Endothelial Src kinase regulates membrane recycling from the lateral border recycling compartment during leukocyte transendothelial migration

Abstract

When leukocytes cross endothelial cells during the inflammatory response, membrane from the recently described lateral border recycling compartment (LBRC) is selectively targeted around diapedesing leukocytes. This "targeted recycling" is critical for leukocyte transendothelial migration. Blocking homophilic PECAM interactions between leukocytes and endothelial cells blocks targeted recycling from the LBRC and blocks diapedesis. However, the cellular signaling pathways that trigger targeted recycling are not known. We show that targeted recycling from the LBRC is dependent on Src kinase. The selective Src kinase inhibitor PP2 blocked targeted recycling and blocked diapedesis by over 70%. However, Src kinase inhibition did not affect the structure or normal constitutive recycling of membrane from the LBRC in the absence of leukocytes. PECAM, a Src kinase substrate, traffics between the LBRC and the endothelial surface at the cell border. However, virtually all of the PECAM in the cell that was phosphorylated on tyrosine residues was found in the LBRC. These findings demonstrate that Src kinase activity is critical for the targeted recycling of membrane from the LBRC to the site of transendothelial migration and that the PECAM in the LBRC is qualitatively different from the PECAM on the surface of endothelial cells.

Figure 1. Transmigration requires src kinase activity

TEM was inhibited by PP2 in a dose dependent manner. Genestein also inhibited TEM at concentrations greater than 100 uM. The data are shown as mean transmigration ± SEM, of 3 fields counted from 2 replicates per condition from three separate experiments. PP2 treatment differed significantly from the nonblocking PP3 condition. Higher concentrations of Genistein blocked transmigration significantly as compared to PP3 (** p<0.005, * p < 0.001, based on one-way ANOVA and Bonferonni correction to compare treated conditions to PP3 control).

Figure 2. Targeted recycling of PECAM from the LBRC is Src kinase dependent

The recycling PECAM assay was performed in the presence of transmigrating monocytes. Overlay showing a monocyte interacting with the EC junction, recycled PECAM (left panel) is shown in red, monocytes (right panel) are shown in green. Orthogonal section through the field is shown below the overlay, to demonstrate the extent of diapedesis. (A) In untreated HUVEC monolayers, PECAM recycling from the LBRC is enriched around the transmigrating monocyte (left). The monocyte shown is actively crossing the HUVEC monolayer (arrow). (B) In PP2 treated monolayers, there is no PECAM enrichment around the monocyte (left), and the orthogonal section shows that the monocyte is blocked on the apical side of the endothelial junction (arrow). Images shown are representative of hundreds of leukocytes observed in 4 independent experiments. (C) Quantitative analysis of monocytes interacting with the endothelial cell junctions. The total number of monocytes that had rings of recycling PECAM around them was counted per field (Materials and Methods). Virtually all of the transmigrating monocytes showed rings of enriched PECAM. In comparison there is an inhibition of targeted recycling of PECAM in the PP2 and Genistein treated HUVEC. Endothelial cells treated with anti-PECAM ab (177) are shown as a control. The data are shown as the average % monocytes with associated PECAM enrichment rings per HPF ± SEM of 30 fields analyzed from each of 2 replicates per condition across 4 separate experiments. PP2 and Genistein treatment (*) differed significantly from the untreated control (p < 0.005) and 177 (**) also blocked ring formation significantly (p < 0.001) as determined by comparing PP2, Genistein and 177 to untreated control via one-way ANOVA and bonferonni correction).

Figure 3. PP2 has no effect on constitutive recycling of PECAM from the LBRC

(A) Sample images of HUVEC monolayers from a recycling assay where PECAM becomes fluorescent as it reaches the cell surface (Materials and Methods). HUVEC were untreated (left) or treated with 100μM PP2 for 2hrs (right). Recycled PECAM shown at the timepoints indicated was quantified to determine the kinetics of recycled PECAM. Untreated cells (solid line, squares) and PP2 treated cells (dotted line, circles) show no differences in constitutive recycling of PECAM from the LBRC (p < 0.005 as determined by nonlinear curve fit and F test). Images shown are representative of 20 fields analyzed per condition for each of 3 independent experiments. The data are an average of normalized curves from 3 separate experiments (where each experiment consisted of 20 fields analyzed for 2 replicates per condition.

Figure 4. Tyrosine phosphorylated PECAM is selectively enriched in the LBRC

(A) HUVEC were cultured in standard HS media (left half of blot) or media containing FBS (right half of blot) in order to enhance baseline PECAM phosphorylation levels. The PECAM on the surface and in the compartment was immunoprecipitated (Materials and Methods). Samples of the total lysate (total), surface fraction (surface), and LBRC fraction (internal) were analyzed by western blots using anti-PECAM and anti-phosphotyrosine ab (P-Tyr). A sample from the final round of surface PECAM immunoprecipitation is shown (test IP) to ensure that all the surface tyrosine phosphorylated PECAM had been depleted. The ratio of tyrosine phosphorylated PECAM to total PECAM is shown each sample. In both HS and FBS cultured cells, the LBRC fraction had a greater amount of tyrosine phosphorylated PECAM. (B) The assay was repeated on HS cultured HUVEC and the surface PECAM was exhaustively immunoprecipitated (Materials and Methods) using a more stringent method (lanes 1-4). By the fourth round of immunoprecipitation, all of the surface PECAM has been depleted from the lysate (lane 4). The PECAM in the LBRC is preferentially tyrosine phosphorylated (final lane). Blots shown are representative of 3 independent experiments.