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Blood, Vol. 107, Issue 5, 1933-1942, March 1, 2006
Neutrophils, lymphocytes, and monocytes exhibit diverse behaviors in transendothelial and subendothelial migrations under coculture with smooth muscle cells in disturbed flow
Blood Chen et al.
107: 1933
Supplemental materials for: Chen et al
Files in this Data Supplement:
- Table S1. Primer sequences and the number of reaction cycles used for RT-PCR. F, forward; R, reverse (PDF, 35.4 KB)
- Table S2. The effects of neutralizing antibodies against various adhesion molecules and chemokines on the transmigration times of neutrophils, PBLs, and monocytes and their subendothelial movements in each of the areas under VSF and under static condition -
The EC/SMC co-cultures were incubated with neutralizing antibodies (20 µg/ml) against the indicated adhesion molecules and chemokines for 1 hr prior to adhesion and transmigration assays under VSF. EC/SMC treated with IgG or without antibody treatment was used as controls. Purified neutrophils, PBLs, or monocytes were perfused over the EC/SMC under the VSF for 20 min, and video records were made of the adhesion and transmigration behaviors in each of the areas under VSF and under static condition. The transmigration times of purified WBCs, as well as the total distances (T), net distances (N), and randomness (T/N) of subendothelial migration of WBCs were measured by computerized image analysis, as described in Materials and Methods. An example is provided in online supplemental Movie S7 to show inhibition of the subendothelial migration of transmigrated PBLs in area d in the VSF chamber by the use of antibody to VCAM-1. Data shown are mean ± SEM of 25 cells from three independent experiments. *P < 0.05 vs. EC/SMC treated with control IgG or without antibody treatment.
- Figure S1. Inhibitory effects of neutralizing antibodies against various adhesion molecules and chemokines on the adhesion of neutrophils, PBLs, and monocytes to and their transmigration through the ECs co-cultured with SMCs in each of the areas under VSF -
The EC/SMC co-cultures were incubated with neutralizing antibodies (20 µg/ml) against the indicated adhesion molecules and chemokines for 1 hr prior to adhesion and transmigration assays under VSF. EC/SMC treated with IgG or without antibody treatment was used as controls. Purified neutrophils, PBLs, and monocytes were perfused over the EC/SMC for 20 min and the adhesion and transmigration of the WBCs in each of the areas were determined, as described in Materials and Methods. Data represent % inhibition, which was calculated from the number densities (in cells/mm2) of adherent (black bars, plotted upward) or transmigrated (gray bars, plotted downward) WBCs with antibody treatment (CE) and without antibody treatment (CC): % inhibition = 100 [(CC-CE)/CC]. Data are expressed as mean ± SEM, n = 4. *P < 0.05 vs. EC/SMC treated with control IgG or without antibody treatment.
- Figure S2. Identification of the phenotype of SMCs used in the experiments (PDF, 78.7 KB) -
SMCs were cultured in a medium containing 0.5% FBS (a) or 10% FBS as described in Materials and Methods (b) for 24 hr. The cell lysates were analyzed by Western blot using antibodies against SM-MHC, SM
 -actin, h-caldesmon, and calponin (Sigma). An antibody against actin was used as control. Results are representative of duplicate experiments with similar results.
- Video 1. On-line microscopic observation of the flow patterns in the VSF channel (MOV, 2.18 MB)
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Time-lapse video microscope was used to visualize the flow patterns (top view) in the VSF channel, as described in Materials and Methods. Flow is from right to left (indicated by the flashing yellow arrowhead) and is made visible with the marker particles. Flow separation occurs in the region distal to the step. The particles were transported in the bulk flow along the curved streamlines with decreasing velocities towards the wall near the reattachment point. Some of the particles moved in a retrograde direction (upstream) towards the step in the eddy, while others moved forward to rejoin the mainstream with increasing velocities. In the recirculation eddy, the particles moved upstream from the reattachment point with increasing velocities and then decelerated when close to the wall of the step. The particles were carried away from the surface of chamber by upward curved streamlines as they approached the step. A still image of this video clip and the schematic drawing of the side view of the streamlines deduced are shown in Figure 1B. This video clip runs at the same speed (real time) as visualized under microscope.
- Video 2. Determinations of rolling velocity and transmigration time of WBCs on ECs co-cultured with SMCs under the VSF (MOV, 1.21 MB)
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The behavior of purified neutrophils perfused over the ECs co-cultured with SMCs under VSF was recorded as examples to show the methods of determination of their rolling velocity and transmigration time. The video clip showing the top view indicates that the processes of rolling (green circle) and transmigration (red circle) of the WBCs can be readily detected in the VSF chamber using phase contrast microscopy. The WBCs on the EC monolayer were detected as bright round spots, whereas those that had transmigrated and located beneath the monolayer were detected as dark images with irregular shapes. The rolling velocity of WBCs was calculated by dividing the rolling distance by the time elapsed for every 10 s and then averaged. Only WBCs that rolled without stopping during the entire 10-s period were included in the analysis. The transmigration time of WBCs was measured by following individual WBCs from the time they firmly attached on the EC monolayer (phase bright appearance) till they turned phase dark in appearance after passing through the monolayer. The transmigration time of neutrophils in area c was considerably shorter than in area b. This video clip runs at 80 times of normal speed. Pink dash line: reattachment point. Yellow arrow: flow direction.
- Video 3. Transmigration of purified neutrophils across the ECs co-cultured with SMCs in areas c and d under VSF (MOV, 1.08 MB)
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Purified neutrophils were perfused over the EC/SMC under the VSF and the process of their transmigration was recorded with the time-lapse video microscope (top view), as described in “Materials and Methods.” The neutrophils beneath the EC monolayer moved in a relatively random and restricted manner unrelated to the flow direction in areas c (red circle) and d (green circle) in the VSF chamber. The movement of neutrophils near the reattachment point (pink dash line; area c) was more confined, as compared with area d. Parts of this video clip are shown in Figure 3. This video clip runs at 80 times of normal speed. Yellow arrow: flow direction.
- Video 4. Transmigration of purified PBLs across the ECs co-cultured with SMCs in areas c and d under VSF (MOV, 1.09 MB)
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Purified PBLs were perfused over the EC/SMC under the VSF and the process of their transmigration was recorded with the time-lapse video microscope (top view), as described in “Materials and Methods.” The PBLs beneath the EC monolayer became elongated and moved preferentially along the flow direction, which was particularly remarkable in area d (green circle). However, some of the PBLs around the flow reattachment point (pink dash line; area c) displayed a relatively random pattern (red circle). Parts of this video clip are shown in Figure 3. This video clip runs at 80 times of normal speed. Yellow arrow: flow direction.
- Video 5. Transmigration of purified monocytes across the ECs co-cultured with SMCs in areas c and d under VSF (MOV, 1.08 MB)
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Purified monocytes were perfused over the EC/SMC under the VSF and the process of their transmigration was recorded with the time-lapse video microscope (top view), as described in Materials and methods. The purified monocytes beneath the EC monolayer had very low migratory ability in both areas c (red circle) and d (green circle), as compared with the neutrophils (online supplemental Movie S3) and PBLs (online supplemental Movie S4). Parts of this video clip are shown in Figure 3. This video clip runs at 80 times of normal speed. Pink dash line: reattachment point. Yellow arrow: flow direction.
- Video 6. Antibody to ICAM-1 inhibited neutrophil transmigration across the ECs co-cultured with SMCs under VSF (MOV, 1.12 MB)
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The EC/SMC co-cultures were incubated with either the control IgG or the neutralizing antibody against ICAM-1 for 1 hr prior to adhesion and transmigration assays under VSF. The behavior of purified neutrophils perfused over the EC/SMC in the reattachment flow area c was recorded to exemplify the effect of neutralizing antibody against ICAM-1 on the transmigration of neutrophils. The video clip showing the top view indicates that the processes of neutrophil transmigration (red and green circles) can be readily detected using phase contrast microscopy. The neutrophils on the EC monolayer were detected as bright round spots, whereas those that had transmigrated and located beneath the monolayer were detected as dark images with irregular shapes. The transmigration time of neutrophils was measured by following individual neutrophils from the time they firmly attached on the EC monolayer (phase bright appearance) till they turned phase dark in appearance after passing through the monolayer. The transmigration time of neutrophils across the EC monolayers of the co-culture treated with ICAM-1-antibody (red circle) was considerably longer than that treated with control IgG (green circle). This video clip runs at 80 times of normal speed. Pink dash line: reattachment point. Yellow arrow: flow direction.
- Video 7. Antibody to VCAM-1 inhibited PBL migration underneath the ECs co-cultured with SMCs under VSF (MOV, 1.10 MB)
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The EC/SMC co-cultures were incubated with either the control IgG or the neutralizing antibody against VCAM-1 for 1 hr prior to adhesion and transmigration assays under VSF. The behavior of purified PBLs perfused over the EC/SMC in the laminar flow area d was recorded to exemplify the effect of neutralizing antibody against VCAM-1 on the PBL subendothelial migration. The PBLs (green circle) beneath the EC monolayer of the co-culture treated with control IgG were elongated and moved preferentially along the flow direction. In contrast, the EC/SMC treated with antibody against VCAM-1 significantly inhibited the movement of PBLs (red circle) beneath the EC monolayers. This video clip runs at 80 times of normal speed. Yellow arrow: flow direction.
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