The P2Y1 receptor-mediated leukocyte adhesion to endothelial cells is inhibited by melatonin.

Extracellular ATP (released by endothelial and immune cells) and its metabolite ADP are important pro-inflammatory mediators via the activation of purinergic P2 receptors (P2Y and P2X), which represent potential new targets for anti-inflammatory therapy. Endothelial P2Y1 receptor (P2Y1R) induces endothelial cell activation triggering leukocyte adhesion. A number of data have implicated melatonin as a modulator of immunity, inflammation, and endothelial cell function, but to date no studies have investigated whether melatonin modulates endothelial P2YR signaling. Here, we evaluated the putative effect of melatonin on P2Y1R-mediated leukocyte adhesion to endothelial cells and TNF-α production, using mesenteric endothelial cells and fresh peripheral blood mononuclear cells isolated from rats. Endothelial cells were treated with the P2Y1R agonist 2MeSATP, alone or in combination with melatonin, and then exposed to mononuclear cells. 2MeSATP increased leukocyte adhesion to endothelial cells and TNF-α production in vitro, and melatonin inhibited both effects without altering P2Y1R protein expression. In addition, assays with the Ca2+ chelator BAPTA-AM indicate that the effect of melatonin on 2MeSATP-stimulated leukocyte adhesion depends on intracellular Ca2+ modulation. P2Y1R is considered a potential target to control chronic inflammation. Therefore, our data unveiled a new endothelial cell modulator of purinergic P2Y1 receptor signaling.

Leukocyte receptor expression in chronic periodontitis.

BACKGROUND AND OBJECTIVE: Microbial recognition in the periodontium through specific leukocyte receptors gives rise to the response which in susceptible individuals can lead to periodontal diseases. The aim of this study was to explore the expression of leukocyte receptors in the gingival tissues of chronic periodontitis patients and to analyse differences between diseased and control sites (sites with probing pocket depth <4 mm). MATERIAL AND METHODS: Thirty-seven chronic periodontitis patients were included in the study. Gingival biopsies were harvested from diseased and control sites and processed by flow cytometry for the determination of the expression of 16 leukocyte receptors (CD4, CD8, CD11b, CD14, CD16, CD19, CD25, CD28, CD49d, CD49e, CD62, CD71, CD80, CCR7, Ly6G and HLA-DR). RESULTS: Expression of all studied receptors was higher in test compared with control sites (p < 0.005). Sampled sites with less bleeding on probing exhibited higher expression of CD16 and CD14 receptors (p = 0.020 and 0.011, respectively). CONCLUSIONS: This study points towards considerable differences in the expression of leukocyte receptors between diseased and control sites in the same periodontal patients.

Intracellular signalling during neutrophil recruitment.

Recruitment of leucocytes such as neutrophils to the extravascular space is a critical step of the inflammation process and plays a major role in the development of various diseases including several cardiovascular diseases. Neutrophils themselves play a very active role in that process by sensing their environment and responding to the extracellular cues by adhesion and de-adhesion, cellular shape changes, chemotactic migration, and other effector functions of cell activation. Those responses are co-ordinated by a number of cell surface receptors and their complex intracellular signal transduction pathways. Here, we review neutrophil signal transduction processes critical for recruitment to the site of inflammation. The two key requirements for neutrophil recruitment are the establishment of appropriate chemoattractant gradients and the intrinsic ability of the cells to migrate along those gradients. We will first discuss signalling steps required for sensing extracellular chemoattractants such as chemokines and lipid mediators and the processes (e.g. PI3-kinase pathways) leading to the translation of extracellular chemoattractant gradients to polarized cellular responses. We will then discuss signal transduction by leucocyte adhesion receptors (e.g. tyrosine kinase pathways) which are critical for adhesion to, and migration through the vessel wall. Finally, additional neutrophil signalling pathways with an indirect effect on the neutrophil recruitment process, e.g. through modulation of the inflammatory environment, will be discussed. Mechanistic understanding of these pathways provide better understanding of the inflammation process and may point to novel therapeutic strategies for controlling excessive inflammation during infection or tissue damage.

Interstitial leukocyte migration and immune function.

The trafficking of leukocytes into and within lymphoid and peripheral tissues is central to immune cell development, immunosurveillance and effector function. Interstitial leukocyte trafficking is the result of amoeboid polarization and migration, guided by soluble or tissue-bound chemoattractant signals for positioning and local arrest. In contrast to other migration modes, amoeboid movement is particularly suited for scanning cellular networks and tissues. Here, we review mechanisms of leukocyte migration and sensing involved in diapedesis, tissue-based interstitial migration and egress, immune cell positioning in inflammation, and emerging therapeutic interference strategies.

Bighorn sheep beta2-integrin LFA-1 serves as a receptor for Mannheimia haemolytica leukotoxin.

Mannheimia haemolytica is an important cause of pneumonia in bighorn sheep (BHS; Ovis canadensis). Leukotoxin (Lkt), the primary virulence determinant of M. haemolytica, induces cytolysis of all subsets of leukocytes. Previously, we have shown that CD18, the beta subunit of beta2-integrins, mediates Lkt-induced cytolysis. However, it is not clear whether CD18 of all three beta2-integrins, LFA-1, Mac-1, and CR4, mediates Lkt-induced cytolysis. The objective of this study was to determine whether BHS LFA-1 (CD11a/CD18) serves as a receptor for Lkt. Plasmids encoding cDNA for BHS CD11a and CD18 were cotransfected into Lkt-resistant HEK-293 cells. Flow cytometric analysis of transfectants confirmed cell surface expression of BHS LFA- 1, Lkt-LFA-1 binding and Lkt-induced intra-cellular calcium elevation. More importantly, the transfectants were efficiently lysed by Lkt in a concentration-dependent manner. Collectively, these results indicate that BHS LFA-1 serves as a functional receptor for M. haemolytica Lkt.

Catch bonds in adhesion.

One of the most exciting discoveries in biological adhesion is the recent and counter-intuitive observation that the lifetimes of some biological adhesive bonds, called catch bonds, are enhanced by tensile mechanical force. At least two types of adhesive proteins have been shown to form catch bonds--blood proteins called selectins and a bacterial protein called FimH. Both mediate shear-enhanced adhesion, in which cells bind more strongly at high shear than at low shear. Single-molecule experiments and cell-free assays have now clearly demonstrated that catch bonds exist and mediate shear-enhanced adhesion. However, the mechanics of cellular organelles also contribute to shear-enhanced adhesion by modulating the force applied to catch bonds. This review examines how individual catch bond behavior contributes to shear-enhanced cellular adhesion for the two best-understood examples. The lessons from these systems offer design principles for understanding other types of shear-enhanced adhesion and for engineering nanostructured force-dependent adhesives out of catch bonds.

Putative alternative trans-splicing of leukocyte adhesion-GPCR pre-mRNAs generates functional chimeric receptors.

The EGF-TM7 receptors, a subfamily of adhesion-GPCRs mostly restricted to leukocytes, are known to express multiple functional protein isoforms through extensive alternative cis-splicing. Here, we demonstrate that EGF-TM7 pre-mRNAs also undergo the rare trans-splicing, leading to the generation of functional chimeric receptors. RT-PCR and in silico analyses of EMR2 transcripts identified unique fragments containing the EGF-like motif 3 of a closely related EGF-TM7 gene, CD97, in addition to the alternative cis-spliced products. The sequence swapping is restricted to the EGF-3 exon, generating unique EMR2(1-2-3*-5) and EMR2(1-2-3*-4-5) molecules, which are functional in ligand-binding as the wild-type EMR2(1-2-3-4-5) and CD97(1-2-3-4-5) receptors. Our results suggest that human leukocytes employ trans-splicing as well as cis-splicing to increase the repertoire of functional adhesion-GPCRs.

Surface engineering of quantum dots for in vivo vascular imaging.

Quantum dot-antibody bioconjugates (QD-mAb) were synthesized incorporating PEG cross-linkers and Fc-shielding mAb fragments to increase in vivo circulation times and targeting efficiency. Microscopy of endothelial cell cultures incubated with QD-mAb directed against cell adhesion molecules (CAMs), when shielded to reduce Fc-mediated interactions, were more specific for their molecular targets. In vitro flow cytometry indicated that surface engineered QD-mAb labeled leukocyte subsets with minimal Fc-mediated binding. Nontargeted QD-mAb nanoparticles with Fc-blockade featured 64% (endothelial cells) and 53% (leukocytes) lower nonspecific binding than non-Fc-blocked nanoparticles. Spectrally distinct QD-mAb targeted to the cell adhesion molecules (CAMs) PECAM-1, ICAM-1, and VCAM-1 on the retinal endothelium in a rat model of diabetes were imaged in vivo using fluorescence angiography. Endogenously labeled circulating and adherent leukocyte subsets were imaged in rat models of diabetes and uveitis using QD-mAb targeted to RP-1 and CD45. Diabetic rats exhibited increased fluorescence in the retinal vasculature from QD bioconjugates to ICAM-1 and VCAM-1 but not PECAM-1. Both animal models exhibited leukocyte rolling and leukostasis in capillaries. Examination of retinal whole mounts prepared after in vivo imaging confirmed the fluorescence patterns seen in vivo. Comparison of the timecourse of retinal fluorescence from Fc-shielded and non-Fc-shielded bioconjugates indicated nonspecific uptake and increased clearance of the non-Fc-shielded QD-mAb. This combination of QD surface design elements offers a promising new in vivo approach to specifically label vascular cells and biomolecules of interest.

Dynamics of in silico leukocyte rolling, activation, and adhesion.

BACKGROUND: We present a multilevel, agent based, in silico model that represents the dynamics of rolling, activation, and adhesion of individual leukocytes in vitro. Object-oriented software components were designed, verified, plugged together, and then operated in ways that represent the molecular and cellular mechanisms believed responsible for leukocyte rolling and adhesion. The result is an in silico analogue of an experimental in vitro system. The experimentally measured, phenotypic attributes of the analogue were compared and contrasted to those of leukocytes in vitro from three different experimental conditions. RESULTS: The individual in silico dynamics of "rolling" on simulated P-selectin, and separately on simulated VCAM-1, were an acceptable match to individual in vitro distance-time and velocity-time measurements. The analogues are also able to represent the transition from rolling to adhesion on P-selectin and VCAM-1 in the presence of GRO-alpha chemokine. The individual in silico and in vitro behavioral similarities translated successfully to population level measures. These behavioral similarities were enabled in part by subdividing the functionality of the analogue's surface into 600 independent, "cell"-controlled, equally capable modules of comparable functionality. CONCLUSION: The overlap in phenotypic attributes of our analogue with those of leukocytes in vitro confirm the considerable potential of our model for studying the key events that determine the behavioral outcome of individual leukocytes during rolling, activation, and adhesion. Our results provide an important foundation and framework for future in silico research into plausible causal links between well-documented, subcellular molecular level events and the variety of systemic phenotypic attributes that distinguish normal leukocyte adhesion from abnormal disease-associated adhesion.

Mannheimia (Pasteurella) haemolytica leukotoxin utilizes CD18 as its receptor on bighorn sheep leukocytes.

Pneumonia caused by Mannheimia (Pasteurella) haemolytica is a highly fatal disease of bighorn sheep (Ovis canadensis). Leukotoxin (Lkt), secreted by M. haemolytica, is an important virulence factor of this organism, and is cytolytic to bighorn sheep leukocytes. Previously, we have shown that CD18, the beta subunit of beta2 integrins, serves as the receptor for Lkt on bovine leukocytes. Furthermore, anti-CD18 antibodies inhibit Lkt-induced cytotoxicity of bighorn sheep leukocytes. Therefore, we hypothesized that Lkt utilizes CD18 as its receptor on bighorn sheep leukocytes. Confirmation of bighorn sheep CD18 as a receptor for Lkt requires the demonstration that the recombinant expression of bighorn sheep CD18 in Lkt-nonsusceptible cells renders them susceptible to Lkt. Therefore, we transfected cDNA encoding CD18 of bighorn sheep into a Lkt-nonsusceptible murine cell line. Cell surface expression of bighorn sheep CD18 on the transfectants was tested by flow cytometry with anti-CD18 antibodies. Transfectants stably expressing bighorn sheep CD18 on their surface were subjected to flow cytometric analysis for detection of Lkt binding, and cytotoxicity assays for detection of Lkt-induced cytotoxicity. Leukotoxin bound to the transfectants. More importantly, the transfectants were effectively lysed by Lkt in a concentration-dependent manner, whereas the parent cells were not. These results clearly indicate that M. haemolytica Lkt utilizes CD18 as a receptor on bighorn sheep leukocytes. Identification of CD18 as a receptor for Lkt on bighorn sheep leukocytes should enhance our understanding of the pathogenesis of pneumonia, which in turn should help in the development of control measures against this fatal disease of bighorn sheep.

Bioactive hydrogel substrates: probing leukocyte receptor-ligand interactions in parallel plate flow chamber studies.

The binding of activated integrins on the surface of leukocytes facilitates the adhesion of leukocytes to vascular endothelium during inflammation. Interactions between selectins and their ligands mediate rolling, and are believed to play an important role in leukocyte adhesion, though the minimal recognition motif required for physiologic interactions is not known. We have developed a novel system using poly(ethylene glycol) (PEG) hydrogels modified with either integrin-binding peptide sequences or the selectin ligand sialyl Lewis X (SLe(X)) within a parallel plate flow chamber to examine the dynamics of leukocyte adhesion to specific ligands. The adhesive peptide sequences arginine-glycine-aspartic acid-serine (RGDS) and leucine-aspartic acid-valine (LDV) as well as sialyl Lewis X were bound to the surface of photopolymerized PEG diacrylate hydrogels. Leukocytes perfused over these gels in a parallel plate flow chamber at physiological shear rates demonstrate both rolling and firm adhesion, depending on the identity and concentration of ligand bound to the hydrogel substrate. This new system provides a unique polymer-based model for the study of interactions between leukocytes and endothelium as well as a platform to develop improved scaffolds for cardiovascular tissue engineering.

The pancreatic islet endothelial cell: emerging roles in islet function and disease.

The pancreatic islets are one of the most vascularized organs of the body. This likely reflects the requirements of the organ for a rich supply of nutrients and oxygen to the tissue, as well as the need for rapid disposal of metabolites and secreted hormones. The islet endothelium is richly fenestrated to facilitate trans-endothelial transport of secreted hormones, has a unique expression of surface markers, and produces a number of vasoactive substances and growth factors. The islet endothelial cells play a critical role in the early phase of type 1 diabetes mellitus by increasing the expression of surface leucocyte-homing receptors, thereby enabling immune cells to enter the endocrine tissue and cause beta-cell destruction. Following transplantation, pancreatic islets lack a functional capillary system and need to be properly revascularized. Insufficient revascularization may severely affect the transport properties of the islet endothelial system, resulting in a dysfunctional islet graft.

Antioxidant effects of N-acetylserotonin: possible mechanisms and clinical implications.

This paper will review our recent data relevant to the antioxidant effects of N-acetylserotonin (NAS), the immediate precursor of melatonin, the pineal gland indole. Mechanisms of the antioxidant effects of NAS might involve interaction with melatonin type 3 receptors and nonreceptor mechanisms such as stimulation of glutathione peroxidase, an antioxidant enzyme; inhibition of lipid peroxidation; suppression of phospholipase A2 activation; attenuation of tumor necrosis factor-alpha production; prevention of pathological opening of the mitochondrial permeability transition pores; and inhibition of sepiapterin reductase, the key enzyme of biosynthesis of tetrahydrobiopterin, the essential cofactor of nitric oxide synthase. NAS actions on some of these enzymes might be receptor-mediated. Protective effects of NAS against oxidative damage are independent from the effect of melatonin and, depending on the model, are 5 to 20 times stronger than that of melatonin. Antioxidant effect of NAS might underpin its cognition-enhancing, antiaging, antidepressant, antihypertensive, and antitumor effects. NAS and its derivatives might be useful in protection against oxidative stress-related disorders (cell death, mutagenesis, aging) and diseases (sepsis, cancer, postischemic trauma, Alzheimer's disease, parkinsonism).

Integrin CD11a cytoplasmic tail interacts with the CD45 membrane-proximal protein tyrosine phosphatase domain 1.

Leucocyte adhesion receptor integrin CD11aCD18 and the transmembrane receptor-like protein tyrosine phosphatase (RPTP) CD45 mediate immune synapse formation and signalling during antigen presentation. Previous cocapping studies on human naïve T cells demonstrate an interaction between CD11aCD18 and CD45. CD45 cross-linking also has an effect on the ligand-binding activity of CD11aCD18. However, the mode of interaction between CD11aCD18 and CD45 remains unclear. Herein, yeast two-hybrid analysis identified a partial CD45 cytoplasmic tail interacting with that of CD11a. The CD45 cytoplasmic tail comprises a membrane proximal (Mp) region, protein tyrosine phosphatase domain 1 (D1), spacer, D2, and carboxyl terminus. CD45 Mp-D1 was found to be the main interacting region for the CD11a cytoplasmic tail. In contrast, the full-length CD45 cytoplasmic tail interacted weakly with that of CD11a. It has been reported that CD45 Mp-D1 but not the full-length cytoplasmic tail forms a homodimer whose enzymatic activity is inhibited. Our in vitro binding and enzymatic assays showed that the homodimeric CD45 cytoplasmic tail interacts with that of CD11a. The biological function of CD45 dimerization and its association with CD11a remains to be investigated.

A novel inhibitor of N-ethylmaleimide-sensitive factor decreases leukocyte trafficking and peritonitis.

Endothelial exocytosis is an early stage in the process of leukocyte trafficking. N-ethylmaleimide-sensitive factor (NSF) plays a critical role in regulating exocytosis. We hypothesized that inhibitors of NSF decrease endothelial exocytosis and vascular inflammation. We designed a novel fusion polypeptide consisting of a human immunodeficiency virus transactivator of transcription (TAT) protein transduction domain joined to a NSF homohexamerization domain. We show that this TAT-NSF polypeptide inhibits the ATPase activity and the disassembly activity of NSF. Furthermore, the TAT-NSF polypeptide decreases endothelial cell exocytosis and reduces leukocyte adherence to endothelial cells in culture. Finally, the TAT-NSF polypeptide inhibits leukocyte rolling on murine venules in vivo and inhibits leukocyte trafficking into the peritoneal cavity in a murine model of experimental peritonitis. These data suggest that NSF is a critical regulator of leukocyte trafficking in vivo. Novel compounds that inhibit the exocytic machinery in endothelial cells may be useful anti-inflammatory drugs.

Polyunsaturated fatty acids interfere with formation of the immunological synapse.

Polyunsaturated fatty acids (PUFAs) exert inhibitory effects on T cell-mediated immune responses. Activation of T cells in vivo depends on formation of an immunological synapse (IS) at the T cell/antigen-presenting cell (APC) interface. Here, we analyzed effects of PUFA treatment on the formation of the IS and APC-induced human T cell activation. In T cells treated with the PUFA eicosapentaenoic (EPA; 20:5,n-3) and arachidonic acid (20:4,n-6), stimulated by superantigen-presenting cells or APCs, relocalization to the IS of distinct molecules [F-actin, talin, leukocyte functional antigen-1alpha, clusters of differentiation (CD)3epsilon] was inhibited markedly compared with cells treated with saturated fatty acid, whereas relocalization of protein kinase Ctheta to the IS remained unaffected. CD3-induced, sustained phosphorylation of nucleotide exchange factor Vav, which controls cytoskeletal rearrangements underlying IS formation, was significantly reduced in EPA-treated Jurkat and peripheral blood T cells. In addition, T cell raft disruption by methyl-beta-cyclodextrin treatment and experiments with a chimeric linker for activation of T cell proteins, which is resistant to PUFA effects on lipid rafts, revealed modifications of lipid rafts as a crucial factor for PUFA-mediated inhibition of APC-stimulated cytoskeletal rearrangements. Furthermore, the efficiency of T cell/APC conjugate formation was significantly reduced with EPA-treated T cells, as was stimulation of CD69 expression, which is not altered following antibody-mediated T cell activation. In conclusion, PUFA treatment of T cells qualitatively and quantitatively alters IS formation, thereby extending T cell signaling defects to pathways that are not intrinsically altered in PUFA-treated T cells when stimulated by antibodies.

Promotion of leukocyte adhesion by a novel interaction between vitronectin and the beta2 integrin Mac-1 (alphaMbeta2, CD11b/CD18).

OBJECTIVE: The leukocyte integrin Mac-1 (alphaMbeta2, CD11b/CD18) binds a number of ligands and counter-receptors and thereby is a major determinant in regulation of leukocyte adhesion and extravasation. Vitronectin (VN) is an adhesion-promoting factor that is abundantly present as matrix molecule in vascular diseases such as atherosclerosis. Until now, only an indirect interaction between Mac-1 and VN via the urokinase receptor (urokinase plasminogen activator receptor) was known. We now propose that Mac-1 and VN can directly interact with each other. METHODS AND RESULTS: In an in vitro system with purified components, Mac-1 specifically bound the multimeric matrix form of VN but not the monomeric plasma form. Using various competitors, the interaction domains in Mac-1 and VN were localized. Mac-1-expressing but not untransfected Chinese hamster ovary cells adhered strongly on VN. Introduction of a GFFKR deletion in the alphaM subunit of Mac-1, which increases the constitutive activation of the integrin, led to increased adhesion on VN. Peripheral human blood neutrophils adhered and migrated on multimeric VN in a Mac-1-dependent manner. CONCLUSIONS: These results show that there is a specific integrin-affinity-regulated interaction between Mac-1 and the matrix form but not the plasma form of VN that may significantly participate in leukocyte adhesion and extravasation.

Mild hyperthermia down-regulates receptor-dependent neutrophil function.

Mild hypothermia impairs resistance to infection and, reportedly, impairs phagocytosis and oxidative killing of unopsonized bacteria. We evaluated various functions at 33 degrees-41 degrees C in neutrophils taken from volunteers. Adhesion on endothelial cells was determined using light microscopy. Adhesion molecule expression and receptors, phagocytosis, and release of reactive oxidants were assessed using flow cytometric assays. Adhesion protein CD11b expression on resting neutrophils was temperature-independent. However, up-regulation of CD11b with tumor necrosis factor (TNF)-alpha was increased by hypothermia and decreased with hyperthermia. Neutrophil adhesion to either resting or activated endothelial cells was not temperature-dependent. Bacterial uptake was inversely related to temperature, more so with Escherichia coli than Staphylococcus aureus. Temperature dependence of phagocytosis occurred only wi thopsonized bacteria. Hypothermia slightly increased N-formyl-L-methionyl-L-leucyl-phenylalanine receptors on neutrophils: hyperthermia decreased expression, especially with TNF-alpha. N-formyl-L-methionyl-L-leucyl-phenylalanine-induced H2O2 production was inversely related to temperature, especially in the presence of TNF-alpha. Conversely, phorbol-13-myristate-12-acetate, an activator of protein kinase C, induced an extreme and homogenous release of reactive oxidants that increased with temperature. In contrast to nonreceptor-dependent phagocytosis and oxidative killing, several crucial receptor-dependent neutrophil activities show temperature-dependent regulation, with hypothermia increasing function. The temperature dependence of neutrophil function is thus more complicated than previously appreciated.