Scott I. Simon

Chemically distinct transition states govern rapid dissociation of single L-selectin bonds under force

Carbohydrate–protein bonds interrupt the rapid flow of leukocytes in the circulation by initiation of rolling and tethering at vessel walls. The cell surface carbohydrate ligands are glycosylated proteins like the mucin P-selectin glycoprotein ligand-1 (PSGL-1), which bind ubiquitously to the family of E-, P-, and L-selectin proteins in membranes of leukocytes and endothelium. The current view is that carbohydrate–selectin bonds dissociate a few times per second, and the unbinding rate increases weakly with force. However, such studies have provided little insight into how numerous hydrogen bonds, a Ca2+ metal ion bond, and other interactions contribute to the mechanical strength of these attachments. Decorating a force probe with very dilute ligands and controlling touch to achieve rare single-bond events, we have varied the unbinding rates of carbohydrate–selectin bonds by detachment with ramps of force/time from 10 to 100,000 pN/sec. Testing PSGL-1, its outer 19 aa (19FT), and sialyl LewisX (sLeX) against L-selectin in vitro on glass microspheres and in situ on neutrophils, we found that the unbinding rates followed the same dependence on force and increased by nearly 1,000-fold as rupture forces rose from a few to ≈200 pN. Plotted on a logarithmic scale of loading rate, the rupture forces reveal two prominent energy barriers along the unbinding pathway. Strengths above 75 pN arise from rapid detachment (<0.01 sec) impeded by an inner barrier that requires a Ca2+ bond between a single sLeX and the lectin domain. Strengths below 75 pN occur under slow detachment (>0.01 sec) impeded by the outer barrier, which appears to involve an array of weak (putatively hydrogen) bonds.

Optical and acoustical dynamics of microbubble contrast agents inside neutrophils.

Acoustically active microbubbles are used for contrast-enhanced ultrasound assessment of organ perfusion. In regions of inflammation, contrast agents are captured and phagocytosed by activated neutrophils adherent to the venular wall. Using direct optical observation with a high-speed camera and acoustical interrogation of individual bubbles and cells, we assessed the physical and acoustical responses of both phagocytosed and free microbubbles. Optical analysis of bubble radial oscillations during insonation demonstrated that phagocytosed microbubbles experience viscous damping within the cytoplasm and yet remain acoustically active and capable of large volumetric oscillations during an acoustic pulse. Fitting a modified version of the Rayleigh-Plesset equation that describes mechanical properties of thin shells to optical radius-time data of oscillating bubbles provided estimates of the apparent viscosity of the intracellular medium. Phagocytosed microbubbles experienced a viscous damping approximately sevenfold greater than free microbubbles. Acoustical comparison between free and phagocytosed microbubbles indicated that phagocytosed microbubbles produce an echo with a higher mean frequency than free microbubbles in response to a rarefaction-first single-cycle pulse. Moreover, this frequency increase is predicted using the modified Rayleigh-Plesset equation. We conclude that contrast-enhanced ultrasound can detect distinct acoustic signals from microbubbles inside of neutrophils and may provide a unique tool to identify activated neutrophils at sites of inflammation.

Neutrophil-derived IL-1β Is Sufficient for Abscess Formation in Immunity against Staphylococcus aureus in Mice

Neutrophil abscess formation is critical in innate immunity against many pathogens. Here, the mechanism of neutrophil abscess formation was investigated using a mouse model of Staphylococcus aureus cutaneous infection. Gene expression analysis and in vivo multispectral noninvasive imaging during the S. aureus infection revealed a strong functional and temporal association between neutrophil recruitment and IL-1β/IL-1R activation. Unexpectedly, neutrophils but not monocytes/macrophages or other MHCII-expressing antigen presenting cells were the predominant source of IL-1β at the site of infection. Furthermore, neutrophil-derived IL-1β was essential for host defense since adoptive transfer of IL-1β-expressing neutrophils was sufficient to restore the impaired neutrophil abscess formation in S. aureus-infected IL-1β-deficient mice. S. aureus-induced IL-1β production by neutrophils required TLR2, NOD2, FPR1 and the ASC/NLRP3 inflammasome in an α-toxin-dependent mechanism. Taken together, IL-1β and neutrophil abscess formation during an infection are functionally, temporally and spatially linked as a consequence of direct IL-1β production by neutrophils.

Dynamic regulation of LFA-1 activation and neutrophil arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow

Neutrophil recruitment during acute inflammation is triggered by G-protein-linked chemotactic receptors that in turn activate β2 integrin (CD18), deemed a critical step in facilitating cell capture and arrest under the shear force of blood flow. A conformational switch in the I domain allosteric site (IDAS) and in CD18 regulates LFA-1 affinity for endothelial ligands including intercellular adhesion molecule 1 (ICAM-1). We examined the dynamics of CD18 activation in terms of the efficiency of neutrophil capture of ICAM-1, and we correlated this with the membrane topography of 327C, an antibody that recognizes the active conformation of CD18 I-like domain. Adhesion increased in direct proportion to chemotactic stimulus rising 7-fold over a log range of interleukin-8 (IL-8). A threshold dose of ∼75 pm IL-8, corresponding to ligation of only ∼10–100 receptors, was sufficient to activate ∼20,000 CD18 and a rapid boost in the capture efficiency on ICAM-1. This was accompanied by a rapid redistribution of active LFA-1, but not Mac-1, into membrane patches, a necessary component for optimum adhesion efficiency. Shear-resistant arrest on a monolayer of ICAM-1 was reversed within minutes of chemotactic stimulation correlating with a shift from high to low affinity CD18 and dispersal of patches of active CD18. Mobility of active CD18 into high avidity patches was dependent on phosphatidylinositol 3-kinase activity and not F-actin polymerization. The data reveal that the number of chemotactic receptors bound and the topography and lifetime of high affinity LFA-1 tightly regulate the efficiency of neutrophil capture on ICAM-1.

Ultrasonic analysis of peptide- And antibody-targeted microbubble contrast agents for molecular imaging of α vβ 3-expressing cells

The goal of targeted ultrasound contrast agents is to significantly and selectively enhance the detection of a targeted vascular site. In this manuscript, three distinct contrast agents targeted to the alphavbeta3 integrin are examined. The alphavbeta3 integrin has been shown to be highly expressed on metastatic tumors and endothelial cells during neovascularization, and its expression has been shown to correlate with tumor grade. Specific adhesion of these contrast agents to alphavbeta3-expressing cell monolayers is demonstrated in vitro, and compared with that of nontargeted agents. Acoustic studies illustrate a backscatter amplitude increase from monolayers exposed to the targeted contrast agents of up to 13-fold (22 dB) relative to enhancement due to control bubbles. A linear dependence between the echo amplitude and bubble concentration was observed for bound agents. The decorrelation of the echo from adherent targeted agents is observed over successive pulses as a function of acoustic pressure and bubble density. Frequency-domain analysis demonstrates that adherent targeted bubbles exhibit high-amplitude narrowband echo components, in contrast to the primarily wideband response from free microbubbles. Results suggest that adherent targeted contrast agents are differentiable from free-floating microbubbles, that targeted contrast agents provide higher sensitivity in the detection of angiogenesis, and that conventional ultrasound imaging techniques such as signal subtraction or decorrelation detection can be used to detect integrin-expressing vasculature with sufficient signal-to-noise.

A Mouse Model of Post-Arthroplasty Staphylococcus aureus Joint Infection to Evaluate In Vivo the Efficacy of Antimicrobial Implant Coatings

Background Post-arthroplasty infections represent a devastating complication of total joint replacement surgery, resulting in multiple reoperations, prolonged antibiotic use, extended disability and worse clinical outcomes. As the number of arthroplasties in the U.S. will exceed 3.8 million surgeries per year by 2030, the number of post-arthroplasty infections is projected to increase to over 266,000 infections annually. The treatment of these infections will exhaust healthcare resources and dramatically increase medical costs. Methodology/Principal Findings To evaluate novel preventative therapeutic strategies against post-arthroplasty infections, a mouse model was developed in which a bioluminescent Staphylococcus aureus strain was inoculated into a knee joint containing an orthopaedic implant and advanced in vivo imaging was used to measure the bacterial burden in real-time. Mice inoculated with 5×103 and 5×104 CFUs developed increased bacterial counts with marked swelling of the affected leg, consistent with an acute joint infection. In contrast, mice inoculated with 5×102 CFUs developed a low-grade infection, resembling a more chronic infection. Ex vivo bacterial counts highly correlated with in vivo bioluminescence signals and EGFP-neutrophil fluorescence of LysEGFP mice was used to measure the infection-induced inflammation. Furthermore, biofilm formation on the implants was visualized at 7 and 14 postoperative days by variable-pressure scanning electron microscopy (VP-SEM). Using this model, a minocycline/rifampin-impregnated bioresorbable polymer implant coating was effective in reducing the infection, decreasing inflammation and preventing biofilm formation. Conclusions/Significance Taken together, this mouse model may represent an alternative pre-clinical screening tool to evaluate novel in vivo therapeutic strategies before studies in larger animals and in human subjects. Furthermore, the antibiotic-polymer implant coating evaluated in this study was clinically effective, suggesting the potential for this strategy as a therapeutic intervention to combat post-arthroplasty infections.

Dynamics of Neutrophil Infiltration during Cutaneous Wound Healing and Infection Using Fluorescence Imaging

Neutrophil influx is an early inflammatory response that is essential for the clearance of bacteria and cellular debris during cutaneous wounding. A non-invasive real-time fluorescence imaging technique was developed to examine the kinetics of enhanced green fluorescence protein-polymorphonuclear leukocyte (EGFP-PMN) influx within a wound. We hypothesized that infection or systemic availability would directly regulate the dynamics of EGFP-PMN recruitment and the efficiency of wound closure. Neutrophil recruitment increased dramatically over the first 24 hours from 10(6) at 4 hours up to a maximum of 5 x 10(6) EGFP-PMNs at 18 hours. A high rate of EGFP-PMN turnover was evidenced by approximately 80% decrease in EGFP signal within 6 hours. In response to wound colonization by Staphylococcus aureus or injection of GM-CSF, systemic PMNs increased twofold above saline control. This correlated with an increase in EGFP-PMN recruitment up to approximately 10(7) within the wound. Despite this effect by these distinct inflammatory drivers, wound closure occurred at a rate similar to the saline-treated control group. In summary, a non-invasive fluorescence-based imaging approach combined with genetic labeling of neutrophils provides a dynamic inner view of inflammation and the kinetics of neutrophil infiltration into the wounded skin over extended durations.

Shear-Dependent Capping of L-Selectin and P-Selectin Glycoprotein Ligand 1 by E-Selectin Signals Activation of High-Avidity β2-Integrin on Neutrophils

Two adhesive events critical to efficient recruitment of neutrophils at vascular sites of inflammation are up-regulation of endothelial selectins that bind sialyl Lewisx ligands and activation of β2-integrins that support neutrophil arrest by binding ICAM-1. We have previously reported that neutrophils rolling on E-selectin are sufficient for signaling cell arrest through β2-integrin binding of ICAM-1 in a process dependent upon ligation of L-selectin and P-selectin glycoprotein ligand 1 (PSGL-1). Unresolved are the spatial and temporal events that occur as E-selectin binds to human neutrophils and dynamically signals the transition from neutrophil rolling to arrest. Here we show that binding of E-selectin to sialyl Lewisx on L-selectin and PSGL-1 drives their colocalization into membrane caps at the trailing edge of neutrophils rolling on HUVECs and on an L-cell monolayer coexpressing E-selectin and ICAM-1. Likewise, binding of recombinant E-selectin to PMNs in suspension also elicited coclustering of L-selectin and PSGL-1 that was signaled via mitogen-activated protein kinase. Binding of recombinant E-selectin signaled activation of β2-integrin to high-avidity clusters and elicited efficient neutrophil capture of β2-integrin ligands in shear flow. Inhibition of p38 and p42/44 mitogen-activated protein kinase blocked the cocapping of L-selectin and PSGL-1 and the subsequent clustering of high-affinity β2-integrin. Taken together, the data suggest that E-selectin is unique among selectins in its capacity for clustering sialylated ligands and transducing signals leading to neutrophil arrest in shear flow.

CD11c/CD18 Expression Is Upregulated on Blood Monocytes During Hypertriglyceridemia and Enhances Adhesion to Vascular Cell Adhesion Molecule-1

Objective—Atherosclerosis is associated with monocyte adhesion to the arterial wall that involves integrin activation and emigration across inflamed endothelium. Involvement of &bgr;2-integrin CD11c/CD18 in atherogenesis was recently shown in dyslipidemic mice, which motivates our study of its inflammatory function during hypertriglyceridemia in humans. Methods and Results—Flow cytometry of blood from healthy subjects fed a standardized high-fat meal revealed that at 3.5 hours postprandial, monocyte CD11c surface expression was elevated, and the extent of upregulation correlated with blood triglycerides. Monocytes from postprandial blood exhibited an increased light scatter profile, which correlated with elevated CD11c expression and uptake of lipid particles. Purified monocytes internalized triglyceride-rich lipoproteins isolated from postprandial blood through low-density lipoprotein–receptor–related protein-1, and this also elicited CD11c upregulation. Laboratory-on-a-chip analysis of whole blood showed that monocyte arrest on a vascular cell adhesion molecule-1 (VCAM-1) substrate under shear flow was elevated at 3.5 hours and correlated with blood triglyceride and CD11c expression. At 7 hours postprandial, blood triglycerides decreased and monocyte CD11c expression and arrest on VCAM-1 returned to fasting levels. Conclusion—During hypertriglyceridemia, monocytes internalize lipids, upregulate CD11c, and increase adhesion to VCAM-1. These data suggest that analysis of monocyte inflammation may provide an additional framework for evaluating individual susceptibility to cardiovascular disease.

Ultrasound radiation force modulates ligand availability on targeted contrast agents.

Radiation force produced by low-amplitude ultrasound at clinically relevant frequencies remotely translates freely flowing microbubble ultrasound contrast agents over distances up to centimeters from the luminal space to the vessel wall in order to enhance ligand-receptor contact in targeting applications. The question arises as to how the microbubble shell might be designed at the molecular level to fully take advantage of such physical forces in targeted adhesion for molecular imaging and controlled therapeutic release. Herein, we report on a novel surface architecture in which the tethered ligand is buried in a polymeric overbrush. Our results, with biotin-avidin as the model ligand-receptor pair, show that the overbrush conceals the ligand, thereby reducing immune cell binding and increasing circulation persistence. Targeted adhesion is achieved through application of ultrasound radiation force to instantly reveal the ligand within a well-defined focal zone and simultaneously bind the ligand and receptor. Our data illustrate how the adhesive properties of the contrast agent surface can be reversibly changed, from stealth to sticky, through the physical effects of ultrasound. This technique can be combined with any ligand-receptor pair to optimize targeted adhesion for ultrasonic molecular imaging.