Human CD99L2 Regulates a Unique Step in Leukocyte Transmigration

Abstract

CD99-like 2 (CD99L2 [L2]) is a highly glycosylated 52-kDa type 1 membrane protein that is important for leukocyte transendothelial migration (TEM) in mice. Inhibiting L2 using function-blocking Ab significantly reduces the recruitment of leukocytes to sites of inflammation in vivo. Similarly, L2 knockout mice have an inherent defect in leukocyte transmigration into sites of inflammation. However, the role of L2 in inflammation has only been studied in mice. Furthermore, the mechanism by which it regulates TEM is not known. To study the relevance to human inflammation, we studied the role of L2 on primary human cells in vitro. Our data show that like PECAM and CD99, human L2 is constitutively expressed at the borders of endothelial cells and on the surface of leukocytes. Inhibiting L2 using Ab blockade or genetic knockdown significantly reduces transmigration of human neutrophils and monocytes across endothelial cells. Furthermore, our data also show that L2 regulates a specific, sequential step of TEM between PECAM and CD99, rather than operating in parallel or redundantly with these molecules. Similar to PECAM and CD99, L2 promotes transmigration by recruiting the lateral border recycling compartment to sites of TEM, specifically downstream of PECAM initiation. Collectively, our data identify a novel functional role for human L2 in TEM and elucidate a mechanism that is distinct from PECAM and CD99.

Figure 1.. Genetic ablation of L2 arrests leukocytes partially through the endothelium in vivo.

The ears of wild-type (WT) and L2 knockout (L2 −/−) mice were treated with croton oil or carrier control. Five hours post stimulation, mice were euthanized, and ears were harvested. Wholemount sections were stained with antibody against PECAM, Collagen IV and Neutrophils. Sections were examined by confocal microscopy. (A) Neutrophil arrest position was ascertained by examination of orthogonally oriented confocal image stacks rotated to give a cross-sectional view. Positions were scored relative to the vessel and basement membrane, as shown in the Schematic. Position 1 (luminal), position 2 (apical), position 3 (Migrating Across EC), position 4 (Arrested at basement membrane), position 5 (Migrating through basement membrane), and position 6 (extravasated). (B) Representative images of WT and L2 ^−/− mouse ears with and without inflammatory stimulus. PMN are green, vessels are red and basement membrane is purple. Images are representative of 3 individual experiments. Scale bar= 50 μm (C) Neutrophil position of arrest was quantified in wild type and CD99L2 knockout mice post inflammatory stimulus. At least 8 fields per sample were imaged and quantified. Quantified data shows the average percent of neutrophil arrest site of 3 individual experiments based on the diagram in (A). Error bars show standard error of mean (SEM) *, P<0.001Student t-test.

Figure 2.. Genetic ablation of endothelial L2 disrupts leukocyte TEM but not leukocyte adherence or rolling in vivo.

FVB/n WT and L2 −/− mice received bone marrow transfers from FVB/n LysM-GFP. (A) WT mice and mice deficient in endothelial L2, respectively, were administered IL-1β intrascrotally and imaged live using intravital microscopy. Still images are sequential frames from a typical video. See videos 1 and 2. Solid white arrows label neutrophils undergoing TEM over time. Open arrows show neutrophils that remain arrested over time. Time stamp shows elapsed time since IL-1β minutes. Scale bar = 25 μm (B-D) Quantification of rolling flux, adhesion, and TEM events in IL-1β stimulated control and endothelial L2 knockout mice. Error bars show SEM. Sequential frames and quantified data are representative of 3 mice per group. *P<0.0001 Student’s t-test.

Figure 3.. L2 is expressed on human leukocytes and along endothelial cell borders.

Leukocytes were prepared from peripheral blood collected from six healthy donors. (A) Lysates of primary human endothelial cells were analyzed using western blot analysis. Antibodies against CD99 and CD99L2 were used to probe for CD99 and CD99L2 respectively. Antibody against CD99 did not recognize CD99L2. Antibody against CD99L2 did not recognize CD99. (B) Antibodies against CD99 and CD99L2 were separately pre-bound to intact HUVEC, washed, and used to immunoprecipitate their respective antigens from the lysates. Anti-CD99 did not pull down CD99L2 and anti-CD99L2 did not pull down CD99. (C) Leukocytes were prepared from peripheral blood collected from six healthy donors. Histograms showing expression of CD99L2 and PECAM on leukocyte subsets. Histograms are representative of the 6 donors. FMO = Fluorescence minus one control. (D) Averaged mean fluorescence intensity (MFI) of leukocyte subsets expressing PECAM and CD99L2 among the 6 donors. (E) Confluent HUVECs were labeled with antibodies against VE-cadherin, PECAM, CD99 or CD99L2. Panels show positive junctional staining for each protein. (F) Confluent HUVEC were activated with TNFα (20 ng/mL in culture media) for four hours and stained for ICAM or L2. ICAM showed increased expression upon TNFα activation while L2 expression did not change. These panels are representative of 3 experiments. Scale bar = 50 μm.

Figure 4.. Inhibition of L2 function reduces TEM of human monocytes and neutrophils across human endothelial cells.

(A) Quantitative TEM assay on TNFα activated HUVECs cultured on collagen in 96-well plates. Antibodies against CD99 and/or L2 were used to block transmigration. Percent transmigration was quantified by counting number of neutrophils beneath the endothelial cell monolayer and dividing by total neutrophils (above and below). (B) Quantification of transmigration assay showing reduction in transmigration of monocytes when blocking CD99 and L2 simultaneously similarly to blocking CD99 and L2 individually. (C) HUVECs were pretreated with adenovirus expressing shRNA against the non-coding region of human L2. Some cultures later received adenovirus expressing full-length L2-eGFP cDNA to re-express it. Western blot shows successful knock down of endothelial human L2 (lane 2) and re-expression of L2 (lane 3). GAPDH was used as loading control. (D) Quantification of TEM assay showed reduced TEM of human monocytes across human endothelial cells when expression of human endothelial L2 is knocked down by shRNA (L2 shRNA) similar to inhibition of L2 function using antibodies against L2 (αL2). TEM is restored upon re-expression of human endothelial L2 (L2shRNA+L2-eGFP). Negative control in all panels is non-blocking anti-VE-Cadherin mAb (αVE-cad). All assays are average transmigration of 3 individual experiments. Error bars show SEM. *P<0.0001 student t-test compared to control.

Figure 5.. Endothelial L2 interacts homophilically with leukocyte L2 to facilitate TEM.

(A) Quantitative TEM assays were run as in figure 4. Antibody against L2 or PECAM and non-blocking control anti-VE-cadherin were preincubated with either EC (closed bar) or monocytes (open bar) and non-bound antibody was washed away before combining the cells. (B) Soluble L2-Fc was generated by expression of a fusion construct of the extracellular domain of L2 to human Fc. Fc portion was confirmed using ELISA. DNAM-Fc served as positive control for ELISA. Soluble L2-Fc was analyzed by western blot to confirm that L2-Fc runs at the appropriate size. DNAM-Fc served as negative control for anti-L2 blot. (C) Quantification of TEM assay showing reduced TEM of both monocytes and PMN in the presence of soluble L2-Fc is comparable to that of blocking TEM with antibodies against L2. (D) Quantification of adhesion in the presence of anti-L2 or soluble L2-Fc. (E) Images of binding assays showing that primary peripheral blood mononuclear cells can bind to wells coated with soluble CD99L2. Blocking CD99L2 on leukocytes prevented binging. Scale bar= 25μm (F) Cells/field were used to measure the binding of leukocytes to the coated wells. Error bars show SEM or 3 independent experiments; *p<0.0001. In (C) and (D), Control was anti-VE-cadherin mAb. In all panels the concentration of mAb or L2-Fc was 20 μg/ml. All TEM assays are averaged percent transmigration of 3 individual experiments. Errors bars show SEM. *P<0.01 (5A; student t-test) relative to control.; *p<0.0001 (5C student t-test) relative to control.

Figure 6.. CD99L2 functions at a step in TEM between steps regulated by PECAM and CD99.

(A) Representative orthogonal images of TEM blocked by antibodies against PECAM, L2, and CD99. VE-cadherin (green) marks endothelial monolayer; PMN stained red by anti-CD18 (B – D) Quantification of sequential TEM assay. Transmigration was inhibited by blocking protein interactions sequentially. The color of the left half of the bar represents the first antibody incubation and color of the right half of the bar represents the second antibody incubation. The heights of the bars represent the final TEM count after the second incubation. (B) TEM resumes to control levels after washing away blocking antibodies and treating with non-blocking anti VE-cadherin. (C) Continued inhibition of TEM was seen when blocking PECAM, CD99 or L2 antibody was washed away as in (B) but added back again. (D) Overall TEM of monocytes when first blocking with the antibody against the indicated molecule, then washing it away and blocking with antibody against the indicated second molecule. Note that all of the incubations described for figures B, C, and D were performed at the same time on the same batches of leukocytes and endothelial cells. The data have been separated into three panels to facilitate description. All data are mean +/− SEM of 3 independent experiments. *p<0.0001 vs. VE-Cad/VE-Cad control; ANOVA. Scale bar= 10 μm

Figure 7.. L2 clustering promotes continued TR to sites of TEM.

(A) Targeted recycling assay was performed on HUVEC monolayers pretreated with control antibody or anti-L2. Monolayers were fixed after 8 – 10 min allowing recruitment of enriched LBRC (red) around migrating leukocyte (green). (B) Quantification of initial LBRC enrichment around migrating leukocytes blocked by anti-L2 is similar to that of control leukocytes transmigrating for the same time. (C) Quantification of TEM assay run in parallel to the targeted recycling assay shows TEM of monocytes was truly blocked. (D-F) Targeted recycling assay run as in A-C but visualizing LBRC movement downstream of L2 blockade. Blocking L2 interaction prevented subsequent targeted recycling of LBRC. Cross-linking L2 (Crosslinked) restores the signal to recruit the LBRC and TEM goes to completion. (See text for details.) Solid arrowheads indicate recruitment of enriched LBRC around migrating monocyte. Open arrowheads indicate lack of recruitment of enriched LBRC. A and D are representative of 3 experiments. Data are mean +/− SEM for 3 independent experiments. *p<0.001 vs. Control; student t-test. Scale bar= 25 μm.