Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flow

Abstract

Flowing leukocytes roll on P-selectin that is mobilized from secretory granules to the surfaces of endothelial cells after stimulation with histamine or thrombin. Before it is internalized, P-selectin clusters in clathrin-coated pits, which enhances its ability to support leukocyte rolling. We found that thrombin and histamine induced comparable exocytosis of P-selectin on endothelial cells. However, compared with histamine, thrombin decreased the recruitment of P-selectin into clathrin-coated pits, slowed the internalization of P-selectin, and reduced the number and stability of neutrophils rolling on P-selectin. Significantly more RhoA was activated in thrombin- than in histamine-stimulated endothelial cells. Inhibitors of RhoA or its effector, Rho kinase, reversed thrombin's ability to inhibit the internalization and adhesive function of P-selectin in endothelial cells. Experiments with transfected cells confirmed that the inhibitory actions of thrombin and Rho kinase on P-selectin required its cytoplasmic domain. Thus, a signaling event affects both the function and clearance of a protein that enters the constitutive clathrin-mediated endocytic pathway.

Figure 1.

Thrombin stimulation of HUVEC reduces the internalization rate of P-selectin and the number of neutrophils rolling on P-selectin. (A) HUVEC were either unstimulated (time 0) or were stimulated with thrombin or histamine for 4, 9, or 14 min. The cells were rapidly chilled, and the steady-state levels of P-selectin on the cell surface were measured by specific binding of a saturating concentration of ¹²⁵I-labeled mAb S12. The data represent the mean ± SEM of three experiments. (B) HUVEC were stimulated with histamine, thrombin, TRAP, or both thrombin and histamine. After 4 min, the internalization rate of P-selectin was determined by the ability of acidic buffer to remove surface-bound ¹²⁵I-mAb G1 as described in Materials and methods. The cell-bound radioactivity remaining at each time point represents the amount of internalized P-selectin, and is plotted as a percentage of the initial cell-bound radioactivity. The data represent the mean ± SEM of at least four experiments. The internalization rate determined from the initial slope was significantly higher for HUVEC stimulated with histamine than for HUVEC stimulated with thrombin or with thrombin plus histamine (P < 0.02). (C) Neutrophils were perfused over confluent HUVEC. At the indicated time, histamine, thrombin, TRAP, or both thrombin and histamine was perfused into the flow chamber. The number of rolling neutrophils was measured at 1-min intervals. The data represent the mean ± SEM of 10 experiments. At each time point after addition of agonist, the number of rolling neutrophils was significantly greater on HUVEC stimulated with histamine than on HUVEC stimulated with thrombin or with thrombin plus histamine (P < 0.05). (D) HUVEC were stimulated with histamine or thrombin, and neutrophils were immediately perfused into the flow chamber at the indicated wall shear stress. After 5 min, the number of rolling neutrophils was measured. The data represent the mean ± SEM of at least four experiments. (E) HUVEC were stimulated with histamine or thrombin, and neutrophils were immediately perfused into the flow chamber at 1 dyn/cm² in the presence or absence of the indicated mAb. After 5 min, the number of rolling neutrophils was measured. The data represent the mean ± SEM of at least seven experiments. (F) Neutrophils incubated with histamine or thrombin were perfused over adsorbed sP-selectin (60 sites/μm²) in the presence or absence of the indicated mAb. After 5 min, the number of rolling neutrophils was measured. The data represent the mean ± SEM of three experiments. Significant p-values as measured by the unpaired t test are indicated.

Figure 2.

Neutrophils roll less stably on thrombin- than on histamine-stimulated HUVEC. (A) Frame by frame velocities of representative neutrophils rolling on thrombin- or histamine-stimulated HUVEC at 1 dyn/cm² in the presence or absence of anti–β2 integrin mAb IB4. (B and C) Mean velocities and variances of velocities for neutrophil populations rolling on thrombin- or histamine-activated HUVEC at 1 dyn/cm² in the presence or absence of IB4. Significant p-values as measured by the unpaired t test are indicated. The data represent the mean ± SEM for 20–50 cells, each measured for up to 5 s.

Figure 3.

Less cell surface P-selectin colocalizes with α-adaptin in thrombin- than in histamine-stimulated HUVEC. HUVEC preincubated with isotonic or hypertonic medium were stimulated with thrombin or histamine for 4 min. They were fixed and incubated with biotinylated polyclonal antibodies to P-selectin, followed by streptavidin conjugated to Alexa-488. After permeabilization, the cells were incubated with an mAb to α-adaptin or to caveolin-1, followed by donkey anti–mouse Ig conjugated to Cy-3.Using a confocal microscope, an optical section at the middle and apical portion of each cell was examined for staining of P-selectin (green) or α-adaptin or caveolin-1 (red). (A) Representative images revealed partial colocalization of α-adaptin with P-selectin (yellow) in histamine-stimulated HUVEC but less colocalization in thrombin-stimulated HUVEC. (B) The degree of colocalization was quantified by measuring the percentage of green pixels (P-selectin) that colocalized with red pixels (α-adaptin or caveolin-1) as described in Materials and methods. The data represent the mean ± SEM of at least three experiments, with at least five cells counted in each experiment. Significant p-values as measured by the unpaired t test are indicated.

Figure 3.

Less cell surface P-selectin colocalizes with α-adaptin in thrombin- than in histamine-stimulated HUVEC. HUVEC preincubated with isotonic or hypertonic medium were stimulated with thrombin or histamine for 4 min. They were fixed and incubated with biotinylated polyclonal antibodies to P-selectin, followed by streptavidin conjugated to Alexa-488. After permeabilization, the cells were incubated with an mAb to α-adaptin or to caveolin-1, followed by donkey anti–mouse Ig conjugated to Cy-3.Using a confocal microscope, an optical section at the middle and apical portion of each cell was examined for staining of P-selectin (green) or α-adaptin or caveolin-1 (red). (A) Representative images revealed partial colocalization of α-adaptin with P-selectin (yellow) in histamine-stimulated HUVEC but less colocalization in thrombin-stimulated HUVEC. (B) The degree of colocalization was quantified by measuring the percentage of green pixels (P-selectin) that colocalized with red pixels (α-adaptin or caveolin-1) as described in Materials and methods. The data represent the mean ± SEM of at least three experiments, with at least five cells counted in each experiment. Significant p-values as measured by the unpaired t test are indicated.

Figure 4.

Disruption of clathrin-coated pits with hypertonic medium decreases rolling of selectin ligand-coupled microspheres on both histamine- and thrombin-activated HUVEC. HUVEC preincubated with isotonic or hypertonic medium for 15 min were stimulated with thrombin or histamine for 4 min. The cells were maintained in the same buffer. The internalization rate of P-selectin (A) or the number of rolling 2-GSP-6–coupled microspheres (B) was measured. The data represent the mean ± SEM of at least three experiments. Significant p-values as measured by the unpaired t test are indicated.

Figure 5.

Thrombin-stimulated HUVEC activate more RhoA but not more p38 than histamine-stimulated HUVEC. (A) Confluent HUVEC were untreated or stimulated with thrombin or histamine for 5 min. Cell lysates were subjected to Western blotting with antibodies to p38 MAPK or to phospho-p38 MAPK. A representative immunoblot from one experiment is depicted. For clarity, only the relevant bands on the blot are shown. (B) Densitometry of stained protein bands was used to quantify the levels of total p38 and phospho-p38. The percentage of phospho-p38 relative to total p38 in stimulated HUVEC was normalized to that of untreated cells and expressed as relative p38 activity. The data represent the mean ± SEM of three experiments. No statistically significant differences (P < 0.05 with unpaired t test) among treatment groups were observed. (C) Confluent HUVEC preincubated with or without 2.5 μg/ml C3T for 16 h were stimulated with thrombin or histamine for 10 min. The amount of activated and total RhoA in cell lysates was measured as described in Materials and methods. A representative immunoblot from one experiment is depicted. For clarity, only the relevant bands on the blot are shown. (D) The percentage of activated RhoA relative to total RhoA in stimulated HUVEC was normalized to that of untreated cells and expressed as relative RhoA activity. The data represent the mean ± SEM of five experiments. Significant p-values as measured by the unpaired t test are indicated.

Figure 5.

Thrombin-stimulated HUVEC activate more RhoA but not more p38 than histamine-stimulated HUVEC. (A) Confluent HUVEC were untreated or stimulated with thrombin or histamine for 5 min. Cell lysates were subjected to Western blotting with antibodies to p38 MAPK or to phospho-p38 MAPK. A representative immunoblot from one experiment is depicted. For clarity, only the relevant bands on the blot are shown. (B) Densitometry of stained protein bands was used to quantify the levels of total p38 and phospho-p38. The percentage of phospho-p38 relative to total p38 in stimulated HUVEC was normalized to that of untreated cells and expressed as relative p38 activity. The data represent the mean ± SEM of three experiments. No statistically significant differences (P < 0.05 with unpaired t test) among treatment groups were observed. (C) Confluent HUVEC preincubated with or without 2.5 μg/ml C3T for 16 h were stimulated with thrombin or histamine for 10 min. The amount of activated and total RhoA in cell lysates was measured as described in Materials and methods. A representative immunoblot from one experiment is depicted. For clarity, only the relevant bands on the blot are shown. (D) The percentage of activated RhoA relative to total RhoA in stimulated HUVEC was normalized to that of untreated cells and expressed as relative RhoA activity. The data represent the mean ± SEM of five experiments. Significant p-values as measured by the unpaired t test are indicated.

Figure 6.

Inhibiting Rho kinase in thrombin-stimulated HUVEC increases the internalization rate of P-selectin and the number of neutrophils rolling on P-selectin. (A) HUVEC preincubated with or without 2.5 μg/ml C3T for 16 h were stimulated with histamine or thrombin. After 4 min, the internalization rate of P-selectin was measured. The internalization rate determined from the initial slope was significantly higher for HUVEC stimulated with thrombin in the presence of C3T than for HUVEC stimulated with thrombin in the absence of C3T (P < 0.02). (B) HUVEC preincubated with or without 10 μM Y27632 for 1 h were stimulated with histamine or thrombin. After 4 min, the internalization rate of P-selectin was measured. The internalization rate determined from the initial slope was significantly higher for HUVEC stimulated with thrombin in the presence of Y27632 than for HUVEC stimulated with thrombin in the absence of Y27632 (P < 0.01). (C) HUVEC preincubated with or without C3T or Y27632 were stimulated with histamine or thrombin. After 5 min, the number of rolling neutrophils was measured. (D and E) HUVEC preincubated with or without C3T or Y27632 were stimulated with thrombin. After 5 min, the mean rolling velocity and the variance of velocity of neutrophils was measured. For all panels, the data represent the mean ± SEM of at least three experiments.

Figure 7.

Thrombin- and Rho kinase–mediated inhibition of the internalization rate and adhesive function of P-selectin requires the cytoplasmic domain of P-selectin. Transfected CHO cells expressing matched densities of wild-type or tail-truncated P-selectin (50–60 sites/μm²) were preincubated with or without 10 μM Y27632 for 1 h and treated with 4 U/ml thrombin for 4 min. (A) The internalization rate of P-selectin was measured. The internalization rate determined from the initial slope was significantly higher for CHO cells stimulated with thrombin in the presence of Y27632 than for CHO cells stimulated with thrombin in the absence of Y27632 (P < 0.05). (B) The number of rolling neutrophils was measured. (A and B) The data represent the mean ± SEM of at least five experiments. Significant p-values as measured by the unpaired t test are indicated.