Effect of Glucose on Cell Rolling
Hyperglycemia is mimicked in our experiments by culturing neutrophils (differentiated of HL–60 cells) in glucose rich media. To measure the effect of hyperglycemia on neutrophil rolling adhesion and neutrophil phenotype in general, we tested neutrophils cultured at three different glucose concentrations: 8, 19 and 25 mM. Neutrophil rolling experiments were carried out at a range of physiologically relevant shear stresses (0.17, 0.41, 0.83 and 1.24 Pa) to determine the physiological relevance of any effects observed.
As a control, we observed a linear relationship between mean cell rolling velocity and the applied shear stress as has been reported in the literature.[20] This result holds true at all glucose concentrations. However, under hyperglycemia conditions, higher glucose concentration results in increased cell rolling velocity (Figure 1a). The slope of rolling velocity as a function of glucose concentration also increases, demonstrating that the change in neutrophil rolling velocity is most evident under high shear stress. Therefore, hyperglycemic conditions lead to an increase in neutrophil rolling velocity which may have immunological consequences in vivo such as increasing the difficulty of extravasation into tissues.
There are two possible mechanisms that could lead to such increase in rolling velocity under hyperglycemic exposure: 1. a decrease in PSGL–1 expression on neutrophil surface leading to weaker adhesive interactions with the P-selectin coated surface and/or 2. an increase in cell size where shear flow exerts a greater force to move the cells. Under different glucose concentration at different shear stresses, we did not observe significant changes in cell size (Figure 1b). We used the projection area of a cell as a proxy for cell size. The lack of significant changes of cell size across different glucose concentrations indicates that glucose alone is not changing the phenotypic size of the cell. Furthermore, cells maintain the same size over a large range of shear stresses, indicating that the cells remain rigid and shear stresses used in the experiment are not deforming the cells.
Next, we examine whether the effect of glucose on cell rolling velocity require chronic glucose exposure or is acute glucose shock sufficient to induce such effect. We mimicked the effects of acute hyperglycemia exposure on neutrophils by exposing cells cultured under various glucose concentrations at the time of rolling experiments only (on the minute timescale). Our result indicates that neither the cell rolling velocity (Figure 1c) or cell size (Figure 1d) are affected by acute glucose exposure over a wide range (6.4 mM to 30.8 mM) glucose concentrations. This result shows the importance of chronic glucose exposure (culturing media for days) in affecting neutrophil phenotypes.
Lastly, we examine whether chronic exposure to non-metabolic sugar such as mannitol induces similar effects as glucose. We replaced glucose with mannitol under the exact cell culturing and rolling conditions. The cell rolling velocity increased only slightly (<10%) at high mannitol concentration (Figure 1e) compared to an increase of up to 400% in the case of glucose (Figure 1a). Furthermore, different concentrations of mannitol induced no change in the cell size across the whole range of shear stress (Figure 1f). This result shows that chronic exposure specifically to glucose has a significant effect on cell rolling behaviour. Exposure to mannitol also serves as a control for osmotic pressure on the cell caused by the increase of sugar concentration, which in our case, did not change the size of the cell and hence, insignificant effect on the cell rolling velocity. From these evidences, it is more likely that such large effect of glucose on cell rolling velocity is the result of changes to cell surface protein (i.e. PSGL–1) expression than physical size changes. Such change may be the result of increased growth rate under high glucose concentration. Indeed, the rate of cell growth increases with glucose in the culturing media, which is particularly true when considering that the insulin resistant glucose transporter GLUT1 is expressed in neutrophils. [21]
Effect of Pro-inflammatory Cytokines on Cell rolling
Cells cultured in media containing the diabetic cocktail (hyperglycemic media plus TNF-α, IL–6 and insulin, see Table 1) were compared to those cultured under only hyperglycemic conditions in rolling experiments. We observed a small increase in cell rolling velocity over a range of shear stresses for neutrophils cultured under these conditions (Figure 2a). However, the effect of the diabetic cocktail compounds on cell size is quite significant (Figure 2b). Here, we observe a significant decrease in cell projection area (up to 25%) upon chronic exposure to the diabetic cocktail compared to hyperglycemic exposure alone. When plotting the cell size against mean rolling velocity (Figure 2c), two clusters corresponding to hyperglycemic and diabetic cocktail conditions clearly emerge. The decrease of cell size is generally accompanied by a decrease in rolling velocity, as the shear force acting on a smaller cell is lower. However, the fact that the smaller cells under diabetic cocktail condition roll faster than larger cells under just hyperglycemic condition came as a surprise. Because of our surface passivation and functionalization, only P-selectin are presented on the surface, enabling only adhesive interactions between P-selectin and PSGL–1. We are directly probing the rolling adhesion behaviour due to the P-selectin, PSGL–1 interaction. Hence a further decrease of PSGL–1 expression under the diabetic cocktail condition comparing to hyperglycemic condition is the most likely cause for the observed behaviour. This is also supported by the need for chronic exposure to hyperglycemia and the lack of effect upon acute exposure which does not allow for the time to affect surface protein expression.