Activation Of Adhesion Receptors Research Articles

Assessment of Novel Erythroid Signaling Pathways Modulating Hydroxyurea-Mediated Reduction of Vaso-Occlusive Adhesive Interactions in Early Precursors of Sickle Red Blood Cells (RBCs)

Sickle cell disease (SCD) affects ~100,000 Americans and millions worldwide, with healthcare costs in the U.S. exceeding $1 billion annually to treat frequent and unpredictable vaso-occlusive episodes (VOEs). In addition to fetal hemoglobin induction, hydroxyurea (HU) reduces the frequency of VOEs by increasing nitric oxide (NO) bioavailability and decreasing adhesion receptor expression and RBC-endothelial interactions. Very late antigen-4 (VLA-4), the best characterized adhesion receptor in SCD, is highly expressed on circulating reticulocytes (immature RBCs) and white blood cells (WBCs) during VOEs and in SCD patients with severe disease phenotypes and, decreased in response to HU therapy. Like other integrins, VLA-4 is functionally regulated by cell signaling pathways to modulate activity and binding affinity to a wide variety of ligands that are elevated in the SCD micro-environment. There is highly intriguing evidence showing HU modulates adhesion by upregulating a NO/cGMP-dependent pathway to decrease adhesion receptor activity however specific mechanisms remain unclear. The objective of this study was to assess rapid cell signaling pathways involved in HU-mediated reduction in VLA-4 binding to endothelial vascular cell adhesion molecule-1 (VCAM-1). Whole blood (WB) was obtained from patients with SCD at benign clinic visits according to IRB-approved protocols. WB density-gradient centrifugation was used to isolate hematopoietic stem cells (HSCs) and WBCs from the buffy coat layer and, a CD71 monoclonal antibody (Ab) coupled with magnetic beads to isolate reticulocytes from the packed RBC layer. HSCs were matured to erythroblasts (EBs) in appropriate cell culture media and flow cytometric analyses assessed purity of blood samples using CD235a (exclusive WBC marker) and CD45 (exclusive RBC marker) Abs . Blood samples were pretreated with and without a therapeutically relevant hydroxyurea concentration (50mcM) followed by perfusion through micro-fluidic channels coated with VCAM-1 during pulsatile, shear flow (1.67Hz, 1.0 dyne/cm 2). Additionally, EBs were treated with HU combined and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1- one (ODQ; NO-dependent guanylate cyclase inhibitor). Adhered cells were quantified to establish an adhesion index. The non-parametric Wilcoxon matched pairs signed rank test was used to test the statistical differences between groups. Data are presented as mean ± standard error of mean. A p-value < 0.05 was considered statistically significant. There was a significant decrease in WB (p=0.01) and reticulocyte (p<0.0001) adhesion to VCAM-1 in HU-treated samples however, there was no effect on WBC (p>0.99) adhesion suggesting reticulocytes reduce the adhesive burden of blood cells in WB adhesion assays and modulation of VLA-4 binding differs in sickle RBCs when compared to WBCs. More recently, preliminary data from my lab demonstrated that EBs pretreated with HU reduces VLA-4 binding to VCAM-1 (from 126.5 ± 29.42 to 109.5 ± 29.09) by cGMP-dependent mechanisms (HU + ODQ; from 109.5 ± 29.09 to 120.2 ± 42.80). Laboratory-based static and flow adhesion assays have been used for decades to describe adhesion in SCD since earlier studies established that sickle RBCs are more adherent than non-sickle blood cells and, adhesion correlates with SCD severity. For example, the adhesion assay described above has been used to demonstrate that VLA-4 binding can stratify SCD patients based on disease severity and predict impending VOEs. Additionally, reticulocyte and HbF levels strongly correlate with VLA-4 binding to VCAM-1 and, VLA-4-mediated adhesion is increased during patient-reported VOEs and decreased in HU-treated SCD patients. This is the first SCD study demonstrating the role of HU in modulating VLA-4 binding to endothelial VCAM-1 through rapid erythroid signaling pathways. There is also compelling evidence involving downregulation of VLA-4 activity/binding affinity through NO/cGMP dependent mechanisms and enhancement of VLA-4 binding by kinase-dependent pathways. HU has also been shown to modulate adhesion by upregulating a NO/cGMP-dependent pathway to decrease adhesion receptor activity. Collectively, these data suggest that VLA-4 mechanisms may be targeted to therapeutically inhibit vaso-occlusive adhesive events that promote VOEs in SCD.

IPMK Loss Inhibits Cellular Motility and Contractility

Cellular adhesion is dependent on the type of adhesion receptors displayed on the cell surface, and activation of adhesion receptors initiates downstream signaling cascades that are context dependent. Cellular expression of adhesion receptors is modulated by a complex array of extracellular and intracellular signals, and we have found that Inositol Phosphate Multikinase (IPMK) is a transcriptional regulator of integrin β1 protein in murine embryonic fibroblast (MEF) cells. Further, we show that although loss of IPMK dramatically reduces levels of both active and inactive integrin β1 protein, activation of the focal adhesion proteins FAK and Src are increased while total protein levels are unchanged, suggesting that IPMK is a negative regulator of focal adhesion complex activity. Importantly, IPMK regulation of integrin gene expression is activity dependent. Using native extracellular matrix mimicking fibers as substrate, generated using STEP (Spinneret-based Tunable Engineered Parameters) technique, we show that loss of this enzyme inhibits migration (0.68 μm/min in Wild Type cells vs 0.35 μm/min in IPMK Knock-out cells) due to decreased cell-matrix adhesion and reduced cellular contractility. Using nanonet force microscopy (NFM), we calculated the forces of migrating MEF WT cells to be 226.8 nN which was significantly higher than those of the IPMK KO cells (115.6 nN). Attempts to rescue IPMK pharmacologically resulted in increased contractility (154.3 nN) but not to the same degree as that of WT cells. To further elucidate the impact of IPMK on adhesion strength, we stretched the cells suspended between fibers until their adhesion bonds failed and the cells detached from the adhered fibers. WT cells demonstrated higher failure force (45.4 nN) as opposed to KO cells (36.2 nN). Based on these findings, we hypothesize that IPMK modulates cell migration and contractility by regulating gene expression of cell-matrix molecules.

Cell-Cell Contact and Receptor Tyrosine Kinase Signaling.

The behavior of cells within tissues is governed by the activities of adhesion receptors that provide spatial cues and transmit forces through intercellular junctions, and by growth-factor receptors, particularly receptor tyrosine kinases (RTKs), that respond to biochemical signals from the environment. Coordination of these two activities is essential for the patterning and polarized migration of cells during morphogenesis and for homeostasis in mature tissues; loss of this coordination is a hallmark of developing cancer and driver of metastatic progression. Although much is known about the individual functions of adhesion and growth factor receptors, we have a surprisingly superficial understanding of the mechanisms by which their activities are coordinated.

Myristoylated Alanine Rich C Kinase Substrate (MARCKS) is essential to β2-integrin dependent responses of equine neutrophils

Neutrophil infiltration is a prominent feature in a number of pathologic conditions affecting horses including recurrent airway obstruction, ischemia-reperfusion injury, and laminitis. Cell signaling components involved in neutrophil migration represent targets for novel anti-inflammatory therapies. In order to migrate into tissue, neutrophils must respond to chemoattractant signals in their external environment through activation of adhesion receptors (i.e. integrins) and reorganization of the actin cytoskeleton. Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS), a highly conserved actin-binding protein, has a well demonstrated role in cytoskeletal dependent cellular functions (i.e. adhesion, spreading, and migration), but the details of MARCKS involvement in these processes remain vague. We hypothesized that MARCKS serves as a link between the actin cytoskeleton and integrin function in neutrophils. Using a MARCKS-specific inhibitor peptide known as MANS on equine neutrophils in vitro, we demonstrate that inhibition of MARCKS function significantly attenuates β2-integrin-dependent neutrophil functions including migration, adhesion, and immune complex-mediated respiratory burst. The MANS peptide did not, however, inhibit the β2-integrin-independent PMA mediated respiratory burst. These results attest to the essential role of MARCKS function in regulating neutrophil responses, and strongly implicate MARCKS as a potential regulator of β2-integrins in neutrophils.

Dynamic analysis of platelet deposition to resolve platelet adhesion receptor activity in whole blood at arterial shear rate

Platelet activation is traditionally quantified using turbidimetric aggregometry, which reflects integrin αIIbβ3 activity, an important determinant of platelet function during pathophysiological thrombus formation. However, aggregometry does not recreate the shear conditions prevailing during thrombosis in vivo. Here we describe novel whole-frame analysis of real-time video microscopy to quantify platelet adhesion receptor activity under shear in parallel-plate flow chambers. We demonstrate that the rate of change of surface coverage (designated Platelet Population Mobility, PM) quantifies platelet mobility. On collagen, PM falls exponentially to a low level, corresponding to firm platelet adhesion, while on other substrates, PM remains high. Different receptor-specific thrombogenic surfaces reveal that the PM time constant reflects real-time changes in integrins αIIbβ3 and α2β1 activity. This ensemble kinetic analysis has the potential to provide valuable diagnostic information about platelet thrombus formation with both academic and clinical applications.

Human eosinophil adhesion and receptor expression.

Eosinophils migrate from the bone marrow in response to cytokines and chemokines which induce the expression and activation of adhesion receptors. In understanding the recruitment of eosinophils, protocols to identify eosinophil adhesion and receptor expression have been identified. In this summary, the eosinophil peroxidase and fluorescent labeling assays are described as measurements of indirect and direct eosinophil adhesion, respectively. Additional protocols that identify eosinophil receptor expression via immunofluorescent microscopy and flow cytometry are also described.

Fluorescence-Based Tension Probes to Image Mechanics at the Lipid Membrane

Forces have a profound role across all living systems, and many essential biological processes ranging from development and migration to mitosis and meiosis cannot proceed without precisely tuned mechanical signals. Despite the importance of mechanotransduction, the molecular details relating the magnitude, timing, and location of forces to specific biochemical pathways remain poorly understood. The challenge pertains to developing molecular probes that allow one to simultaneously measure biochemical activation and mechanical tension in living systems. Herein, I will describe the synthesis and characterization of fluorescence-based turn-on probes in molecular tension fluorescence microscopy (MTFM) for imaging forces at the lipid membrane of living cells. MTFM probes take advantage of fluorescence quenching as a “ruler”, and an extendable linker as a reversible “spring” with a known constant. Using this technique, a standard fluorescence microscope can be used to quantify molecular tension at the cell membrane of living cells. In this talk, I will describe the development of second and third generation MTFM probes that allow allow one to quantify molecular forces with high spatial and temporal resolution for a wide range of receptors and cell types. I will describe the application of these sensors to image forces associated with a range of mechano-regulatory processes that occur at the lipid membrane of the cell, such as endocytosis, Notch receptor activation, and integrin adhesion receptor activation.

Integrins and extracellular matrix in mechanotransduction

Integrins and extracellular matrix in mechanotransduction Lindsay RamageQueen’s Medical Research Institute, University of Edinburgh, Edinburgh, UKAbstract: Integrins are a family of cell surface receptors which mediate cell–matrix and cell–cell adhesions. Among other functions they provide an important mechanical link between the cells external and intracellular environments while the adhesions that they form also have critical roles in cellular signal-transduction. Cell–matrix contacts occur at zones in the cell surface where adhesion receptors cluster and when activated the receptors bind to ligands in the extracellular matrix. The extracellular matrix surrounds the cells of tissues and forms the structural support of tissue which is particularly important in connective tissues. Cells attach to the extracellular matrix through specific cell-surface receptors and molecules including integrins and transmembrane proteoglycans. Integrins work alongside other proteins such as cadherins, immunoglobulin superfamily cell adhesion molecules, selectins, and syndecans to mediate cell–cell and cell–matrix interactions and communication. Activation of adhesion receptors triggers the formation of matrix contacts in which bound matrix components, adhesion receptors, and associated intracellular cytoskeletal and signaling molecules form large functional, localized multiprotein complexes. Cell–matrix contacts are important in a variety of different cell and tissue properties including embryonic development, inflammatory responses, wound healing, and adult tissue homeostasis. This review summarizes the roles and functions of integrins and extracellular matrix proteins in mechanotransduction.Keywords: ligand binding, α subunit, ß subunit, focal adhesion, cell differentiation, mechanical loading, cell–matrix interaction

Reduction in human melanoma cell adhesion receptor activity by lysophosphatidylcholine (LPC) treatment – functional characterization and signal pathway analyses

Reduction in human melanoma cell adhesion receptor activity by lysophosphatidylcholine (LPC) treatment – functional characterization and signal pathway analyses

Effect of alpha3beta1 and alphavbeta3 integrin glycosylation on interaction of melanoma cells with vitronectin.

The metastatic transformation of melanocytes is associated with altered expression of adhesion molecules, including alpha(v)beta(3) and alpha(3)beta(1) integrins. Integrin alpha(v)beta(3) is a primary vitronectin (VN) receptor, while both integrin types take part in adhesion to VN when they are in complex with uPAR. Although their role in melanoma cell interaction with VN is of great interest, the influence of N-oligosaccharides attached to these glycoproteins is still unappreciated. The present study assesses the role of alpha(v)beta(3) and alpha(3)beta(1) integrins and the influence of their glycosylation status on WM9 and WM239 metastatic melanoma cell interactions with VN. Cell adhesion to and migration on VN were selected as the studied cell behaviour parameters. Function-blocking antibodies and swainsonine (SW) treatment were used in these tests. Both cell lines interacted with VN in an integrin-mediated but cell-line-specific manner. In WM9 cells, migration was not completely inhibited by antibodies against alpha(3)beta(1) or alpha(v)beta(3) integrins, suggesting the participation of other VN receptors. In both cell lines in coprecipitation test the formation of an integrins/uPAR complex was shown. In the presence of SW formation of the complex did not occur, suggesting the participation of glycosylation in this process. Additionally, the adhesion properties of WM9 cells were changed after SW treatment. Our results suggest that in these two metastatic cell lines integrin-linked N-oligosaccharides influence the VN adhesion receptor activity and function.

The effects of HSP72 expression on adhesion receptor activation and phagolysosome function in human PMNs (134.93)

Abstract Heat shock protein 72 (HSP72) expression is a hallmark of functionally suppressed neutrophils from pediatric burn patients. CD11b and CD15 are critical receptors that mediate neutrophil (PMN) adhesion. In this study, we examined the effects of HSP72 on these receptors as well as phagolysosome function. Expression of CD11b, CD15 and pHrodo were measured by flow cytometry. HSP72 expression in PMNs, induced by 60-90 min incubation at 42C, suppressed the upregulation of high affinity CD11b conformation induced by fMLP (~90% reduction vs controls). Additionally, CD15 expression is reduced in HSP72 positive PMNs. GM-CSF treatment (5-25 ng/ml) appears to have a modest ability to preserve PMN function in stressed PMNs. HSP72 positive PMNs also have a reduced capacity phagocytose. Opsonized pHrodo labelled Staphylococcus aureus were incubated with HSP72 positive or negative PMNs. Control samples had significantly more pHrodo positive bacteria (~2-fold increase, controls vs stressed PMNs, P<0.01) than the thermally stressed cells. However, the HSP72 positive PMNs appeared to acidify phagolysosomes to the same extent as control PMNs. Taken together, these data suggest that HSP72 expression reduces adhesion as well as the extent of phagocytosis. These deficits may contribute to the immune suppression found in thermally injured patients. Supported by Shriners of North America.

Dual Role of Melanoma Cell Adhesion Molecule (MCAM)/CD146 in Lymphocyte Endothelium Interaction: MCAM/CD146 Promotes Rolling via Microvilli Induction in Lymphocyte and Is an Endothelial Adhesion Receptor

The melanoma cell adhesion molecule (MCAM)/CD146 is expressed as two isoforms differing by their cytoplasmic domain (MCAM long (MCAM-l) and MCAM short (MCAM-s)). MCAM being expressed by endothelial cells and activated T cells, we analyzed its involvement in lymphocyte trafficking. The NK cell line NKL1 was transfected by MCAM isoforms and submitted to adhesion on both the endothelial cell monolayer and recombinant molecules under shear stress. MCAM-l transfection reduced rolling velocity and increased NKL1 adhesion on the endothelial cell monolayer and VCAM-1. Scanning electron microscopy revealed that MCAM-l induced microvilli formation and extension. In contrast, MCAM short or mock transfection had no effect on adhesion of NKL1 cells and microvilli formation. As shown by mutagenesis, serine 32 of the MCAM-l cytoplasmic tail, belonging to a putative protein kinase C phosphorylation site, was necessary for MCAM-l-actin cytoskeleton interaction and microvilli induction. Accordingly, chelerythrine chloride, a protein kinase C inhibitor, abolished MCAM-l-induced microvilli and rolling of MCAM-l-transfected NKL1 cells. Inhibition of adhesion under shear stress by anti-MCAM Abs suggested that both lymphoid MCAM-l and endothelial MCAM were also directly involved in lymphocyte endothelium interaction. MCAM-l-transfected NKL1 and activated CD4 T cells adhered to rMCAM under shear stress whereas anti-MCAM Ab treatment inhibited this process. Taken together, these data establish that MCAM is involved in the initial steps of lymphocyte endothelium interaction. By promoting the rolling on the inflammation marker VCAM-1 via microvilli induction and displaying adhesion receptor activity involving possible homophilic MCAM-l-MCAM-l interactions, MCAM might be involved in the recruitment of activated T cells to inflammation sites.

Reactive oxygen species and human platelet GP IIb/IIIa receptor activation

This study concerned reactive oxygen species for their potential to activate human platelet GP IIb/IIIa receptors. All cells produce reactive oxygen species — radicals that can abstract electrons and hydrogen atoms from biological molecules to alter cell function. In many cells, radicals contribute to cellular signaling. In platelets, the predominant oxidant effect is platelet activation. Less is known concerning oxidants and GP IIb/IIIa receptor activation. The first aim of the current study was to confirm that although both H2O2 and tert butyl hydroperoxide both predispose platelets to aggregation; neither directly activates GP IIb/IIIa receptors. The second aim was to demonstrate that even in the presence of extracellular redox iron; H2O2 does not activate GP IIb/IIIa receptors. The third aim was to determine if extracellular superoxide anions evoke GP IIb/IIIa activation. Finally, a role for intra-platelet iron in GP IIb/IIIa activation was examined. Intracellular superoxide anions are produced in excess during platelet activation and curiously, they are uniquely able to increase intracellular free iron. This iron can, in a redox manner, generate radicals and these iron dependent species modulate signaling systems, including systems associated with adhesion receptor activation. In the current studies, platelets in suspension were exposed to H2O2 and to tert butyl hydroperoxide, to H2O2 plus ferrous or ferric chloride (± ascorbate to enhance iron redox cycling) and to xanthine plus xanthine oxidase to generate extra-platelet superoxide anions. Intra-platelet iron was increased with iron ionophore 8-hydroxyquinoline. During flow cytometry, intra-platelet oxidant state was assessed with the redox sensitive fluorescent indicator H2DCF, while GP IIb/IIIa activation was assessed using fluorescent antibody PAC-1. Results showed that although all the oxidizing systems examined increased intra-platelet oxidant state, GP IIb/IIIa receptors were not activated by H2O2, by tert butyl hydroperoxide, by H2O2 plus iron (± ascorbate) or by xanthine plus xanthine oxidase. In contrast, iron plus ionophore 8-hydroxyquinoline evoked GP IIb/IIIa activation. Platelet positivity for PAC-1 increased from 2 ± 0.2 to 28 ± 7% (P < 0.005). However this response, although vigorous, was less than 56 ± 8% (P < 0.001) evoked by thrombin 0.1 milliunit/ml. In conclusion, the results indicated that oxidant systems external to platelets did not activate GP IIb/IIIa receptors while increased intra-platelet iron was associated with appearance of cytosolic oxidizing species and with GP IIb/IIIa receptor activation.

Analysis of Chemokine and Adhesion Markers in Waldenstrom Macroglobulinemia.

Background: Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM), indicating continuous entry and egression of malignant cells in and out of the BM. The process of homing and trafficking involves chemokines, cytokines, and adhesion molecules. In this study, we sought to determine the level of chemokines, cytokines and adhesion molecules in the BM and peripheral blood (PB) of patients with WM.Method: We compared BM supernatant to serum samples from 10 patients with WM, including 5 matched samples from the same patients, and PB serum and BM supernatant from 2 normal healthy controls. The Luminex® xMAP® Multiplex (Upstate, VA) allows detection of human cytokines, chemokines, and growth factors using the Beadlite® products (Upstate, VA). We focused on chemokines that regulate migration: Eotaxin, GRO, IP10, MIP1a, MIP1b, MCP1, MCP3, MDC/CCL22, and RANTES; cytokines that regulate proliferation: Interleukin (IL)-2, -4, -6, -8, -10, and -15; and adhesion molecules: ICAM, VCAM. The means fluorescence intensity (MFI) was measured for all molecules. Chemokine and adhesion receptor expression on primary WM cells and WM cell line BCWM.1 was assessed using flow cytometry. Adhesion was measured using the in vitro adhesion assay (EMD Biosciences, CA), and the anti-VLA-4 antibody (10–100ug/ml, BD Pharmingen, CA).Results: We first compared the BM supernatant of patients with WM to healthy donors. Adhesion molecules ICAM (mean MFI 5624 in WM vs 720 in control) and VCAM (mean MFI 9096 in WM vs 2135 in control) were significantly higher in the WM supernatant, p=0.03 and p=0.05 respectively. In addition, the chemokines IP-10 (Mean MFI 8186 in WM vs 1647 in control), MDC/CCL22 (mean MFI 13015 in WM vs 4221 in control), and GRO (mean MFI 7463 in WM vs 1337 in control), that regulate migration, were significantly upregulated in WM BM supernatant. We found higher expression of MIP1a in the PB of WM patients compared to patient BM supernatant (mean MFI 5715 in PB vs 1484 in BM), p=0.02. Similarly, RANTES and IL-10 were significantly higher in patients' PB sera. There was no statistically significant difference between the PB sera of patients and healthy controls. The expression of CXCR3 (receptor of IP-10) and CCR4 (receptor of MDC/CCL22) were highly expressed on BCWM.1 (mean 90% expression). We then determined the expression of adhesion receptors on BM CD19+ cells from patient samples and BCWM.1. The WM cells and cell line expressed very high levels of surface adhesion receptors VLA4 and LFA1 (mean expression 95%). Anti-VLA-4 antibody 10ug/ml completely abrogated adhesion to fibronectin, 6% compared to control.Conclusion: Adhesion molecules VCAM and ICAM and their receptors VLA-4 and LFA-1 were significantly elevated in the bone marrow of WM patients, indicating activation of adhesion receptors and localizing WM cells in the BM. The anti-VLA-4 antibody completely abrogated adhesion in vitro. In addition, the chemokines IP-10, MDC/CCL22 and GRO were upregulated in the BM of patients, which induce homing of cells into the BM. This study, therefore, systematically defines chemokines, cytokines and adhesion markers in WM, providing the preclinical framework for inhibition of adhesion molecules in clinical protocols to improve patient outcome in WM. * HN and XL are co-first authors.

Phosphoinositide 3-kinases and the regulation of platelet function.

A clear understanding of the role of PI (phosphoinositide) 3-kinases in supporting the haemostatic function of platelets has been slow to evolve. In fact, insight into the roles of individual PI 3-kinase isoforms in platelet function remains rudimentary. However, based on in vitro studies using wortmannin and LY294002, there is evidence for an important role for PI 3-kinases in regulating a broad range of functional platelet responses, including primary platelet adhesion, cytoskeletal remodelling and platelet aggregation. One of the critical platelet responses involves affinity regulation of the major platelet integrin alphaIIbbeta3, the primary receptor mediating platelet aggregation and thrombus growth. The input signals regulating integrin alphaIIbbeta3 can be divided into three main groups: (1) G(q)-coupled receptors linked to the activation of PLCbeta (phospholipase Cbeta); (2) G(i)-coupled receptors linked to the regulation of adenylate cyclase and Rap1b; and (3) adhesion receptor signalling involving Src kinase-dependent activation of PLCgamma isoforms. PI 3-kinases have not been demonstrated to play a critical role in G(q)-dependent platelet activation; however, one or more PI 3-kinase isoforms appears to be important for G(i)-dependent activation of Rap1b and adhesion receptor activation of PLCgamma isoforms. Thus distinct co-operative PI 3-kinase signalling mechanisms appear to play an important role in regulating the adhesive function of integrin alphaIIbbeta3.

Signaling mechanism for equine neutrophil activation by immune complexes

Neutrophils (PMN) are critical host defense cells that have a role in the pathophysiology of a variety of inflammatory diseases, particularly those diseases associated with antigen–antibody immune complexes (IC) deposited in tissues. Activation of PMN by IC is most efficient if the IC are presented immobilized on a surface. Adhesion to the immobilized IC is important for subsequent activation of PMN effector functions, such as generation of reactive oxygen metabolites. Adhesion of human PMN to immobilized IC requires the expression and activation of adhesion receptors called integrins. Of the integrins expressed on PMN, the beta 2 family has been found to be of particular importance for PMN function. The mechanism of beta 2 integrin activation during adhesion to IC has been studied in human PMN, but not in equine PMN. We show here that adhesion of equine PMN to immobilized IC requires beta 2 integrins. Like adhesion, IC-induced respiratory burst activity is dependent on beta 2 integrins. Furthermore, the signaling pathway triggering beta 2 integrin-dependent adhesion of equine PMN to IC and subsequent generation of respiratory burst activity is inhibited by the specific phosphatidylinositol 3-kinase (PI3K) antagonists wortmannin and LY294002 with IC 50 (concentration at which 50% inhibition is achieved) similar to the published values for inhibition of PI3K enzymatic activity. In contrast, PMA-induced activation of beta 2 integrin-dependent adhesion and respiratory burst activity are wortmannin and LY294002 insensitive. These data demonstrate that like in human PMN, IC-induced activation of beta 2 integrins and beta 2 integrin-dependent functions in equine PMN is dependent on PI3K activity.

Cytokines Modulate Integrin αvβ3-Mediated Human Endothelial Cell Adhesion and Calcium Signaling

Angiogenesis is a complex process regulated by the interactions of endothelial cells with cytokines and the adhesive protein matrix. The cytokines basic fibroblast growth factor (bFGF) and tumor necrosis factor-α (TNF-α) are two of the modulators of angiogenesis. One mechanism by which these cytokines induce their effects may be through the regulation of integrin adhesion receptor activity, in particular, αvβ3. In this study, we examined the ability of these angiogenic factors to modulate the adhesion of human umbilical vein endothelial cells (HUVECs) to immobilized disintegrins (i.e., rhodostomin and arietin), which are specific in antagonizing integrin αvβ3 in cells. As these disintegrins were immobilized as substrates, they acted as agonists to induce HUVEC adhesion in a dose- and αvβ3-dependent manner. In addition, adhesion also triggered a sustained increase of intracellular free calcium. Furthermore, bFGF-primed HUVECs potentiated, but TNF-α primed cells attenuated, about 50% adhesion events and calcium signaling triggered by immobilized disintegrin compared to naive cells, respectively. The mechanisms of modulating αvβ3-dependent HUVEC adhesion by cytokines may be related to changes of integrin αvβ3 conformation, as demonstrating the antagonistic effect of Mn2+ on decreased adhesion by TNF-α pretreatment, and confirmed with flow cytometric analysis probed by anti-LIBS1 mAb. However, cytokine pretreatment did not alter the expression of this integrin on the cell surface, as determined by flow cytometry. Phosphoinositide-3 kinase may be one of the signaling molecules involved in the enhanced adhesion of bFGF-primed cells.

Adhesion Molecules — Blood Vessels and Blood Cells

Maintaining vascular integrity and defending against invasive pathogens require the rapid regulation of adhesive interactions among blood cells and between blood cells and the vessel wall. Control of these mechanisms depends on the activation of adhesion receptors that are already present on resting blood cells and endothelium, or the expression of new receptors on the cell surface. Some functional overlap of adhesion receptors and their ligands adds a measure of safety to the repair and defense systems, but defective adhesion can occur and cause tissue injury or disrupt homeostasis of the vascular system.The process that allows the extravasation of . . .

Adhesion receptor activation of phosphatidylinositol 3-kinase. von Willebrand factor stimulates the cytoskeletal association and activation of phosphatidylinositol 3-kinase and pp60c-src in human platelets.

The cytoskeleton participates in the coordinated regulation of intracellular signaling molecules, following agonist stimulation of cells. We have demonstrated that von Willebrand factor (vWF) induced the cytoskeletal association and activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in human platelets. The activation of PtdIns 3-kinase coincided with the tyrosine phosphorylation of multiple platelet proteins, as assessed by anti-phosphotyrosine immunoblotting. One of these tyrosine-phosphorylated proteins, pp60c-src, became specifically enriched in the cytoskeletal fraction of vWF-stimulated platelets. The vWF-stimulated cytoskeletal association of PtdIns 3-kinase and pp60c-src required platelet stirring and aggregation, was specifically blocked by an anti-GPIb monoclonal antibody, and was not observed in platelets lacking the glycoprotein Ib/IX complex (Bernard-Soulier syndrome). Pretreatment of normal platelets with 5 mM EDTA (37 degrees C for 90 min) or RGDS (2 mM), which disrupts the binding of various adhesive proteins to platelet integrins and inhibits fibrinogen-mediated platelet aggregation, did not alter the vWF-stimulated activation and cytoskeletal association of PtdIns 3-kinase and pp60c-src. Pretreatment of platelets with acetylsalicylic acid (1 mM) completely abolished vWF-stimulated production of thromboxane A2, dense granule release, and the activation of protein kinase C, without altering the activation and cytoskeletal translocation of PtdIns 3-kinase and pp60c-src. Our results suggest that vWF binding to the platelet adhesion receptor glycoprotein Ib/IX can mediate activation and translocation of both tyrosine and lipid kinase(s) independent of other agonists.

Chapter 14 Adhesive proteins and their cellular receptors

Many, and perhaps most, important cell adhesion receptors and ligands have been identified and characterized. The reagents, systems, and technologies are therefore available for a “reductionist” approach to the roles of cell adhesion in processes such as the interaction of blood with biomedical materials. Key to the success of this approach is the recognition that the same molecules or families of molecules are responsible for many different processes in organisms that have evolved over hundreds of millions of years. For instance, the α IIb β 3 integrin receptor that mediates platelet adhesion and aggregation and is defective in Glanzmann thrombasthenia in humans is very similar to the receptor that mediates muscle attachment and is defective in fruitflies with the myospheriod phenotype. The challenges to the field of cell adhesion are to understand the events that control the activities of adhesion receptors and ligands, the signals that emanate from ligated receptors, and how the signals are processed to influence behavior when cells encounter surfaces or other cells. To exemplify these issues, this chapter focuses on the α v β 3 integrin receptor, an adhesive ligand for the receptor, vitronectin; and modulation of cellular adhesion to vitronectin by high-molecular-weight kininogen. Platelet adhesion to biomaterials is contrasted with the more complex phenomenon of neutrophil adhesion to vascular endothelial cells.