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Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration.

Watson RL, Buck J, Levin LR, Winger RC, Wang J, Arase H, Muller WA - J. Exp. Med. (2015)

Bottom Line: How CD99 signals during this process remains unknown.We show that during TEM, endothelial cell (EC) CD99 activates protein kinase A (PKA) via a signaling complex formed with the lysine-rich juxtamembrane cytoplasmic tail of CD99, the A-kinase anchoring protein ezrin, and soluble adenylyl cyclase (sAC).PKA then stimulates membrane trafficking from the lateral border recycling compartment to sites of TEM, facilitating the passage of leukocytes across the endothelium.

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Affiliation: Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60208.

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CD99 engagement stimulates a second wave of TR to sites of transmigration. (a) TR assays were performed (see Materials and methods) in presence of either anti-CD99 mAb (IgG1) or mouse IgG1 (control). Arrows denote LBRC enrichment. Insets show xz-orthogonal view. (b) Quantification of results. Values represent percent of leukocytes per field of view. (c) LBRC enrichment was quantified as previously described (Mamdouh et al., 2003). In brief, the maximum fluorescence intensity (MFI) around the leukocyte was divided by the MFI of staining along neighboring junctions not in contact with leukocytes. Values greater than one denote enrichment. (d) Quantitative TEM assays were performed in parallel to ensure that anti-CD99 blocked TEM. (e) Two-color TR assays (see Materials and methods) were performed in the continuous presence of anti-CD99 (IgG1) or mouse IgG1 (control). DyLight550 GαM IgG2a (550-GαM IgG2a, first antibody) labeled LBRC membrane (labeled with nonblocking mouse anti-PECAM IgG2a antibody, clone P1.1) that trafficked before the CD99-dependent step of TEM. DyLight488 GαM IgG2a (GαM IgG2a, second antibody) labeled LBRC membrane (labeled with P1.1 antibody) delivered after leukocytes have been arrested by anti-CD99. Time denotes minutes incubated with 488-GαM2a. (f) Quantification of LBRC enrichment was performed for both antibodies as a function of time. In brief, the average MFI around the leukocyte was divided by the MFI of neighboring junctional staining for each antibody. (g) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin (nonblocking, control) or anti-CD99. After 50 min, either GαM (GαM IgG, cross-linking secondary antibody, XL) or goat anti–rabbit (GαRb IgG, control) was added to the cells for 10 min. (h) Two-color TR assays were performed in the presence of anti-CD99 mAb (IgG1, as described above). Before incubation of samples with 488-GαM IgG2a at 37°C, cells were treated with either GαM IgG1 (CD99-specific cross-linking antibody) or GαRb IgG for 0 or 5 min. (i) Degree of LBRC enrichment was quantified. Bars, 10 µm. Images are representative of three (a and h) or four (e) independent experiments. Data represent the mean value of three (b–d, f, and i) or four (g) independent experiments. Error bars denote SEM. **, P < 0.01; ***, P < 0.001; Student’s t test).
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fig1: CD99 engagement stimulates a second wave of TR to sites of transmigration. (a) TR assays were performed (see Materials and methods) in presence of either anti-CD99 mAb (IgG1) or mouse IgG1 (control). Arrows denote LBRC enrichment. Insets show xz-orthogonal view. (b) Quantification of results. Values represent percent of leukocytes per field of view. (c) LBRC enrichment was quantified as previously described (Mamdouh et al., 2003). In brief, the maximum fluorescence intensity (MFI) around the leukocyte was divided by the MFI of staining along neighboring junctions not in contact with leukocytes. Values greater than one denote enrichment. (d) Quantitative TEM assays were performed in parallel to ensure that anti-CD99 blocked TEM. (e) Two-color TR assays (see Materials and methods) were performed in the continuous presence of anti-CD99 (IgG1) or mouse IgG1 (control). DyLight550 GαM IgG2a (550-GαM IgG2a, first antibody) labeled LBRC membrane (labeled with nonblocking mouse anti-PECAM IgG2a antibody, clone P1.1) that trafficked before the CD99-dependent step of TEM. DyLight488 GαM IgG2a (GαM IgG2a, second antibody) labeled LBRC membrane (labeled with P1.1 antibody) delivered after leukocytes have been arrested by anti-CD99. Time denotes minutes incubated with 488-GαM2a. (f) Quantification of LBRC enrichment was performed for both antibodies as a function of time. In brief, the average MFI around the leukocyte was divided by the MFI of neighboring junctional staining for each antibody. (g) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin (nonblocking, control) or anti-CD99. After 50 min, either GαM (GαM IgG, cross-linking secondary antibody, XL) or goat anti–rabbit (GαRb IgG, control) was added to the cells for 10 min. (h) Two-color TR assays were performed in the presence of anti-CD99 mAb (IgG1, as described above). Before incubation of samples with 488-GαM IgG2a at 37°C, cells were treated with either GαM IgG1 (CD99-specific cross-linking antibody) or GαRb IgG for 0 or 5 min. (i) Degree of LBRC enrichment was quantified. Bars, 10 µm. Images are representative of three (a and h) or four (e) independent experiments. Data represent the mean value of three (b–d, f, and i) or four (g) independent experiments. Error bars denote SEM. **, P < 0.01; ***, P < 0.001; Student’s t test).

Mentions: Abolishing PECAM function has been previously shown to inhibit the targeted enrichment of LBRC membrane to sites of TEM, thus preventing TEM (Mamdouh et al., 2003). Because CD99 is also a resident molecule of the LBRC and it functions downstream of PECAM during TEM, we hypothesized that CD99 is required for a subsequent step in TR. To test this, we used a specialized technique to monitor LBRC membrane movement during TEM, known as the TR assay (see Materials and methods; Mamdouh et al., 2003; Mamdouh et al., 2008). In brief, this technique utilizes PECAM as a surrogate marker for the LBRC. We used a Fab fragment of a nonfunctional blocking antibody (mouse anti–human PECAM, clone P1.1 (Liao et al., 1995), to prebind PECAM in the LBRC. Any P1.1 Fab on the surface is saturated with unlabeled anti–mouse IgG at 4°C. We are then able to track the movement of the LBRC during TEM using the identical anti–mouse IgG conjugated to a fluorophore. To test our hypothesis, target recycling assays were performed using ECs pretreated with either anti-CD99 mAb or control IgG. Despite blocking transmigration with anti-CD99, a similar percentage of leukocytes were enriched with membrane from the LBRC (Fig. 1, a–d). (Definition of LBRC enrichment is a >1 fold increase in the staining around a leukocyte compared with a neighboring junction not in contact with the leukocyte.) From this, we concluded that CD99 is not required for the initiation of TR of the LBRC. However, this is not surprising given the phenotype of anti-CD99 blockade.


Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration.

Watson RL, Buck J, Levin LR, Winger RC, Wang J, Arase H, Muller WA - J. Exp. Med. (2015)

CD99 engagement stimulates a second wave of TR to sites of transmigration. (a) TR assays were performed (see Materials and methods) in presence of either anti-CD99 mAb (IgG1) or mouse IgG1 (control). Arrows denote LBRC enrichment. Insets show xz-orthogonal view. (b) Quantification of results. Values represent percent of leukocytes per field of view. (c) LBRC enrichment was quantified as previously described (Mamdouh et al., 2003). In brief, the maximum fluorescence intensity (MFI) around the leukocyte was divided by the MFI of staining along neighboring junctions not in contact with leukocytes. Values greater than one denote enrichment. (d) Quantitative TEM assays were performed in parallel to ensure that anti-CD99 blocked TEM. (e) Two-color TR assays (see Materials and methods) were performed in the continuous presence of anti-CD99 (IgG1) or mouse IgG1 (control). DyLight550 GαM IgG2a (550-GαM IgG2a, first antibody) labeled LBRC membrane (labeled with nonblocking mouse anti-PECAM IgG2a antibody, clone P1.1) that trafficked before the CD99-dependent step of TEM. DyLight488 GαM IgG2a (GαM IgG2a, second antibody) labeled LBRC membrane (labeled with P1.1 antibody) delivered after leukocytes have been arrested by anti-CD99. Time denotes minutes incubated with 488-GαM2a. (f) Quantification of LBRC enrichment was performed for both antibodies as a function of time. In brief, the average MFI around the leukocyte was divided by the MFI of neighboring junctional staining for each antibody. (g) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin (nonblocking, control) or anti-CD99. After 50 min, either GαM (GαM IgG, cross-linking secondary antibody, XL) or goat anti–rabbit (GαRb IgG, control) was added to the cells for 10 min. (h) Two-color TR assays were performed in the presence of anti-CD99 mAb (IgG1, as described above). Before incubation of samples with 488-GαM IgG2a at 37°C, cells were treated with either GαM IgG1 (CD99-specific cross-linking antibody) or GαRb IgG for 0 or 5 min. (i) Degree of LBRC enrichment was quantified. Bars, 10 µm. Images are representative of three (a and h) or four (e) independent experiments. Data represent the mean value of three (b–d, f, and i) or four (g) independent experiments. Error bars denote SEM. **, P < 0.01; ***, P < 0.001; Student’s t test).
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fig1: CD99 engagement stimulates a second wave of TR to sites of transmigration. (a) TR assays were performed (see Materials and methods) in presence of either anti-CD99 mAb (IgG1) or mouse IgG1 (control). Arrows denote LBRC enrichment. Insets show xz-orthogonal view. (b) Quantification of results. Values represent percent of leukocytes per field of view. (c) LBRC enrichment was quantified as previously described (Mamdouh et al., 2003). In brief, the maximum fluorescence intensity (MFI) around the leukocyte was divided by the MFI of staining along neighboring junctions not in contact with leukocytes. Values greater than one denote enrichment. (d) Quantitative TEM assays were performed in parallel to ensure that anti-CD99 blocked TEM. (e) Two-color TR assays (see Materials and methods) were performed in the continuous presence of anti-CD99 (IgG1) or mouse IgG1 (control). DyLight550 GαM IgG2a (550-GαM IgG2a, first antibody) labeled LBRC membrane (labeled with nonblocking mouse anti-PECAM IgG2a antibody, clone P1.1) that trafficked before the CD99-dependent step of TEM. DyLight488 GαM IgG2a (GαM IgG2a, second antibody) labeled LBRC membrane (labeled with P1.1 antibody) delivered after leukocytes have been arrested by anti-CD99. Time denotes minutes incubated with 488-GαM2a. (f) Quantification of LBRC enrichment was performed for both antibodies as a function of time. In brief, the average MFI around the leukocyte was divided by the MFI of neighboring junctional staining for each antibody. (g) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin (nonblocking, control) or anti-CD99. After 50 min, either GαM (GαM IgG, cross-linking secondary antibody, XL) or goat anti–rabbit (GαRb IgG, control) was added to the cells for 10 min. (h) Two-color TR assays were performed in the presence of anti-CD99 mAb (IgG1, as described above). Before incubation of samples with 488-GαM IgG2a at 37°C, cells were treated with either GαM IgG1 (CD99-specific cross-linking antibody) or GαRb IgG for 0 or 5 min. (i) Degree of LBRC enrichment was quantified. Bars, 10 µm. Images are representative of three (a and h) or four (e) independent experiments. Data represent the mean value of three (b–d, f, and i) or four (g) independent experiments. Error bars denote SEM. **, P < 0.01; ***, P < 0.001; Student’s t test).
Mentions: Abolishing PECAM function has been previously shown to inhibit the targeted enrichment of LBRC membrane to sites of TEM, thus preventing TEM (Mamdouh et al., 2003). Because CD99 is also a resident molecule of the LBRC and it functions downstream of PECAM during TEM, we hypothesized that CD99 is required for a subsequent step in TR. To test this, we used a specialized technique to monitor LBRC membrane movement during TEM, known as the TR assay (see Materials and methods; Mamdouh et al., 2003; Mamdouh et al., 2008). In brief, this technique utilizes PECAM as a surrogate marker for the LBRC. We used a Fab fragment of a nonfunctional blocking antibody (mouse anti–human PECAM, clone P1.1 (Liao et al., 1995), to prebind PECAM in the LBRC. Any P1.1 Fab on the surface is saturated with unlabeled anti–mouse IgG at 4°C. We are then able to track the movement of the LBRC during TEM using the identical anti–mouse IgG conjugated to a fluorophore. To test our hypothesis, target recycling assays were performed using ECs pretreated with either anti-CD99 mAb or control IgG. Despite blocking transmigration with anti-CD99, a similar percentage of leukocytes were enriched with membrane from the LBRC (Fig. 1, a–d). (Definition of LBRC enrichment is a >1 fold increase in the staining around a leukocyte compared with a neighboring junction not in contact with the leukocyte.) From this, we concluded that CD99 is not required for the initiation of TR of the LBRC. However, this is not surprising given the phenotype of anti-CD99 blockade.

Bottom Line: How CD99 signals during this process remains unknown.We show that during TEM, endothelial cell (EC) CD99 activates protein kinase A (PKA) via a signaling complex formed with the lysine-rich juxtamembrane cytoplasmic tail of CD99, the A-kinase anchoring protein ezrin, and soluble adenylyl cyclase (sAC).PKA then stimulates membrane trafficking from the lateral border recycling compartment to sites of TEM, facilitating the passage of leukocytes across the endothelium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60208.

Show MeSH
Related in: MedlinePlus