Michael B. Lawrence

CD24 mediates rolling of breast carcinoma cells on P-selectin

P‐selectin mediates rolling of neutrophils and other leukocytes on activated endothelial cells and platelets through binding to P‐selectin glycoprotein ligand‐1 (PSGL‐1). Certain PSGL‐1 negative tumor cell lines can bind P‐selectin under static conditions through the GPI‐linked surface mucin, CD24, but the physiological significance of this interaction and whether it can occur under flow conditions is not known. Here, we show that CD24+ PSGL‐1– KS breast carcinoma cells attach to and roll on recombinant P‐selectin under a continuous wall shear stress, although at a lower density and higher velocity than CD24+ PSGL‐1+ cells, such as HL‐60. Adding excess soluble CD24 or removing CD24 from the cell surface with phosphatidylinositol‐phospholipase C (PI‐PLC) significantly reduced KS cell rolling on P‐selectin. The ability of KS cells to roll on P‐selectin was positively correlated with the CD24 expression level. Comparison with three other CD24+ cell lines established that expression of sialyl‐Lewisx antigen was also necessary for CD24‐mediated rolling on P‐selectin. CD24 purified from KS cells supported rolling of P‐selectin transfectants, but not L‐selectin transfectants. Finally, KS cells rolled on vascular endothelium in vivo in a P‐selectin‐dependent manner. Together our data show that CD24 serves as a ligand for P‐selectin under physiological flow conditions. Interaction of tumor cells with P‐selectin via CD24 may be an important adhesion pathway in cancer metastasis.—Aigner, S., Ramos, C. L., Hafezi‐Moghadam, A., Lawrence, M. B., Friederichs, J., Altevogt, P., and Ley, K. CD24 mediates rolling of breast carcinoma cells on P‐selectin. FASEB J. 12, 1241–1251 (1998)

A direct comparison of selectin-mediated transient, adhesive events using high temporal resolution.

Leukocyte capture and rolling on the vascular endothelium is mediated principally by the selectin family of cell adhesion receptors. In a parallel plate flow chamber, neutrophil rolling on purified selectins or a selectin-ligand substrate was resolved by high speed videomicroscopy as a series of ratchet-like steps with a characteristic time constant (Kaplanski, G., C. Farnarier, O. Tissot, A. Pierres, A.-M. Benoliel, M. C. Alessi, S. Kaplanski, and P. Bongrand. 1993. Biophys. J. 64:1922-1933; Alon, R., D. A. Hammer, and T. A. Springer. 1995. Nature (Lond.). 374:539-542). Under shear, neutrophil arrests due to bond formation events were as brief as 4 ms. Pause time distributions for neutrophils tethering on P-, E-, L-selectin, or peripheral node addressin (PNAd) were compared at estimated single bond forces ranging from 37 to 250 pN. Distributions of selectin mediated pause times were fit to a first order exponential, resulting in a molecular dissociation constant (k(off)) for the respective selectin as a function of force. At estimated single bond forces of 125 pN and below, all three selectin dissociation constants fit the Bell and Hookean spring models of force-driven bond breakage equivalently. Unstressed k(off) values based on the Bell model were 2.4, 2.6, 2.8, 3.8 s(-1) for P-selectin, E-selectin, L-selectin, and PNAd, respectively. Bond separation distances (reactive compliance) were 0.39, 0.18, 1.11, 0.59 A for P-selectin, E-selectin, L-selectin, and PNAd, respectively. Dissociation constants for L-selectin and P-selectin at single bond forces above 125 pN were considerably lower than either Bell or Hookean spring model predictions, suggesting the existence of two regimes of reactive compliance. Additionally, interactions between L-selectin and its leukocyte ligand(s) were more labile in the presence of flow than the L-selectin endothelial ligand, PNAd, suggesting that L-selectin ligands may have different molecular and mechanical properties. Both types of L-selectin bonds had a higher reactive compliance than P-selectin or E-selectin bonds.

Ultrasound Molecular Imaging of Tumor Angiogenesis with an Integrin Targeted Microbubble Contrast Agent

Rationale and Objectives:Ultrasound molecular imaging is an emerging technique for sensitive detection of intravascular targets. Molecular imaging of angiogenesis has strong potential for both clinical use and as a research tool in tumor biology and the development of antiangiogenic therapies. Our objectives are to develop a robust ultrasound contrast agent platform using microbubbles (MB) to which targeting ligands can be conjugated by biocompatible, covalent conjugation chemistry, and to develop a pure low mechanical index (MI) imaging processing method and corresponding quantification method. The MB and the imaging methods were evaluated in a mouse model of breast cancer in vivo. Materials and Methods:We used a cyclic arginine-glycine-aspartic acid (cRGD) pentapeptide containing a terminal cysteine group conjugated to the surface of MB bearing pyridyldithio-propionate (PDP) for targeting &agr;v&bgr;3 integrins. As negative controls, MB without a ligand or MB bearing a scrambled sequence (cRAD) were prepared. To enable characterization of peptides bound to MB surfaces, the cRGD peptide was labeled with FITC and detected by plate fluorometry, flow cytometry, and fluorescence microscopy. Targeted adhesion of cRGD-MB was demonstrated in an in vitro flow adhesion assay against recombinant murine &agr;v&bgr;3 integrin protein and &agr;v&bgr;3 integrin-expressing endothelial cells (bEnd.3). The specificity of cRGD-MB for &agr;v&bgr;3 integrin was demonstrated by treating bEnd.3 EC with a blocking antibody. A murine model of mammary carcinoma was used to assess targeted adhesion and ultrasound molecular imaging in vivo. The targeted MB were visualized using a low MI contrast imaging pulse sequence, and quantified by intensity normalization and 2-dimensional Fourier transform analysis. Results:The cRGD ligand concentration on the MB surface was ∼8.2 × 106 molecules per MB. At a wall shear stress of 1.0 dynes/cm2, cRGD-MB exhibited 5-fold higher adhesion to immobilized recombinant &agr;v&bgr;3 integrin relative to nontargeted MB and cRAD-MB controls. Similarly, cRGD-MB showed significantly greater adhesion to bEnd.3 EC compared with nontargeted MB and cRAD-MB. In addition, cRGD-MB, but not nontargeted MB or cRAD-MB, showed significantly enhanced contrast signals with a high tumor-to-background ratio. The adhesion of cRGD-MB to bEnd.3 was reduced by 80% after using anti-&agr;v monoclonal antibody to treat bEnd.3. The normalized image intensity amplitude was ∼0.8, 7 minutes after the administration of cRGD-MB relative to the intensity amplitude at the time of injection, while the spatial variance in image intensity improved the detection of bound agents. The accumulation of cRGD-MB was blocked by preadministration with an anti-&agr;v blocking antibody. Conclusions:The results demonstrate the functionality of a novel MB contrast agent covalently coupled to an RGD peptide for ultrasound molecular imaging of &agr;v&bgr;3 integrin and the feasibility of quantitative molecular ultrasound imaging with a low MI.

Comparison of PSGL-1 Microbead and Neutrophil Rolling: Microvillus Elongation Stabilizes P-Selectin Bond Clusters

A cell-scaled microbead system was used to analyze the force-dependent kinetics of P-selectin adhesive bonds independent of micromechanical properties of the neutrophils surface microvilli, an elastic structure on which P-selectin ligand glycoprotein-1 (PSGL-1) is localized. Microvillus extension has been hypothesized in contributing to the dynamic range of leukocyte rolling observed in vivo during inflammatory processes. To evaluate PSGL-1/P-selectin bond kinetics of microbeads and neutrophils, rolling and tethering on P-selectin-coated substrates were compared in a parallel-plate flow chamber. The dissociation rates for PSGL-1 microbeads on P-selectin were briefer than those of neutrophils for any wall shear stress, and increased more rapidly with increasing flow. The microvillus length necessary to reconcile dissociation constants of PSGL-1 microbeads and neutrophils on P-selectin was 0.21 microm at 0.4 dyn/cm2, and increased to 1.58 microm at 2 dyn/cm2. The apparent elastic spring constant of the microvillus ranged from 1340 to 152 pN/microm at 0.4 and 2.0 dyn/cm2 wall shear stress. Scanning electron micrographs of neutrophils rolling on P-selectin confirmed the existence of micrometer-scaled tethers. Fixation of neutrophils to abrogate microvillus elasticity resulted in rolling behavior similar to PSGL-1 microbeads. Our results suggest that microvillus extension during transient PSGL-1/P-selectin bonding may enhance the robustness of neutrophil rolling interactions.

Immobilized IL-8 Triggers Progressive Activation of Neutrophils Rolling In Vitro on P-Selectin and Intercellular Adhesion Molecule-1

The chemokine IL-8 is found on the luminal side of vascular endothelial cells, where it is postulated to be immobilized during inflammation. In this study, we observed that immobilized IL-8 can stimulate neutrophils to firmly adhere to a substrate containing ICAM-1 in a static adhesion assay. Soluble IL-8 was then perfused over neutrophils rolling on P-selectin (P-sel) and ICAM-1, confirming that IL-8 in solution can quickly cause rolling neutrophils to arrest. To mimic a blood vessel wall with IL-8 expressed on the luminal surface of endothelial cells, IL-8 was immobilized along with P-sel and ICAM-1 at defined site densities to a surface. Neutrophils rolled an average of 200 μm on surfaces of P-sel, ICAM-1, and IL-8 before firmly adhering through ICAM-1-β2 integrin interactions at 2 dynes/cm2 wall shear stress. Increasing the density of IL-8 from 60 to 350 sites/μm2 on the surface decreased by 50% the average distance and time the neutrophils rolled before becoming firmly adherent. Temporal dynamics of ICAM-1-β2 integrin interactions of rolling neutrophils following IL-8 exposure suggest the existence of two classes of β2 integrin-ICAM-1 interactions, a low avidity interaction with a 65% increase in pause times as compared with P-sel-P-sel glycoprotein ligand-1 interactions, and a high avidity interaction with pause times 400% greater than the selectin interactions. Based on the proportionality between IL-8 site density and time to arrest, it appears that neutrophils may need to sample a critical number of IL-8 molecules presented by the vessel wall before forming a sufficient number of high avidity β2 integrin bonds for firm adhesion.

Influence of Cell Deformation on Leukocyte Rolling Adhesion in Shear Flow

Blood cell interaction with vascular endothelium is important in microcirculation, where rolling adhesion of circulating leukocytes along the surface of endothelial cells is a prerequisite for leukocyte emigration under flow conditions. HL-60 cell rolling adhesion to surface-immobilized P-selectin in shear flow was investigated using a side-view flow chamber, which permitted measurements of cell deformation and cell-substrate contact length as well as cell rolling velocity. A two-dimensional model was developed based on the assumption that fluid energy input to a rolling cell was essentially distributed into two parts: cytoplasmic viscous dissipation, and energy needed to break adhesion bonds between the rolling cell and its substrate. The flow fields of extracellular fluid and intracellular cytoplasm were solved using finite element methods with a deformable cell membrane represented by an elastic ring. The adhesion energy loss was calculated based on receptor-ligand kinetics equations. It was found that, as a result of shear-flow-induced cell deformation, cell-substrate contact area under high wall shear stresses (20 dyn/cm2) could be as much as twice of that under low stresses (0.5 dyn/cm2). An increase in contact area may cause more energy dissipation to both adhesion bonds and viscous cytoplasm, whereas the fluid energy input may decrease due to the flattened cell shape. Our model predicts that leukocyte rolling velocity will reach a plateau as shear stress increases, which agrees with both in vivo and in vitro experimental observations.

The Molecular Mechanics of P- and L-Selectin Lectin Domains Binding to PSGL-1

A laser trap was used to compare the load-dependent binding kinetics between truncated P- and L-selectin to their natural ligand, P-selectin glycoprotein ligand-1 (PSGL-1) over the predicted physiological range of loading rates. Human PSGL-1 was covalently coupled to polystyrene beads. Chimeric selectins were adsorbed to nitrocellulose-coated glass beads on a coverslip. A PSGL-1 bead was held in a laser trap and touched to a vertical surface of the glass bead, allowing a bond to form between selectin and ligand. The surface was moved away from the microsphere, applying load at a constant rate until bond rupture. Rupture force for both selectins increased with loading rate, but significant differences in rupture force between P- and L-selectin were observed only above 460 pN/s. These data are best represented as two energy barriers to unbinding, with the transition from the low to high loading rate regime at 260-290 pN/s. The data also allow the first estimate of a two-dimensional specific on-rate for binding of these two selectins to their natural ligand (1.7 microm2/s). These data suggest that P- and L-selectin lectin domains have very similar kinetics under physiological conditions.

Activation of A2A adenosine receptors inhibits expression of α4/β1 integrin (very late antigen‐4) on stimulated human neutrophils

The α4/β1 integrin very late antigen‐4 (CD49d/CD29) is up‐regulated on circulating neutrophils of septic patients. Although no individual agent mimics this effect of sepsis, we now report that following priming of human neutrophils with lipopolysaccharide or tumor necrosis factor α (TNF‐α), addition of formyl‐Met‐Leu‐Phe (fMLP) results in a “stimulated”, sepsis‐like, four‐ to fivefold rise in CD49d expression. TNF/fMLP stimulation also produced a similar increase in CD49d‐mediated adhesion of neutrophils to a vascular cell adhesion molecule‐1 (VCAM‐1)‐coated surface. Adenosine is a naturally occurring, anti‐inflammatory mediator released from injured or inflamed tissues. We observed that stimulated neutrophil CD49d expression was decreased by activation of A2A adenosine receptors (A2AAR) with the selective agonist 4‐{3‐[6‐amino‐9‐(5‐ethylcarbamoyl‐3,4‐dihydroxy‐tetrahydro‐furan‐2‐yl)‐9H‐purin‐2‐yl]‐prop‐2‐ynyl}‐cyclohexanecarboxylicacid methyl ester (ATL146e; EC50=6.4 nM). ATL146e (100 nM) also reduced the fraction of stimulated neutrophils that adhered to VCAM‐1 from 38 ± 6% to 27 ± 5%. Inhibition of CD49d expression was equally inhibited by ATL146e, added before or after TNF priming, and was reversed by incubation with the A2AAR‐selective antagonist 4‐{2‐[7‐amino‐2‐(2‐furyl) (1, 2, 4)triazolo(2,3‐a) (1, 3, 5)triazin‐5‐yl‐amino]ethyl}‐phenol (ZM241385; 100 nM). A suboptimal ATL146e concentration (1 nM) combined with the type IV phosphodiesterase inhibitor rolipram (100 nM) synergistically decreased stimulated CD49d expression by >50%. The cyclic adenosine monophosphate (cAMP)‐dependent kinase [protein kinase A (PKA)] inhibitor H‐89 (10 μM) reversed the effect of ATL146e on stimulated CD49d expression. Other means of increasing cAMP in neutrophils also decreased stimulated CD49d expression. We conclude that adenosine binding to A2AAR counteracts stimulation of neutrophil CD49d integrin expression and neutrophil binding to VCAM‐1 via a cAMP/PKA‐mediated pathway.

Sonorheometry: A Noncontact Method for the Dynamic Assessment of Thrombosis

Inappropriate blood coagulation plays a central role in the onset of myocardial infarction, stroke, pulmonary embolism, and other thrombotic disorders. The ability to screen for an increased propensity to clot could prevent the onset of such events by appropriately identifying those at risk and enabling prophylactic treatment. Similarly, the ability to characterize the mechanical properties of clots in vivo might improve patient outcomes by better informing treatment strategies. We have developed a technique called sonorheometry. Unlike existing methods, sonorheometry is able to assess mechanical properties of coagulation with minimal disturbance to the delicate structure of a forming thrombus. Sonorheometry uses acoustic radiation force to produce small, localized displacements within the sample. Time delay estimation is performed on returned ultrasound echoes to determine sample deformation. Mechanical modeling and parametric fitting to experimental data yield maps of mechanical properties. Sonorheometry is well suited to both in vitro and in vivo applications. A control experiment was performed to verify that sonorheometry provides mechanical characterization in agreement with that from a conventional rheometer. We also examined thrombosis in blood samples taken from four subjects. This data suggests that sonorheometry may offer a novel and valuable method for assessing the thrombogenicity of blood samples.

Characterizing emergent properties of immunological systems with multi- cellular rule-based computational modeling

The immune system is comprised of numerous components that interact with one another to give rise to phenotypic behaviors that are sometimes unexpected. Agent-based modeling (ABM) and cellular automata (CA) belong to a class of discrete mathematical approaches in which autonomous entities detect local information and act over time according to logical rules. The power of this approach lies in the emergence of behavior that arises from interactions between agents, which would otherwise be impossible to know a priori. Recent work exploring the immune system with ABM and CA has revealed novel insights into immunological processes. Here, we summarize these applications to immunology and, particularly, how ABM can help formulate hypotheses that might drive further experimental investigations of disease mechanisms.