RhoA is required for monocyte tail retraction during transendothelial migration

Abstract

Transendothelial migration of monocytes is the process by which monocytes leave the circulatory system and extravasate through the endothelial lining of the blood vessel wall and enter the underlying tissue. Transmigration requires coordination of alterations in cell shape and adhesive properties that are mediated by cytoskeletal dynamics. We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration. By loading monocytes with C3, an inhibitor of RhoA, we found that RhoA was required for transendothelial migration. We then examined individual steps of transmigration to explore the requirement for RhoA in extravasation. Our studies showed that RhoA was not required for monocyte attachment to the endothelium nor subsequent spreading of the monocyte on the endothelial surface. Time-lapse video microscopy analysis revealed that C3-loaded monocytes also had significant forward crawling movement on the endothelial monolayer and were able to invade between neighboring endothelial cells. However, RhoA was required to retract the tail of the migrating monocyte and complete diapedesis. We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction. Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of beta2 integrin in the unretracted tails.

Figure 1.

(A) Transendothelial migration of monocytes is enhanced by MCP-1. The number of monocytes that transmigrated through an IL-1 activated, and endothelial monolayer increased more than fivefold for THP-1 cells and twofold for primary monocytes when MCP-1 was present in the lower chamber of a transwell chamber. The average number of transmigrated cells per field is plotted as the average from three separate experiments. (B) RhoA is required for transendothelial migration of monocytes. Transendothelial migration of primary monocytes electroporated with C3 is plotted as a percent of transmigration activity achieved by monocytes loaded with GST (control).

Figure 2.

RhoA is not required for adhesion to the endothelium. Fluorescently labeled GST- (control) or C3-loaded monocytes were plated onto resting or IL-1–stimulated endothelial monolayers grown in 96 well plates. After 15 min, the cocultures were washed with PBS, and adhesion of the monocytes was quantitated by measuring the fluorescence. Data plotted are the average of triplicate wells from a typical experiment.

Figure 3.

Inhibition of RhoA causes a defect in tail retraction. (A) Primary monocytes were loaded with GST, C3, or RBD and plated on coverslips in serum-free media for 45 min before fixation and staining for F-actin. (B) Monocytes were loaded with GST, C3, or RBD and plated on coverslips in media containing 10% autologous serum for 45 min, followed by staining for F-actin (red) and tubulin (green). Bar, 20 μm.

Figure 4.

Morphology of monocytes loaded with C3 when plated on endothelial cells. (A) Either GST- or C3-loaded monocytes were fluorescently labeled with CMFDA and cultured with IL-1–activated endothelial cells for 45 min before fixation. (B) Either GST- or C3-loaded monocytes were cultured with IL-1–activated endothelial cells for 45 min before fixation and staining for F-actin. Arrows indicate the location of monocytes in the cocultures. Bars, 20 μm.

Figure 5.

Time-lapse analysis of monocyte movement. Monocytes loaded with either GST or C3 were cocultured with IL-1–activated endothelial monolayers, and video microscopy data were collected. Frames were taken every 20 s for 25 min. The sequences shown represent cell movement over 6.5 min. (A) GST-treated monocytes. (B) C3-loaded monocytes. (C) The movement of the x/y center of 10 GST- or C3-loaded monocytes was tracked for 15 frames. Each line represents the movement of a single monocyte over a 5-min period. (D) The ability of monocytes to invade the monolayer or to complete diapedesis was quantitated from analysis of video sequences from three separate experiments. Bars, 20 μm. Supplemental video is available at http://www.jcb.org/cgi/content/full/200103048/DC1.

Figure 6.

p160ROCK is both necessary and sufficient for RhoA-mediated tail retraction. (A) Monocytes were plated onto coverslips in the presence of 10% autologous serum with or without the p160ROCK inhibitor, Y-27632 (10 μM). The graph represents data obtained from monocytes plated onto coverslips with serum containing media and a 1.0–10 μM range of Y-27632 for 45 min. Cells were then fixed, stained for F-actin, and the percentage of cells with tails was scored. The data plotted represent the average from three separate experiments. After 45 min incubation, cells were fixed and stained for F-actin to reveal cell morphology. (B) Monocytes were electroporated with C3 + CA ROCK and compared with those electroporated with C3 + GST. Cells were plated on coverslips with serum-containing media for 45 min before fixation. Cells were stained for F-actin for morphological assessment. (C) Fluorescently labeled monocytes were pretreated with 10 μM Y-27632 for 15 min and then washed and added to activated endothelial monolayers in the presence of 1 μM Y-27632 for 20 min before fixation. Images reveal the morphology of only the monocytes in the coculture. Cells on the left of each pair of images were on top of the endothelial monolayer, as judged by the focal plane. Cells on the right were underneath the monolayer (controls), or caught between neighboring endothelial cells (Y-27632). Bars, 20 μm.

Figure 7.

Microtubules are not the target of C3 for tail retraction. (Top) Monocytes that were loaded with C3 and plated for 20 min before the incubation with 1 μM nocodazole for an additional 20 min. (Bottom) Monocytes that were plated on coverslips in the presence or absence of 3 μM taxol for 45 min before fixation and staining for F-actin (red) and tubulin (green). Cells were then fixed and stained for F-actin (red) and tubulin (green). Bar, 20 μm.

Figure 8.

The contractility inhibitors, BDM and ML-7, do not induce tail formation. Monocytes were plated in serum containing media in the presence of various contractility inhibitors. After 45 min, cells were fixed and stained for F-actin to reveal cell morphology. (A) Control; (B) 20 mM BDM; (C) 20 μM ML-7; (D) 10 μM Y-27632. Bar, 20 μm.

Figure 9.

p160ROCK increases adhesion of monocytes to individual adhesion molecules. (A) For adhesion to activated endothelial monolayers, monocytes were fluorescently labeled, after treatment with Y-27632 or loading with CA ROCK or GST. Adhesion was detected by fluorimetry. For adhesion to ICAM-1 and VCAM, monocytes were added to plates coated with the adhesion molecules in the presence or absence of Y-27632, or after loading with CA ROCK or GST. At times ranging 5–60 min, cells were fixed and stained with Coomassie blue, followed by microscopic analysis. The data represent the number of cells counted in 10 separate fields from triplicate wells in a representative experiment. (B) The morphology of the adherent cells was recorded from the cell samples described in A. Y-27632 dramatically increased cell spreading on both ICAM-1 and VCAM substrates, whereas CA ROCK had little affect.

Figure 10.

C3-loaded monocytes mislocalize β2 integrin to the unretracted tail. Monocytes were loaded with GST or C3 and plated on coverslips in serum-containing media for 45 min before fixation and immunofluorescent localization of β2 integrin. The β2 integrin was concentrated at the leading edge of polarized GST control cells, whereas its expression was reduced in regions where the cell was retracting (arrows). In contrast, high levels of β2 integrin are visible in the unretracted tails of C3-loaded monocytes. Bars, 20 μm.