Rolling of neutrophils over stimulated endothelial cells is a prerequisite to firm attachment and subsequent transendothelial migration during the inflammatory response. The selectin family of adhesion molecules are thought to mediate rolling by binding counter-receptors that present carbohydrates, such as sialyl Lewis(x) (sLe[x]). Recently we described a cell-free system for rolling using sLe(x)-coated microspheres and E-selectin molecules on inert substrates. We showed that sLe(x)-coated microspheres rolled over E-selectin-IgG chimera substrates with dynamics that are similar to those of leukocytes rolling over stimulated endothelium. In this paper we provide a thorough quantitative description of the dynamics of adhesion for this system. We find that particle rolling velocity increases with increasing wall shear stress and decreases with increasing E-selectin or sLe(x) surface densities. Large changes in the average rolling velocity can occur with small changes in sLe(x) or E-selectin density; however, rolling velocity is more sensitive to E-selectin surface coverage than to the number of sLe(x) molecules on the microspheres. Aided by dimensional analysis, we show that decreasing the wall shear stress or increasing either receptor (E-selectin) or ligand (sLe[x]) surface coverage results in an equivalent decrease in particle rolling velocity. In addition, we find that different Lewis carbohydrates are more effective in mediating rolling on E-selectin, with effectiveness following the trend sialyl Lewis(a) > sialyl Lewis(x) >> sulfated Lewis(x) >> Lewis(x). Rolling velocity fluctuated with time for all carbohydrate-selectin pairs tested, and the magnitude of the velocity fluctuations was linearly proportional to the mean rolling velocity for all combinations of E-selectin site density, sLe(x) site density, wall shear stress, and carbohydrate chemistry tested.

Selections mediate transient adhesion of neutrophils to stimulated endothelial cells at sites of inflammation by binding counter-receptors that present carbohydrates such as sialyl Lewis(x). We have developed a cell-free adhesion assay using sialyl Lewis(x)-coated microspheres and E-selection-IgG chimera-coated substrates to investigate the premise that rolling primarily results from functional properties of selection-carbohydrate bonds, whereas cellular morphology and signaling act as secondary effects. Sialyl Lewis(x)-coated microspheres attach to and roll over E-selectin-IgG chimera-coated substrates between the physiological wall shear stresses of 0.7 and 2 dynes/cm2. Rolling velocities vary with time and depend on E-selectin-IgG chimera site density and wall shear stress. Our results show that sialyl Lewis(x) is a minimal functional recognition element required for rolling on E-selectin under flow.

The selectin family of adhesion molecules mediates attachment and rolling of neutrophils to stimulated endothelial cells. This step of the inflammatory response is a prerequisite to firm attachment and extravasation. We have reported that microspheres coated with sialyl Lewis(x) (sLe(x)) interact specifically and roll over E-selectin and P-selectin substrates (Brunk et al., 1996; Rodgers et al 2000). This paper extends the use of the cell-free system to the study of the interactions between L-selectin and sLe(x) under flow. We find that sLe(x) microspheres specifically interact with and roll on L-selectin substrates. Rolling velocity increases with wall shear stress and decreases with increasing L-selectin density. Rolling velocities are fast, between 25 and 225 microm/s, typical of L-selectin interactions. The variability of rolling velocity, quantified by the variance in rolling velocity, scales linearly with rolling velocity. Rolling flux varies with both wall shear stress and L-selectin site density. At a density of L-selectin of 800 sites/microm(2), the rolling flux of sLe(x) coated microspheres goes through a clear maximum with respect to shear stress at 0.7 dyne/cm(2). This behavior, in which the maintenance and promotion of rolling interactions on selectins requires shear stress above a threshold value, is known as the shear threshold effect. We found that the magnitude of the effect is greatest at an L-selectin density of 800 sites/microm(2) and gradually diminishes with increasing L-selectin site density. Our study is the first to reveal the shear threshold effect with a cell free system and the first to show the dependence of the shear threshold effect on L-selectin site density in a reconstituted system. Our ability to recreate the shear threshold effect in a cell-free system strongly suggests the origin of the effect is in the physical chemistry of L-selectin interaction with its ligand, and largely eliminates cellular features such as deformability or topography as its cause.

Leukocytes must bind to vascular endothelium under conditions of flow to perform their appropriate physiological functions, which require trafficking into and out of the tissue space surrounding blood vessels. Trafficking into tissues is required of neutrophils during the acute inflammatory response, and during trafficking of lymphocytes into lymphoid tissue. Egress from blood lumen to tissue involves a series of adhesion-dependent steps, each of which involve different leukocyte adhesion receptors and counterreceptors on the endothelium. Transient adhesion, or rolling, of leukocytes over endothelial cells is a prerequisite to firm attachment and transendothelial migration (1,2).

Objective: Suboptimal magnesium status is likely widespread in the United States and increasing evidence links it to many chronic diseases. Therapeutically addressing magnesium status can be challenging, as higher supplementation often leads to bowel intolerance. This study evaluated the absorption, cellular uptake, and clinical effectiveness of a timed-release formulation containing dimagnesium malate with vitamins B6, B12, and folate (MagSRT TM ) in a standard clinical population. Methods: A standard clinical population of 91 adults participated in a placebo-controlled study carried out at two clinics; 53 individuals received MagSRT TM , containing 500 mg dimagnesium malate and vitamins B6, B12, and folate, while the remaining individuals received a placebo. Baseline serum magnesium, red blood cell (RBC) magnesium, and magnesium status questionnaire scores were collected prior to trial initiation. Serum magnesium was measured 4 and 8 hours after participants ingested 2 supplemental tablets (250 mg magnesium) or 2 placebo tablets. After 30 days, RBC magnesium was evaluated and participants completed the magnesium status questionnaire. A subset of MagSRT TM participants (24) continued the trial for 90 days. Both RBC magnesium and the magnesium status questionnaire were evaluated at 90 days. Results: More than 75% of trial participants presented with suboptimal serum and RBC magnesium status at baseline, while the magnesium status questionnaire predicted 100% of participants to have suboptimal magnesium status. MagSRT TM was well tolerated by 91% of magnesium intervention participants. RBC magnesium increased 6% and 30% over 30 and 90 days, respectively, suggesting magnesium absorption and uptake into red blood cells over time. Overall symptomatology, assessed through a magnesium status questionnaire, improved 28% over 30 days and 63% over 90 days. Conclusion: A standard adult clinical population presented with both qualitative and quantitative evidence of compromised magnesium status at the beginning of the trial. Supplementation with MagSRT TM , a timed-release dimagnesium malate supplement containing vitamins B6, B12, and folate, for at least 30 days significantly improved magnesium status symptoms and increased RBC magnesium with minimal gastrointestinal symptoms.