Neutrophils, lymphocytes, and monocytes exhibit diverse behaviors in transendothelial and subendothelial migrations under coculture with smooth muscle cells in disturbed flow

Abstract

Atherosclerosis develops at regions of the arterial tree exposed to disturbed flow. The early stage of atherogenesis involves the adhesion of leukocytes (white blood cells [WBCs]) to and their transmigration across endothelial cells (ECs), which are located in close proximity to smooth muscle cells (SMCs). We investigated the effects of EC/SMC coculture and disturbed flow on the adhesion and transmigration of 3 types of WBCs (neutrophils, peripheral blood lymphocytes [PBLs], and monocytes) using our vertical-step flow (VSF) chamber, in which ECs were cocultured with SMCs in collagen gels. Such coculture significantly increased the adhesion and transmigration of neutrophils, PBLs, and monocytes under VSF, particularly in the reattachment area, where the rolling velocity of WBCs and their transmigration time were decreased, as compared with the other areas. Neutrophils, PBLs, and monocytes showed different subendothelial migration patterns under VSF. Their movements were more random and shorter in distance in the reattachment area. Coculture of ECs and SMCs induced their expressions of adhesion molecules and chemokines, which contributed to the increased WBC adhesion and transmigration. Our findings provide insights into the mechanisms of WBC interaction with the vessel wall (composed of ECs and SMCs) under the complex flow environments found in regions of prevalence for atherogenesis.

Figure 1.

Schematic configuration and flow pattern in VSF channel. (A) Schematic diagram of the flow channel and test section. ECs were seeded on the collagen gel in the presence of SMCs. The dimensions of the test section are described in “Materials and methods.” The thickness (t) of the gel was measured to be approximately 0.03 mm. (B, top) Phase-contrast photomicrograph (top view) of experimental flow patterns in the VSF channel. (B, bottom) Schematic drawing of the side view of the streamlines in the VSF deduced from the top-view photomicrograph. Flow is from left to right and made visible with marker particles, as described in “Materials and methods.” Flow separation occurs in the region distal to the step, forming 4 specific flow areas: (a) the stagnant flow area, (b) the area of the recirculation eddy, (c) the reattachment flow area, and (d) the area where the flow has developed again to laminar flow, as denoted in the bottom part of the figure. The span of the areas (represented as length) and the mean shear stress (MSS) in each of the areas were measured as indicated. The movie for online microscopic observation of the flow patterns is provided in Video S1.

Figure 2.

Coculture of ECs with SMCs increased the adhesion of neutrophils, PBLs, and monocytes and their subsequent transmigration under VSF, especially in the vicinity of flow reattachment. ECs were seeded on the collagen gel in the absence (EC/Gel) or presence (EC/SMCs) of SMCs. Purified neutrophils (A,D,G), PBLs (B,E,H), or monocytes (C,F,I) were perfused over the EC monolayers for 20 minutes under the VSF, and their adhesion (A-C) to and transmigration (D-F) across the EC monolayer were counted in each of the areas a, b, c, and d, as described in “Materials and methods.” The transmigration-adherence ratio (G-I) was calculated from the numbers of transmigrated cells relative to adherent cells. As positive controls, EC/Gel and EC/SMC treated with IL-1β (5 ng/mL) for 4 hours caused increases in WBC adhesion and transmigration (data not shown). Data are expressed as mean ± SEM from 3 independent experiments. ^*P < .05 versus control EC/Gel in the same area. ^#P < .05 for comparing area c with areas a, b, and d (P < .05 for each comparison).

Figure 3.

Tracings of the subendothelial migration paths of the transmigrated neutrophils, PBLs, and monocytes in each of the areas under VSF and under static condition. Purified neutrophils, PBLs, and monocytes were perfused over the EC/SMC coculture, and the migration of arrested WBCs was traced for 20 minutes in areas a, b, c, and d under VSF or under static condition after their transmigration across the EC monolayer to the underside. This figure shows the results of a representative experiment containing 8 cells transmigrated in area a and 15 cells transmigrated in areas b, c, and d, as well as under static condition. The positions of the centers of the cells were determined at 20-second intervals from 0 to 20 minutes, and their paths of travel were processed with image analysis software NIH Image 1.60b7. Examples for online microscopic observations of the movements of transmigrated neutrophils, PBLs, and monocytes in areas c and d are provided in Videos S3, S4, and S5, respectively.

Figure 4.

Coculture of ECs and SMCs induced their expressions of adhesion molecules and chemokines relevant to WBC recruitment. ECs were seeded on the collagen gel in the absence (EC/Gel) or presence (EC/SMC) of SMCs. SMCs were also embedded in the collagen gels without coculture of ECs (NC/SMC). The cells cultured on Petri dishes were used as controls (Control). In additional experiments, EC/Gel and EC/SMC were treated with IL-1β for 4 hours. (A) Specificity of ECs and SMCs. ECs and SMCs were purified from their cocultures, and their specificity was examined by Western blot analysis of their lysates using antibodies against PECAM-1 and SMα-actin as markers for ECs and SMCs, respectively. (B-C) Coculture of ECs and SMCs induced their expressions of adhesion molecules and chemokines. The mRNA levels of selected adhesion molecules and chemokines in ECs (B) and SMCs (C) from different experimental conditions were determined by RT-PCR analysis, as described in “Materials and methods.” Amplification of cDNA was performed in parallel samples using human GAPDH primers. Results are representative of triplicate experiments with similar results. (D) Coculture with SMCs increased ICAM-1, VCAM-1, and E-selectin expressions on ECs. ECs isolated from EC/SMC or EC/Gel were analyzed by flow cytometry, as described in “Materials and methods.” The EC/SMC shows a shift of fluorescence intensity profile for ICAM-1, VCAM-1, and E-selectin, as compared with the EC/Gel. ECs incubated with only FITC-conjugated antibody were used as blank controls. Results are representative of duplicate experiments with similar results. (E) Coculture of ECs and SMCs increased the release of chemokines into conditioned media and collagen gels. The soluble forms of the chemokines in both the media and collagen gels of EC/Gel, NC/SMC, and EC/SMC experiments were determined by ELISA assay, as described in “Materials and methods.” The collagen gel incubated with only the media was used as negative controls (NC/Gel). The results shown are mean ± SEM from 3 independent experiments. ^*P < .05 for EC/SMC versus NC/Gel, EC/Gel, and NC/SMC samples.

Figure 5.

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 cocultured with SMCs in the reattachment flow area c under VSF. The EC/SMC cocultures were incubated with neutralizing antibodies (20 μg/mL) against the indicated adhesion molecules and chemokines for 1 hour prior to adhesion and transmigration assays under VSF. EC/SMC treated with IgG or without antibody treatment were used as controls. Purified neutrophils, PBLs, and monocytes were perfused over the EC/SMC for 20 minutes, and the adhesion and transmigration of the WBCs in the reattachment flow area c were determined, as described in “Materials and methods.” Data represent percentage of inhibition, which was calculated from the number densities (in cells/mm²) of adherent (▪) or transmigrated () WBCs with antibody treatment (C_E) and without antibody treatment (C_C): % inhibition = 100 [(C_C - C_E)/C_C]. Data are expressed as mean ± SEM, n = 4. ^*P < .05 versus EC/SMC treated with control IgG or without antibody treatment.