-1. High affinity interaction of PSGL-1 with P-selectin requires sulfation of tyrosines 46, 48, and 51 (human) or 54 and 56 (mouse). We tested binding of two anti-human (KPL1 and PL1) and two anti-mouse (4RA10 and 2PH1) PSGL-1 mAbs to synthetic peptides of N-terminus of human and mouse PSGL-1 and found binding to be independent of tyrosine sulfation. In peptide-blocking experiments, sulfated and nonsulfated human and mouse peptides competed with antibody binding to PSGL-1 expressed on myeloid cells. Arylsulfatase treatment significantly reduced P-selectin binding but had no effect on antibody binding. Our data show, in three independent assay systems, that function-blocking antibodies to mouse or human PSGL-1 do not require sulfation of N-terminal tyrosines for binding.

Sulfation is an essential component of the selectin ligands, potentially mediated by members of a new family of carbohydrate sulfotransferases. In this study, we assessed the contributions of CHST1, CHST2, CHST3, and CHST4 in producing functional l-selectin ligands. Human umbilical vein endothelial cells predominantly expressed CHST1 and CHST2 transcripts with low levels of CHST3 mRNA, while cytokine activation up-regulated CHST2 expression and induced low-level CHST4 expression. A human umbilical vein endothelial cell line, EA.hy926, displayed functional l-selectin ligands that correlated with CHST1 and CHST2 expression in the absence of CHST4 expression. Increased CHST1 or CHST2 expression by a cell line expressing low-level l-selectin ligand activity during in vitro flow chamber assays increased rolling leukocyte numbers, reduced rolling velocities, and enhanced leukocyte rolling under higher shear stresses. These results suggest that CHST1 and CHST2 contribute to the generation of optimal l-selectin ligands in vascular endothelial cells at sites of inflammation.

Biopsy specimens from patients with inflammatory bowel disease have demonstrated an up-regulation of P-selectin, suggesting a role for P-selectin in intestinal inflammation. We examined the role of P-selectin in experimental intestinal inflammation using mice deficient in P-selectin alone or in combination with either ICAM-1 or E-selectin. Colitis was induced using acetic acid or trinitrobenzene sulfonic acid (TNBS). Damage scores and neutrophil infiltration 24 h post acetic acid were not different between wild-type and P-selectin- or P-selectin/ICAM-1-deficient mice, whereas P/E-selectin-deficient mice had enhanced leukocyte recruitment and damage. At 72 h an attenuation in damage scores and a slight decrease in neutrophil infiltration was observed in the P- and P/ICAM-deficient animals. The P/E-selectin-deficient mice maintained enhanced leukocyte recruitment and damage. In wild-type mice P-selectin expression was elevated 48 and 72 h post acetic acid-induced inflammation. Surprisingly, P-selectin or P-selectin/ICAM-1 deficiency did not improve the inflammation induced by TNBS over 7 days. In fact, increased mortality was observed. Anti-adhesion therapy may play only a limited, beneficial role and often a detrimental role in intestinal inflammation. J. Leukoc. Biol. 66: 67–74; 1999.

Neutrophil (PMN) migration into the peritoneal cavity in response to fecal peritonitis is an important mechanism of host defense against bacterial invasion. We show that the murine C-X-C (PMN-specific) chemokine, macrophage inflammatory protein-2 (MIP-2), on intraperitoneal injection in mice, causes PMN migration into the peritoneum. MIP-2 mRNA and protein were expressed by peritoneal leukocytes after cecal ligation and puncture (CLP) in mice and neutralization of MIP-2 reduced peritoneal PMN migration. A prerequisite for neutrophil-endothelial adhesion and subsequent migration from the circulation is selectin-mediated rolling. Pretreatment of mice with an anti-P-selectin antibody before intraperitoneal injection of MIP-2 significantly reduced peritoneal PMN migration. However, there are no reports that a C-X-C chemokine can up-regulate endothelial selectins. We postulated that MIP-2, when injected intraperitoneally, interacts with a cell that is known to release factors that up-regulate endothelial selectins. A likely candidate is the mast cell, which contains histamine and tumor necrosis factor α (TNF-α), and both of these factors induce selectins. Intraperitoneally injected MIP-2 caused an early significant increase in peritoneal TNF-α, whereas histamine levels were unaffected. In a subsequent experiment, mast cell-deficient mice and their normal controls were then injected intraperitoneally with MIP-2 or underwent CLP. Significantly fewer PMNs migrated into the peritoneal cavity in the mast cell-deficient mice after MIP-2 injection or CLP. Thus, our findings indicate that mast cells and MIP-2 are necessary for PMN migration into the peritoneum in response to intra-abdominal infection, and that MIP-2 appears to facilitate this through an increase in TNF-α release. J. Leukoc. Biol. 65: 249–255; 1999.

Leukocyte emigration is essential in both lymphocyte homing, as a central part of immune surveillance, and in leukocyte invasion at sites of inflammation. The emigration of leukocytes requires the interplay of adhesion molecules of the selectin and integrin families and chemokines. Selectindependent cell-cell interaction is essential in localizing leukocytes within tissues by promoting the rolling of leukocytes along the endothelial cell surface before development of tight adhesion and subsequent transendothelial migration. Selectins also play an active role in the initiation of intracellular signaling pathways and regulation of cell-cell interactions involving monocytes, lymphocytes, platelets, and endothelial cells. This review focuses mainly on the emerging evidence of biochemical signaling mechanisms involved in the regulation of selectin-dependent leukocyte activation and adhesion, as well as the critical role played by selectins as leukocyte stimulatory molecules. This evidence has serious implications regarding the development of immune and inflammatory responses. This article will also review key structural features of the selectin receptors. A summary is provided of our current understanding of the specific molecular interaction occurring between these adhesion molecules and their counter-receptors, focusing on the critical roles they may play in the regulation of functional responses.

Cell adhesion molecules provide the foundation for cell communication, trafficking, and immune surveillance central to host defense. These adhesion molecules which include selecting, integrins and members of the Ig superfamily, provide a recognition system between leukocytes, endothelial cells and matrix molecules. Leukocyte-endothelial interactions initiate recruitment at sites of injury, infection and inflammation. Cell-cell and cell-matrix interactions also influence leukocyte phenotype and function. Dysregulation of these adhesion and signal transduction pathways can contribute to continued recruitment and persistent leukocyte activation with unresolved inflammation. Based on the pivotal role adhesive interactions play, the adhesion molecules provide potential targets for intervention. Selected synthetic fibronectin peptides, which inhibit leukocyte integrin binding and signal transduction in vitro, block recruitment and activation to limit inflammation in vivo.

Leukocytes usually pass through the blood stream as nonadherent cells, but during an immune response and inflammation, they become adherent in order to migrate through tissues. Three families of adhesion molecules, the immunoglobulin family, the selectins, and the integrins, participate in interactions between leukocytes and tissues. Near sites of inflammation, leukocytes initially interact with the endothelium via selectins, causing them to slow down and to roll along the walls of blood vessels. Next, chemoattractans induce the activation of integrins on leukocytes. Finally, activated integrins mediate leukocyte migration through the endothelium into the inflamed site. Interactions of leukocytes with other cells and various extracellular matrix (ECM) proteins lead to different functional cell responses, including changes in growth, behavior, and differentiation. Many of these interactions are mediated by integrins, which “integrate” ECM protein signals with the cytoskeleton and which also act as true receptors that generate biochemical signals within the cell. Changes in pH, cytoplasmic Ca2+ concentration, phosphorylation, and gene induction have all been observed after integrin engagement. Adhesion-mediated gene induction in monocytes is perhaps the best example that integrins initiate signaling cascades in the cell to deliver information from the ECM all the way to the nucleus. J. Leukoc. Biol. 57: 189–198: 1995.

The time course of endothelial P-selectin, ICAM-1, and E-selectin expression was studied in a feline model of myocardial ischemia and reperfusion. Cats were subjected to 90 min of myocardial ischemia followed by 0, 10, 20, 60, 150, or 270 min of reperfusion. At the end of reperfusion, the coronary vasculature was examined immunohistochemically to localize monoclonal antibodies (mAbs) PB1.3, RR1/1, and Cyl787 directed against P-selectin, ICAM-1, and E-selectin, respectively. Immuno-histochemical localization for P-selectin, recognized by mAb PB1.3, was maximally expressed 20 min after reperfusion in 60 ± 6% of coronary venules (P < 0.05 compared to non-reperfused controls), and covered 59 ± 3% of the endothelial cell perimeter of immunostained coronary venules. Immunolocalization of mAb PB1.3 gradually declined at 60, 150, and 270 min of reperfusion. Immunohistochemical localization of mAb RR1/1 (anti-ICAM-1) in endothelial cells of coronary venules was observed to a modest extent in non-ischemic myocardium and at 10, 20, and 60 min of reperfusion, but was significantly increased following 150 and 270 min of reperfusion (P < 0.05 compared non-reperfused controls). At 270 min post-reperfusion, mAb RR1/1 was seen in 50 ± 4% of coronary venules. Endothelial immunolocalization of mAb Cyl787 (anti-E-selectin) was only observed in 13 ± 1 and 14 ± 3% of coronary venules after 150 and 270 min of reperfusion, respectively, suggesting that pronounced expression of E-selectin does not occur within 270 min after reperfusion. These results demonstrate sequential expression of three major endothelial cell adherence molecules in situ following myocardial ischemia and reperfusion. The timing of endothelial cell expressed P-selectin and ICAM-1 could coordinate neutrophil trafficking during the early stages of reperfusion. J. Leukoc. Biol. 57: 45–55; 1995.

Leukocyte recruitment is ontogenetically regulated during fetal life, with strongly impaired adhesiveness of fetal leukocytes and endothelial cells, during early fetal development. Attenuation of the immune response contributes to the high rate of neonatal infections, particularly in premature infants. Whereas our knowledge of innate immune functions in mature neonates is growing, little is known about the ontogeny of neutrophil recruitment. We investigated neutrophils and ECs in the course of gestation with respect to rolling and adhesive functions. With the use of microflow chambers, we demonstrate that the neutrophilˈs ability to roll and adhere directly correlates with gestational age. These adhesion-related abilities are very rare in extremely premature infants (

Activated platelets expressing P-selectin on their surface are known to adhere to monocytes and neutrophils. We examined the possibility that the leukocytes were activated by their adhesion to activated platelets and demonstrated that P-selectin–dependent platelet adhesion to neutrophils and monocytes induced production of extracellular superoxide anion (O2-) by these leukocytes. Leukocyte membrane glycoproteins containing Ser/Thr-linked carbohydrate chains were responsible for the signal reception leading to the leukocyte activation. Cytokines were shown to influence these processes. For example, treatments of neutrophils with interleukin-8 (IL-8) or granulocyte colony-stimulating factor (G-CSF) potentiated the P-selectin-induced O2- production. Furthermore, interleukin-1 (IL-1) and interferon-γ (IFN-γ) induced surface expression of P-selectin on platelets in the presence of a low concentration of thrombin and consequently enhanced their adhesion capacity to leukocytes. These results indicated that the adhesion of activated platelets to the leukocytes through the interaction between P-selectin and its carbohydrate ligand, sialyl Lewis X (Lex), was a crucial step for the activation of leukocyte function and supported the notion that activated platelets were actively involved in the inflammatory processes. J. Lcukoc. Biol. 56: 583–587; 1994.

Plasma concentrations of lactoferrin relevant to an inflammatory response are known to elicit leukocyteendothelial cell adhesion in mesenteric venules. The objectives of this study were (1) to determine whether exogenously administered lactoferrin causes microvascular and mucosal injury in rat intestine and (2) to assess the contribution of adherent leukocytes to a lactoferrinmediated injury process. Mucosal myeloperoxidase (MPO) activity and vascular protein clearance were monitored in the distal intestine of male Sprague-Dawley rats. Macroscopic erosive lesions of the mucosa and increases in mucosal MPO and intestinal vascular protein were observed 2 h following the lactoferrin infusion, results consistent with granulocyte accumulation and microvascular protein leakage. These lactoferrin-induced alterations were significantly attenuated in animals pretreated with a monoclonal antibody (mAb) directed against P-selectin but not by an E-selectin-specific mAb. In another series of experiments, leukocyte adherence/emigration and leakage of fluorescein isothiocyanate (FITC)-labeled albumin were measured in rat mesenteric venules using intravital video microscopy. Lactoferrin elicited increases in both leukocyte adhesion/emigration and albumin extravasation, which were attenuated by mAbs directed against P-selectin but not E-selectin. These observations indicate that (1) the lactoferrin released by activated neutrophils may lead to significant microvascular and mucosal injury or dysfunction and (2) the lactoferrininduced injury is related to P-selectin-mediated adhesion of leukocytes to microvascular endothelium. Our results raise the possibility that neutrophil-derived lactoferrin contributes to the inflammatory response by promoting further granulocyte accumulation and activation and that mAbs to P-selectin may be therapeutically beneficial in inflammatory disorders. J. Leukoc. Biol. 55: 771–777; 1994.

The accumulation of leukocytes in inflamed tissue results from adhesive interactions between leukocytes and endothelial cells within the microcirculation. These adhesive interactions and the excessive filtration of fluid and protein that accompanies an inflammatory response are largely confined to one region of the microvasculature: postcapillary venules. The nature and magnitude of the leukocyte–endothelial cell adhesive interactions that take place within postcapillary venules are determined by a variety of factors, including expression of adhesion molecules on leukocytes and/or endothelial cells, products of leukocyte (superoxide) and endothelial cell (nitric oxide) activation, and the physical forces generated by the movement of blood along the vessel wall. The contribution of different adhesion molecules to leukocyte rolling, adherence, and emigration in venules is discussed. Emerging views on potential endogenous antiadhesion molecules produced by endothelial cells as well as the influence of alterations in shear rate on leukocyte adhesion are addressed. Finally, the pathophysiological significance of the microvascular responses to inflammation are discussed in terms of adhesion-directed strategies for the treatment of different cardiovascular diseases and circulatory disorders. J. Leukoc. Biol. 55: 662–675; 1994.

The role of CRP as a regulator of inflammation is not fully understood. Structural rearrangement in CRP results in expression of potent proinflammatory actions. Proteolysis of CRP yields the C-terminal peptide Lys201-Pro-Gln-Leu-Trp-Pro206. Here, we investigated the impact of this peptide on neutrophil interactions with endothelial cells and platelets, critical inflammatory events triggering acute coronary artery disease. CRP peptide 201–206 induced L-selectin shedding from human neutrophils and inhibited L-selectin-mediated neutrophil adhesion to TNF-α-activated HCAECs under nonstatic conditions. CRP peptide 201–206 also attenuated shear-induced up-regulation of platelet P-selectin expression, platelet capture of neutrophils, and subsequent homotypic neutrophil adhesion in human whole blood. Anti-CD32 but not anti-CD16 or anti-CD64 mAb effectively prevented the inhibitory actions of CRP peptide 201–206. Substitution of Lys201, Gln203, or Trp205 with Ala in CRP peptide 201–206 resulted in loss of the biological activities, whereas peptides in which Pro202, Leu204, or Pro206 was substituted with Ala retained biological activity. We identified amino acid residues involved in CRP peptide 201–206-FcγRII (CD32) interactions, which mediate potent antineutrophil and antiplatelet adhesion actions, and these findings open up new perspectives for limiting inflammation and thrombosis underlying coronary artery disease.

An acute inflammatory response requires that circulating monocytes bind to and migrate across the endothelium of the vessel wall to gain access to inflamed sites. Various mediators of inflammation - cytokines and chemoattractants - have been shown to initiate and regulate the margination and extravasation of monocytes. This review summarizes evidence that the mechanism underlying the initial adhesion of monocytes to normal and cytokine-stimulated endothelial cells and their subsequent transendothelial migration are successive events in monocyte-endothelial cell interaction. Special emphasis is given to the current knowledge of the contribution of adhesion molecules belonging to the family of 13i- and 132-integrins, the immunoglobulin supergene family, and the selectins to such cellular interaction. The sequence of events that allow monocytes to attach to, migrate over and finally pass the endothelium is discussed in detail.

Review on PSGL-1 effects on signaling in myeloid leukocytes activates following selectin engagement. Cell-cell interactions mediating leukocyte recruitment and inflammation are crucial for host defense. Leukocyte recruitment into injured tissue proceeds in a multistep process. The first contact of leukocytes with endothelial cells (“capturing” or “tethering”) is mediated by selectins and their counter-receptor P-selectin glyco-protein ligand (PSGL)-1. During capture and rolling, leukocytes collect different inflammatory signals, which can activate various pathways. Integration of these signals leads to leukocyte activation, integrin-mediated arrest, cytoskeleton rearrangement, polarization, and transmigration. PSGL-1 on leukocytes also binds to activated platelets, where P-selectin is expressed at locally high site densities following α-granule fusion with the plasma membrane. Here, we review the signaling functions of PSGL-1 and speculate how the different known signaling events might relate to different phases of leukocyte recruitment.

Skin wound healing is mediated by inflammatory cell infiltration that is highly regulated by various adhesion molecules. Mice lacking intercellular adhesion molecule-1 (ICAM-1) delayed skin wound healing and mice lacking both L-selectin and ICAM-1 (L-selectin/ICAM-1−/−) show more delayed wound healing. Deficiency of both endothelial selectins (E-selectin or P-selectin) also delays wound healing. However, the relative contribution and interaction of selectins and ICAM-1 to the wound healing remain unknown. To clarify them, repair of excisional wounds was examined in L-selectin/ICAM-1−/− mice, wild-type mice with both E- and P-selectin blockade, and L-selectin/ICAM-1−/− mice with both E- and P-selectin blockade. Wild-type mice with both E- and P-selectin blockade showed delayed wound healing that was comparable with that in L-selectin/ICAM-1−/− mice. Combined E- and P-selectin blockade in L-selectin/ICAM-1−/− mice resulted in more significant delay. Mice lacking or blocked for adhesion molecules also showed suppressed keratinocyte migration, angiogenesis, granulation tissue formation, leukocyte infiltration, and cytokine expression, including transforming growth factor-β and interleukin-6. Application of basic fibroblast growth factor (bFGF) but not platelet-derived growth factor to the wounds significantly improved wound healing in L-selectin/ICAM-1−/− mice with both E- and P-selectin blockade. bFGF significantly increased the leukocyte infiltration and subsequent fibrogenic cytokine production, as well as keratinocyte migration, angiogenesis, and collagen synthesis despite the loss of four kinds of adhesion molecules. These results indicate that skin wound healing is regulated cooperatively by all selectins and ICAM-1 and may provide critical information for the therapy of skin wounds.

Selectins and their ligands support leuocyte rolling, facilitating the subsequent firm adhesion and migration that occur during inflammation. TBC-1269 (Bimosiamose), a structural mimetic of natural selectin ligands, inhibits P-, E-, and L-selectin in vitro, has anti-inflammatory effects in vivo, and recently underwent phase II clinical trials for childhood asthma and psoriasis. We studied whether the anti-inflammatory effects of TBC-1269 could be related to leukocyte rolling in vivo. Although TBC-1269 inhibited rolling of a murine leukocyte cell line on murine P-selectin in vitro and thioglycollate-induced peritonitis in vivo, it did not alter leukocyte rolling in mouse cremaster venules. TBC-1269 reduced neutrophil recruitment in thioglycollate-induced peritonitis in wild-type and P-selectin−/− mice but not in E-selectin−/− mice. We suggest that the in vivo effects of TBC-1269 may be mediated through E-selectin but do not appear to involve leukocyte rolling.

Immune complex (IC)-induced tissue injury is mediated by inflammatory cell infiltration that is highly regulated by various adhesion mole- cules. To assess the contribution of P-selectin gly- coprotein ligand-1 (PSGL-1) and selectins in the pathogenetic process, the cutaneous reverse-pas- sive Arthus reaction was examined in mice treated with monoclonal antibodies (mAb) to PSGL-1 or P- and/or E-selectin. Edema and hemorrhage were significantly reduced in mice treated with anti-P- selectin mAb compared with control mice while they were not inhibited in mice treated with anti- E-selectin mAb. It is remarkable that blocking PSGL-1 by mAb resulted in significant, further re- duction in edema and hemorrhage compared with blocking anti-P- or anti-E-selectin. However, blockade of E- and P-selectins exhibited more sig- nificant reduction relative to PSGL-1 blockade. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells. Reduced infiltration of neutrophils and mast cells was observed in the peritoneal Arthus reaction and was associated with the decreased production of tumor necrosis factor and interleukin-6. The results of this study indicate that PSGL-1 contrib- utes to the Arthus reaction mainly as a ligand of P-selectin and partly as a ligand of E- and/or L- selectin by regulating neutrophil and mast-cell re- cruitment and that PSGL-1 would be a therapeutic target for human IC-mediated diseases. J. Leukoc. Biol. 76: 374-382; 2004.

Allergic asthma is increasing in incidence and severity in many industrial countries. Leukocyte recruitment into the airways of affected individuals contributes to the severity of the disease. In this study, whole blood from normal, allergic, asthmatic, or allergic-asthmatic subjects was perfused over immobilized adhesion molecules using an in vitro flow chamber system to determine if there were differences in leukocyte recruitment in these patient populations. Leukocytes from allergic-asthmatic subjects showed a threefold increase in recruitment on P-selectin as compared with normal controls. In both patient populations, the accumulated cells were exclusively neutrophils and eosinophils. Increased granulocyte recruitment was specific for P-selectin, as neither purified E-selectin nor vascular cell adhesion molecule-1 (VCAM-1) supported enhanced leukocyte recruitment from allergic-asthmatics. Leukocyte accumulation on P-selectin was completely blocked by an anti-P-selectin or anti-P-selectin glycoprotein ligand-1 (PSGL-1) monoclonal antibody. Flow cytometry revealed that neutrophils and eosinophils from allergic-asthmatic subjects had increased expression of PSGL-1, whereas expression of another adhesion molecule, L-selectin, was unchanged. PSGL-1 expression on peripheral blood mononuclear cells of allergic-asthmatic patients was unaffected. The increased PSGL-1 expression on granulocytes from allergic-asthmatic patients also led to enhanced leukocyte recruitment on interleukin-4-stimulated human umbilical vein endothelial cells, which express P-selectin and VCAM-1. Thus, increased PSGL-1 expression on granulocytes from allergic-asthmatic subjects resulted in increased leukocyte recruitment on P-selectin under flow conditions.

Cell adhesion molecules and their ligands are essential for regulating lymphocyte recirculation and leucocyte emigration into an inflamed or injured tissue. Vascular endothelial selectins as mediators of leucocyte rolling and intercellular cell adhesion molecule-1 (ICAM-1) have been found to be up-regulated on activated endothelium during acute allograft rejection. This study was designed to investigate whether ICAM-1 or selectin-ligand deficiency, or a combination of both, affected graft survival during acute cardiac allograft rejection. To this goal, we performed cardiac transplantation using mice deficient in genes for ICAM-1 or α(1,3)fucosyltransferase Fuc-TVII, representing a model for general absence of selectin-ligand expression, and a newly developed strain with a double mutation in Fuc-TVII and ICAM-1 alleles. Transplantation of a heart from ICAM-1 −/− or Fuc-TVII/ICAM-1 double-mutated mice into allogeneic recipients resulted in limited (2–2.5 days) but nevertheless significant prolongation of the graft survival (P

E-selectin, a member of the selectin family of adhesion molecules, is thought to play an important role in leukocyte-endothelial (EC) interactions during inflammation and atherosclerosis. To critically examine the role of E-selectin in leukocyte-EC interactions in the vascular system, we created a recombinant adenoviral vector containing a human E-selectin cDNA (AdRSVE-sel) and examined the effect of AdRSVE-sel in an ex vivo vascular model of a rat aortic segment. A segment of abdominal aorta was isolated from a male Sprague-Dawley rat transduced with AdRSVE-sel ex vivo. After 72 h, surface expression of transduced E-selectin in the segment was confirmed by Western blotting and immunohistochemistry using anti-E-selectin mAb. Aortic segments were connected to a perfusion system and the adhesion of human polymorphonuclear neutrophils (PMN), and a human monocytic cell line (THP-1) to the EC surface was studied in the presence of a physiological level of flow (0.85 ml/min, approximate luminal surface shear stress=1.76 dyn/cm2). Adhesion of PMN was assessed by scanning electron microscopy and quantified using fluorescently labeled PMN. AdRSVE-sel transduced aortic segments mediated significantly more PMN and THP-1 adhesion than control segments transduced with AdRSVLacZ. Pretreatment of AdRSVE-sel transduced aortic segments with anti-E-selectin mAb inhibited PMN adhesion significantly, as well as THP-1. These data indicate that human E-selectin expressed in rat aortic segments can support the adhesion of human PMN as well as THP-1 under physiological flow conditions. This genetically modified, excised, vascular-segment model provides a useful tool for the study of leukocyte recruitment in the vascular system.

The recently discovered tetraspanin superfamily comprises a group of cell-surface proteins that are suggested to be involved in cell activation and signal transduction as well as in cell adhesion, motility, and metastasis. In this study, we have assessed the expression of two tetraspanins, CD53 and CD63, and two principal leukocyte adhesion molecules, CD11b and CD62L, on human apoptotic neutrophils. After aging of human neutrophils for 20 and 40 h in vitro, apoptosis was analyzed by light microscopy and flow cytometry. The binding of monoclonal antibodies directed against CD11b, CD62L, CD53, and CD63 on apoptotic and nonapoptotic cells was determined by dual-color flow cytometry. Aging of neutrophils in vitro resulted in a significant (P < 0.05) down-regulation of expression of the selectin CD62L, and a significantly increased expression of the two tetraspanins CD53 and CD63. The selective analysis of apoptotic versus nonapoptotic cells proved that both the increased expression of the tetraspanins and the loss of CD62L were restricted to the apoptotic subpopulation. An identical pattern of surface molecule expression was detected at 12 h after induction of apoptosis by an agonistic anti-Fas IgM monoclonal antibody. Further studies are required to clarify whether tetraspanins participate in the recognition of apoptotic circulating or extrava-sated neutrophils by macrophages. J. Leukoc. Biol. 67: 369–373; 2000.

Adhesive interactions between receptors on vascular endothelial cells (EC) and circulating leukocytes are pivotal in regulating leukocyte extravasation. Although primary adhesion of lymphocytes to EC has been primarily attributed to the selectin family of receptors, CD44 can also mediate this function when activated to bind its ligand hyaluronan (HA). Triggering through the T cell receptor induces activated CD44 and CD44-dependent primary adhesion in both human and mouse lymphocytes, and the interaction can mediate the extravasation of activated T cells into an inflamed site. Lymphocytes capable of CD44/HA-dependent primary adhesion are found in peripheral blood of some rheumatologic patients, and their presence is associated with concurrent symptomatic or active disease. Thus, circulating T cells bearing activated CD44 may represent a pathogenically important subpopulation of activated cells that is elevated under conditions of chronic inflammation. Together, these data add to the selectin and immunoglobulin gene families a new receptor/ligand pair and further our understanding of their potential physiological role; i.e., antigen-specific T cell activation together with local vascular inflammation permits the CD44/HA interaction and subsequent T cell extravasation. J. Leukoc. Biol. 66: 315–321; 1999.

Platelet-derived adherence-inhibiting factor (AIF) has been demonstrated to regulate the neutrophil binding to type IV collagen. In this study, we have examined the effect of AIF on neutrophil adherence to confluently cultured human umbilical vein endothelial cells (EC). AIF inhibited neutrophil adherence to thrombin- or tumor necrosis factor α (TNF-α)-stimulated EC by 75 or 43%, respectively, but hardly affected neutrophil adherence to resting EC. It is interesting to note that the inhibitory activity of AIF was reduced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of neutrophils. Pretreatment of neutrophils or EC with AIF inhibited neutrophil adherence to thrombin- or TNF-α-stimulated EC, suggesting that neutrophils and EC have AIF-binding proteins. Using AIF-Sepharose affinity chromatography, AIF-binding proteins containing L-selectin were isolated from 125I-labeled resting neutrophils. However, L-selectin was markedly decreased in the AIF-binding fraction from fMLP-stimulated neutrophils. With the use of AIF-affinity chromatography, P- and E-selectins were obtained in the AIF-binding fractions from resting, thrombin-, and TNF-α-stimulated EC. It is important to note that P- and E-selectin were greatly increased in the AIF-binding fractions from thrombin- and TNF-α-stimulated EC, respectively. Furthermore, AIF was able to bind to L-selectin-IgG chimeric protein and inhibit the binding of chimeric protein to thrombin- or TNF-α-stimulated EC. In addition, AIF inhibited the binding of anti-P- or anti-E-selectin monoclonal antibody to the lysates of thrombin- or TNF-α-stimulated EC. Together these observations indicate that AIF could recognize L-, P-, and E-selectins, and modulate neutrophil adherence to EC through interactions with selectin molecules.

Intravital microscopy was used to examine the role of reduced shear forces on neutrophilendothelium interactions in single 25–40 μm diameter vessels in the feline postischemic mesenteric microvasculature. Neutrophil rolling, neutrophil adhesion, and microvascular permeability alterations were determined in vessels exposed to ischemia/reperfusion in untreated animals and in cats that received feline plasma directly into the superior mesenteric artery to increase intestinal blood flow and venular shear rates. Ischemia, followed by reperfusion, caused a profound increase in venular shear forces during the initial hyperemic response, which then decreased to below control values by 10 min of reperfusion and to less than 50% of control by 60 min of reperfusion. Associated with the decrease in blood flow was a profound increase in neutrophil rolling and neutrophil adhesion and an increase in microvascular permeability. Also, leukocyte rolling velocity decreased dramatically to 10–20% of control values during the first 10 min of reperfusion. Infusion of autologous plasma into the intestinal vasculature to maintain the shear forces at control levels during reperfusion did not affect the flux of rolling neutrophils. The rolling velocity of the neutrophils was not increased despite dramatically improved shear rates. Improved shear rates did reduce the number of adherent cells, resulting in less vascular dysfunction. The reduction in shear forces through inflamed microvessels does not contribute to the increased leukocyte rolling flux but is an essentential, permissive component for neutrophil adhesion and subsequent vascular dysfunction in postischemic microvasculature.

GlyCAM-1 (glycosylation-dependent cell adhesion molecule-1) is one of the sialomucin-like ligands for L-selectin, which is a member of the selectin family and mediates initial adhesion of leukocytes to specialized high endothelial venules in lymph nodes and venules at sites of inflammation. GlyCAM-1, lacking a transmembrane domain, is supposed to be secreted into the blood, To understand the functional role of secreted GlyCAM-1, we performed sandwich enzyme-linked immunosorbent assay to measure GlyGAM-1 plasma levels after inflammatory stimulus. BALB/c mice were injected with complete Freund's adjuvant (CFA) in the hind footpads; serum levels of GlyCAM-1 and L-selectin bound to GlyCAM-1 and several inflammatory cytokines, including interleukin-6 (IL-6), were measured at various intervals. IL-6 showed a significant increase 3 h after CFA stimulation. GlyGAM-1 was increased at 3 h, reached peak levels at 12 h, and gradually decreased thereafter. Levels of L-selectin bound to the plasma GlyCAM-1 changed over a similar time course, reached peak at 12 h after, and then began to decrease. The binding of L-selectin to plasma GlyCAM-1 was completely eliminated with the presence of ethyleneglycol-bis(β-aminoethylether)-N,N'-tetraacetic acid, showing the calcium dependency of this binding. These findings show that GlyGAM-1 release is enhanced by inflammatory stimulation and also suggest that released plasma GlyGAM-1 may trap, at least in part, soluble L-selectin shed from stimulated leukocytes to neutralize each other.

We have examined the role of CD66 in the modulation of neutrophil adhesion and effector function. Engagement of neutrophil CD66 with anti-carcinoembryonic antigen (anti-CEA) Ig results in activation-associated phenomena including shape change, activation of β2-integrins, and priming of the respiratory burst. Anti-CEA Ig-treated neutrophils underwent transient shape change distinct from that induced by formyl-Met-Leu-Phe (fMLP). fMLP stimulated β2-integrin up-regulation and 70% loss of L-selectin, whereas only low-level up-regulation of the β2-integrins, without loss of L-selectin, occurred with anti-CEA Ig. Anti-CEA F(ab’)2 fragments and whole Ig augmented β2-integrin-dependent adhesion. Anti-CEA Ig-induced β2-integrin activation was transient, whereas fMLP-induced activation persisted longer. Although they did not cause a significant increase in respiratory burst activity, CEA Ig and F(ab’)2 fragments of antibody primed neutrophils so that the subsequent fMLP-induced respiratory burst was significantly increased. J. Leukoc. Biol. 58: 40–48; 1995.