Leukocyte Capture Research Articles

P-selectin-dependent leukocyte adhesion is governed by endolysosomal two-pore channel 2

Upon proinflammatory challenges, endothelial cell surface presentation of the leukocyte receptor P-selectin, together with the stabilizing co-factor CD63, is needed for leukocyte capture and is mediated via demand-driven exocytosis from the Weibel-Palade bodies that fuse with the plasma membrane. We report that neutrophil recruitment to activated endothelium is significantly reduced in mice deficient for the endolysosomal cation channel TPC2 and in human primary endothelial cells with pharmacological TPC2 block. We observe less CD63 signal in whole-mount stainings of proinflammatory-activated cremaster muscles from TPC2 knockout mice. We find that TPC2 is activated and needed to ensure the transfer of CD63 from endolysosomes via Weibel-Palade bodies to the plasma membrane to retain P-selectin on the cell surface of human primary endothelial cells. Our findings establish TPC2 as a key element to leukocyte interaction with the endothelium and a potential pharmacological target in the control of inflammatory leukocyte recruitment.

Vasculogenic mimicry structures in melanoma support the recruitment of monocytes

ABSTRACT The progression of cancer is facilitated by infiltrating leukocytes which can either actively kill cancer cells or promote their survival. Our current understanding of leukocyte recruitment into tumors is largely limited to the adhesion molecules and chemokines expressed by conventional blood vessels that are lined by endothelial cells (ECs). However, cancer cells themselves can form their own vascular structures (a process known as vasculogenic mimicry (VM)); but whether they actively participate in the recruitment of leukocytes remains to be elucidated. Herein, we demonstrate that VM-competent human melanoma cell lines express multiple adhesion molecules (e.g. CD44, intercellular adhesion molecule (ICAM)-1 and junction adhesion molecules (JAMs)) and chemokines (e.g. CXCL8 and CXCL12) relevant for leukocyte recruitment. Microfluidic-based adhesion assays revealed that similar to ECs, VM-competent melanoma cells facilitate the rolling and adhesion of leukocytes, particularly monocytes, under conditions of shear flow. Moreover, we identified ICAM-1 to be a key participant in this process. Transwell assays showed that, similar to ECs, VM-competent melanoma cells facilitate monocyte transmigration toward a chemotactic gradient. Gene expression profiling of human melanoma patient samples confirmed the expression of numerous leukocyte capture adhesion molecules and chemokines. Finally, immunostaining of patient tissue microarrays revealed that tumors with high VM content also contained higher numbers of leukocytes (including macrophages). Taken together, this study suggests an underappreciated role of VM vessels in solid tumors via their active participation in leukocyte recruitment and begins to identify key adhesion molecules and chemokines that underpin this process.

An inflammatory vascular endothelium-mimicking microfluidic device to enable leukocyte rolling and adhesion for rapid infection diagnosis

Recruitment of circulating leukocytes to sites of infection is of utmost importance in the development, propagation, and outcome of sepsis. These multi-step processes are mediated by interactions between adhesion receptors of leukocytes and cell adhesion molecules (CAMs) of endothelial cells, such as P-selectin, E-selectin and ICAM-1. However, the potential utility of the CAMs-facilitated leukocyte capture has not been thoroughly investigated as an index of the host response to infection for diagnostic purposes. Here, we report that the systemic infection affects the expression of CAMs ligands on leukocytes, upregulating the expression of P-selectin ligand-1 (PSGL-1) and increasing the number of PSGL-1- and E-selectin ligand-1 (ESL-1)-expressing leukocyte levels in septic blood. We leveraged this finding to determine infection by measuring the increased adhesion of leukocytes to an inflammatory vascular endothelium-mimicking microchannel coated with CAMs. We successfully validated that the proposed method can significantly differentiate infection in bacteremia and endotoxemia models in rats as early as an hour post-infection using a finger-prick volume of blood (50 μL), which were unachievable with the conventional diagnostic methods.

Stabilization of Endothelial Receptor Arrays by a Polarized Spectrin Cytoskeleton Facilitates Rolling and Adhesion of Leukocytes

SUMMARYMultivalent complexes of endothelial adhesion receptors (e.g., selectins) engage leukocytes to orchestrate their migration to inflamed tissues. Proper anchorage and sufficient density (clustering) of endothelial receptors are required for efficient leukocyte capture and rolling. We demonstrate that a polarized spectrin network dictates the stability of the endothelial cytoskeleton, which is attached to the apical membrane, at least in part, by the abundant transmembrane protein CD44. Single-particle tracking revealed that CD44 undergoes prolonged periods of immobilization as it tethers to the cytoskeleton. The CD44-spectrin “picket fence” alters the behavior of bystander molecules—notably, selectins—curtailing their mobility, inducing their apical accumulation, and favoring their clustering within caveolae. Accordingly, depletion of either spectrin or CD44 virtually eliminated leukocyte rolling and adhesion to the endothelium. Our results indicate that a unique spectrin-based apical cytoskeleton tethered to transmembrane pickets—notably, CD44—is essential for proper extravasation of leukocytes in response to inflammation.

Stabilization of Endothelial Receptor Arrays by a Polarized Spectrin Cytoskeleton Facilitates Rolling Adhesion of Leukocytes

Multivalent complexes of endothelial adhesion receptors (e.g. selectins) engage leukocytes to orchestrate their migration to inflamed tissues. Proper anchorage and sufficient density (clustering) of endothelial receptors are required for efficient leukocyte capture. We demonstrate that a polarized spectrin network dictates the stability of the endothelial cytoskeleton, which is attached to the apical membrane, at least in part, by the abundant transmembrane protein CD44. Single-particle tracking revealed that CD44 undergoes prolonged periods of immobilization as it tethers to the cytoskeleton. The CD44-spectrin picket fence alters the behaviour of bystander molecules, notably selectins, inducing their apical accumulation, curtailing their mobility and favouring their clustering within caveolae. Accordingly, depletion of either spectrin or CD44 virtually eliminated leukocyte rolling and adhesion to the endothelium. Our results indicate that a unique spectrin-based apical cytoskeleton tethered to transmembrane pickets, notably CD44, is essential for proper extravasation of leukocytes in response to inflammation.

Dependence of leukocyte capture on instantaneous pulsatile flow

Atherosclerosis, an artery disease, is currently the leading cause of death in the United States in both men and women. The first step in the development of atherosclerosis involves leukocyte adhesion to the arterial endothelium. It is broadly accepted that blood flow, more specifically wall shear stress (WSS), plays an important role in leukocyte capture and subsequent development of an atherosclerotic plaque. What is less known is how instantaneous WSS, which can vary by up to 5 Pa over one cardiac cycle, influences leukocyte capture. In this paper we use direct numerical simulations (DNS), performed using an in-house code, to illustrate that leukocyte capture is different whether as a function of instantaneous or time-averaged blood flow. Specifically, a stenotic plaque is modeled using a computational fluid dynamics (CFD) solver through fully three-dimensional Navier-Stokes equations and the immersed boundary method. Pulsatile triphasic inflow is used to simulate the cardiac cycle. The CFD is coupled with an agent-based leukocyte capture model to assess the impact of instantaneous hemodynamics on stenosis growth. The computed wall shear stress agrees well with the results obtained with a commercial software, as well as with theoretical results in the healthy region of the artery. The analysis emphasizes the importance of the instantaneous flow conditions in evaluating the leukocyte rate of capture. That is, the capture rate computed from mean flow field is generally underpredicted compared to the actual rate of capture. Thus, in order to obtain a reliable estimate, the flow unsteadiness during a cardiac cycle should be taken into account.

A cellular platform to enable targeted brain delivery of T cells to glioblastoma.

2053 Background: Poor T cell homing hinders the development of effective cell therapy for central nervous system (CNS) malignancies. Lessons learnt from inflammatory brain diseases can give insight into how to overcome the blood brain barrier (BBB) blockade created by cancer. Activated Leukocyte Cell Adhesion Molecule (ALCAM; CD166) is a pathological adhesion molecule upregulated in the endothelium of a number of inflammatory/infiltrative CNS diseases, such as multiple sclerosis. Antibodies blocking ALCAM decrease leukocyte access to the brain and are currently being tested in a clinical trial for MS. Methods: We studied the difference in the dynamic signature of adhesion molecules in the “anergic” brain tumor endothelium and that of infiltrative brain conditions. Consequently, we mapped the ALCAM minimal binding region to domain 3 (D3) of CD6 and created an artificial molecule with the intent of creating a novel cellular platform to reverse endothelial anergy, through ALCAM specific binding. Results: GBM endothelium fails to launch the second wave of adhesion molecules necessary for firm T leukocyte capture and effective BBB transmigration. Engineered D3 on the T cell crosslinked to ALCAM on endothelial cells in proximity ligation assays (PLA; < 40nm) during TEM. Under shear stress, D3 T cells showed a global improved ALCAM specific trafficking kinetics: higher capture on ALCAM+ endothelium, rolling with slower velocity, and better TEM. In an ex vivo model of BBB, D3 T cells exhibited higher transmigratory ability. We discovered that signaling through the D3 endodomain phosphorylated pZAp70 recruiting Talin that enables LFA-1 (ICAM-ligand) open confirmation, mediating effective TEM. Lastly, in an orthotopic model of GBM, D3 T cells homed more and accumulated at the tumor site compared to NT controls. And, testing Her2 CAR T cells on D3 platform showed an advantageous homing after IV administration which was reflected in tumor control and better survival. Conclusions: We created a cellular platform that enables targeted brain delivery of T cells. This platform serves as a gateway to the effective cellular therapeutics for brain malignancies but potentially as a delivery system for complex biologics for other pathological conditions.

Advances in rare cell isolation: an optimization and evaluation study

BackgroundRare nucleated CD45 negative cells in peripheral blood may be malignant such as circulating tumor cells. Untouched isolation thereof by depletion of normal is favored yet still technological challenging. We optimized and evaluated a novel magnetic bead-based negative selection approach for enhanced enrichment of rare peripheral blood nucleated CD45 negative cells and investigated the problem of rare cell contamination during phlebotomy.MethodsFirstly, the performance of the magnetic cell separation system was assessed using leukocytes and cultivated fibroblast cells in regard to depletion efficiency and the loss of cells of interest. Secondly, a negative selection assay was optimized for high performance, simplicity and cost efficiency. The negative selection assay consisted of; a RBC lysis step, two depletion cycles comprising direct magnetically labelling of leukocytes using anti-CD45 magnetic beads followed by magnetic capture of leukocytes using a duopole permanent magnet. Thirdly, assay evaluation was aligned to conditions of rare cell frequencies and comprised cell spike recovery, cell viability and proliferation, and CD45 negative cell detection. Additionally, the problem of CD45 negative cell contamination during phlebotomy was investigated.ResultsThe depletion factor and recovery of the negative selection assay measured at most 1600-fold and 96%, respectively, leaving at best 1.5 × 104 leukocytes unseparated and took 35 min. The cell viability was negatively affected by chemical RBC lysis. Proliferation of 100 spiked ovarian cancer cells in culture measured 37% against a positive control. Healthy donor testing revealed findings of nucleated CD45 negative cells ranging from 1 to 22 cells /2.5 × 107 leukocytes or 3.5 mL whole blood in 89% (23/26) of the samples.ConclusionOur assay facilitates high performance at shortest assay time. The enrichment assay itself causes minor harm to cells and allows proliferation. Our findings suggest that rare cell contamination is unavoidable. An unexpected high variety of CD45 negative cells have been detected. It is hypothesized that a rare cell profile may translate into tumor marker independent screening.

Rapid capture and labeling of cells on single domain antibodies-functionalized flow cell

Current techniques to characterize leukocyte subgroups in blood require long sample preparation times and sizable sample volumes. A simplified method for leukocyte characterization using smaller blood volumes would thus be useful in diagnostic settings. Here we describe a flow system comprised of two functionalized graphene oxide (GO) surfaces that allow the capture of distinct leukocyte populations from small volumes blood using camelid single-domain antibodyfragments (VHHs) as capture agents. We used site-specifically labeled leukocytes to detect and identify cells exposed to fungal challenge. Combining the chemical and optical properties of GO with the versatility of the VHH scaffold in the context of a flow system provides a quick and efficient method for the capture and characterization of functional leukocytes.

Nanoscale Imaging Reveals a Tetraspanin-CD9 Coordinated Elevation of Endothelial ICAM-1 Clusters.

Endothelial barriers have a central role in inflammation as they allow or deny the passage of leukocytes from the vasculature into the tissue. To bind leukocytes, endothelial cells form adhesive clusters containing tetraspanins and ICAM-1, so-called endothelial adhesive platforms (EAPs). Upon leukocyte binding, EAPs evolve into docking structures that emanate from the endothelial surface while engulfing the leukocyte. Here, we show that TNF-α is sufficient to induce apical protrusions in the absence of leukocytes. Using advanced quantitation of atomic force microscopy (AFM) recordings, we found these structures to protrude by 160 ± 80 nm above endothelial surface level. Confocal immunofluorescence microscopy proved them positive for ICAM-1, JAM-A, tetraspanin CD9 and f-actin. Microvilli formation was inhibited in the absence of CD9. Our findings indicate that stimulation with TNF-α induces nanoscale changes in endothelial surface architecture and that—via a tetraspanin CD9 depending mechanism—the EAPs rise above the surface to facilitate leukocyte capture.

Stiff substrates enhance monocytic cell capture through E-selectin but not P-selectin.

The stiffening of blood vessel walls is associated with inflammatory diseases, including atherosclerosis, diabetes, and obesity. These diseases are driven by the excessive recruitment of inflammatory leukocytes out of the bloodstream and into tissues, but whether vascular stiffening plays a direct role in this process is not clear. In this study, we investigated the possibility that leukocyte capture from blood flow is enhanced on stiffer substrates. We modeled blood flow in vitro by perfusing monocytic cells over hydrogels that matched the stiffness of healthy and diseased arteries. The hydrogels were coated with either E-selectin or P-selectin, which are the endothelial adhesion proteins known to mediate immune cell capture from flow. Interestingly, we discovered that cell attachment to P-selectin coated gels was not dependent on substrate stiffness, while attachment through E-selectin was enhanced on stiffer gels. Specifically we found that on E-selectin coated gels, cells attached in greater numbers, remained attached for longer time periods, and rolled more slowly on stiff gels than soft gels. These results suggest that vascular stiffening could promote leukocyte adhesion to vessel walls where E-selectin is expressed, but may have less of an effect when P-selectin is also present.

OR22: LIGATION OF HLA CLASS II MOLECULES BY HLA ANTIBODIES INDUCES ENDOTHELIAL CELL PERMEABILITY AND MONOCYTE TRANSENDOTHELIAL MIGRATION

Aim Leukocyte extravasation plays a major rolein the development of allograft rejection and transplant vasculopathy and consists of multiple processes, including leukocyte capture, firm adhesion, transmigration, and loss of endothelial cell integrity causing barrier dysfunction. Ligation of HLA class I molecules by antibodies (Ab) elicits signaling events in endothelial cells (EC) leading to EC cell migration, proliferation and leukocyte recruitment. We hypothesize that ligation of HLA class II molecules on EC by Ab contributes to chronic rejection by transducing intracellular signaling cascades leading to cytoskeletal remodeling, increase in EC permeability and monocyte transmigration. Methods Class II expression on primary EC was achieved by infection with adenoviral recombinant Class II Transactivator (CIITA) subcloned in pAd/PL-DEST. EC were treated with murine mAb against class II (F26C6G1). Stress fiber formation was assessed using Texas Red-phalloidin staining. Protein phosphorylation was assessed by Western blot. Changes in EC permeability were measured by VE-cadherin staining and FITC-dextran permeability. Monocyte MM6 recruitment to EC was measured by immunofluorescence microscopy and monocyte transendothelial migration was determined using a transwell assay. Results Anti-class II Ab significantly increased ERK phosphorylation, stimulated stress fiber formation, reduced VE-cadherin localization in EC junctions and enhanced FITC-dextran permeability. ERK inhibitor UO126 pretreatment abrogated class II-induced stress fiber formation, reversed VE-cadherin expression in EC junctions, and attenuated class II-stimulated FITC-dextran permeability. In addition, treatment of EC with anti-class II antibodies induced a 2-4-fold increase in the recruitment and transmigration of monocytes, which was attenuated by treatment with the ERK inhibitor U0126 or recombinant PSGL1 that blocks monocyte capture. Conclusions Ab ligation of HLA class II molecules on EC stimulates activation of the ERK signaling pathway and actin cytoskeleton remodeling leading to barrier dysfunction and monocyte transendothelial migration. Elucidation of the class II signaling pathways has the potential to identify novel therapeutic targets for treatment of chronic rejection.

α(1,3) Fucosyltransferases IV and VII Are Essential for the Initial Recruitment of Basophils in Chronic Allergic Inflammation

Basophils act as initiator cells for the development of IgE-mediated chronic allergic inflammation (IgE-CAI). However, detailed mechanisms of initial recruitment of basophils into the skin have yet to be clarified. Selectins mediate leukocyte capture and rolling on the vascular endothelium for extravasation. Counter-receptor activity of selectins is regulated by α(1, 3) fucosyltransferases (FTs) IV and VII. To clarify the contribution of selectin ligands regulated by FTs for initial basophil recruitment, IgE-CAI was induced in mice deficient in FT-IV and/or FT-VII genes. Although FT-IV(-/-) and FT-VII(-/-) mice exhibited comparable skin responses to wild-type mice, the FT-IV(-/-)/FT-VII(-/-) mice showed significantly impaired inflammation. Although the transfer of basophils to FcRγ(-/-) mice induced IgE-CAI, this induction was completely absent when basophils from FT-IV(-/-)/FT-VII(-/-) mice were transferred. L-selectin, but not P- and E-selectin, blocking Abs inhibited skin inflammation in vivo. P-selectin glycoprotein-1 (PSGL-1) antibody also ameliorated skin inflammation, and basophils were bound to L-selectin in a PSGL-1-dependent manner, which was regulated by FT-IV/VII. Functional PSGL-1 generated by basophil FT-IV/VII and its subsequent binding to L-selectin could be one of the essential steps required for initial basophil recruitment and the development of IgE-CAI in mice.

Reconfigurable microfluidics combined with antibody microarrays for enhanced detection of T-cell secreted cytokines

Cytokines are small proteins secreted by leukocytes in blood in response to infections, thus offering valuable diagnostic information. Given that the same cytokines may be produced by different leukocyte subsets in blood, it is beneficial to connect production of cytokines to specific cell types. In this paper, we describe integration of antibody (Ab) microarrays into a microfluidic device to enable enhanced cytokine detection. The Ab arrays contain spots specific to cell-surface antigens as well as anti-cytokine detection spots. Infusion of blood into a microfluidic device results in the capture of specific leukocytes (CD4 T-cells) and is followed by detection of secreted cytokines on the neighboring Ab spots using sandwich immunoassay. The enhancement of cytokine signal comes from leveraging the concept of reconfigurable microfluidics. A three layer polydimethylsiloxane microfluidic device is fabricated so as to contain six microchambers (1 mm × 1 mm × 30 μm) in the ceiling of the device. Once the T-cell capture is complete, the device is reconfigured by withdrawing liquid from the channel, causing the chambers to collapse onto Ab arrays and enclose cell/anti-cytokine spots within a 30 nl volume. In a set of proof-of-concept experiments, we demonstrate that ∼90% pure CD4 T-cells can be captured inside the device and that signals for three important T-cell secreted cytokines, tissue necrosis factor-alpha, interferon-gamma, and interleukin-2, may be enhanced by 2 to 3 folds through the use of reconfigurable microfluidics.

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (≈ 5.0 − 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography (3), that topography limits adhesive bond formation with other surfaces (9), and that physiological contact forces (≈ 5.0 - 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface (3, 7). We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface (6). It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface (4). In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo (2). The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa (5). The thickness and structure of the glycocalyx have been studied using electron microscopy (8), and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

Leukocyte capture and modulation of cell-mediated immunity during human sepsis: an ex vivo study

Promising preclinical results have been obtained with blood purification therapies as adjuvant treatment for sepsis. However, the mechanisms by which these therapies exert beneficial effects remain unclear. Some investigators have suggested that removal of activated leukocytes from the circulation might help ameliorate remote organ injury. We designed an extracorporeal hemoadsorption device capable of capturing both cytokines and leukocytes in order to test the hypothesis that leukocyte capture would alter circulating cytokine profiles and influence immunological cell-cell interactions in whole blood taken from patients with sepsis. We performed a series of ex vivo studies in 21 patients with septic shock and 12 healthy volunteers. Blood circulated for four hours in closed loops with four specially designed miniaturized extracorporeal blood purification devices including two different hemoadsorption devices and a hemofilter in order to characterize leukocyte capture and to assess the effects of leukocyte removal on inflammation and immune function. Hemoadsorption was selective for removal of activated neutrophils and monocytes. Capture of these cells led to local release of certain cytokines, especially IL-8, and resulted in complex cell-cell interactions involved in cell-mediated immunity. Inhibition of cell adherence reversed the cytokine release and the effects on lymphocyte function. Monocyte and neutrophil capture using a sorbent polymer results in upregulation of IL-8 and modulation of cell-mediated immunity. Further studies are needed to understand better these cellular interactions in order to help design better blood purification therapies.

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane "partner" proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact with ADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8-ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting.

Channel Surface Patterning of Alternating Biomimetic Protein Combinations for Enhanced Microfluidic Tumor Cell Isolation

Here, we report a new method for multicomponent protein patterning in a microchannel and also a technique for improving immunoaffinity-based circulating tumor cell (CTC) capture by patterning regions of alternating adhesive proteins using the new method. The first of two proteins, antiepithelial cell adhesion molecule (anti-EpCAM), provides the specificity for CTC capture. The second, E-selectin, increases CTC capture under shear. Patterning regions with and without E-selectin allows captured leukocytes, which also bind E-selectin and are unwanted impurities in CTC isolation, to roll a short distance and detach from the capture surface. This reduces leukocyte capture by up to 82%. The patterning is combined with a leukocyte elution step in which a calcium chelating buffer effectively deactivates E-selectin so that leukocytes may be rinsed away 60% more efficiently than with a buffer containing calcium. The alternating patterning of this biomimetic protein combination, used in conjunction with the elution step, reduces capture of leukocytes while maintaining a high tumor cell capture efficiency that is up to 1.9 times higher than the tumor cell capture efficiency of a surface with only anti-EpCAM. The new patterning technique described here does not require mask alignment and can be used to spatially control the immobilization of any two proteins or protein mixtures inside a sealed microfluidic channel.

Novel aspects of leukocyte trafficking

The inflammatory response to the Toll‐like receptor (TLR) 4 ligand lipopolysaccharide (LPS) involves a sequence of intravascular leukocyte capture, rolling and adhesion, followed by their emigration into surrounding tissue which is accompanied by plasma protein extravasation (PPE). Typically, such events are observed hours after LPS injection, but we have chosen to investigate the earlier stages of leukocyte trafficking during murine experimental endotoxaemia.Using intravital microscopy of the mesentery, we observed a rapid increase in neutrophil emigration and PPE after i.p. LPS injection, reaching a plateau after 2h. The same response was seen with the TLR2 ligand zymosan, but not the non‐specific neutrophil activator ammonium thioglycolate, implicating TLR‐specific mechanisms in early leukocyte trafficking. LPS‐induced trafficking was associated with activation of peritoneal mast cells, shown by selective uptake of the dye ruthenium red from the superfusion buffer, and was inhibited by pre‐treatment with cromolyn sodium.To conclude, our data suggest that mast cells play a role in LPS‐induced peritoneal inflammation in terms of leukocyte emigration. Further work is underway to determine the sequential mechanisms of cell types and mediators involved in these hyperacute stages of LPS‐induced inflammation.Work supported by the BBSRC.