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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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Inhibiting sAC or PKA blocks leukocyte transmigration. (a) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin or anti-CD99, as well as DMSO, PKI, KH7, or ddAdo. Before fixation, either GαM IgG (cross-linking antibody, XL) or GαRb IgG (control) was added to samples for 10 min (b and c) PBMCs were added to HUVECs pretreated with DMSO, anti-CD99, PKI, KH7, or ddAdo. Samples were stained with anti–VE-cadherin (EC) and anti-CD18 (leukocyte). Confocal images were taken to assess the site of blockade. Leukocytes were scored as being above the endothelium, blocked partway through, or migrated below HUVEC monolayers. (d and e) HUVECs were pretreated with anti–VE-cadherin, anti-PECAM, or anti-CD99. PBMCs were allowed to transmigrate for 1 h. Cells were then fixed, stained, imaged, and analyzed (as described above). (f) Eluate control TEM assays were performed as previously described (Mamdouh et al., 2009). In brief, HUVECs were pretreated with anti–VE-cadherin, anti-CD99, PKI, KH7, ddAdo, or DMSO for the duration of the incubation in blocking experiments. Cells were then washed and fresh media was added to samples. HUVECs were incubated at 37°C for 1 h (the duration of the normal blocking experiments). The media was collected from each well. The eluate media contains all of the inhibitor that would have eluted out of the cultures over the duration of the blocking experiment. PBMCs were resuspended in the eluate media, added to untreated HUVECs, and incubated at 37°C for 1 h. Cells were subsequently fixed and analyzed. Bars, 10 µm. Images are representative of two (e) or three (c) independent experiments. Numerical values are the average of two (d and f) or three (a and b) independent experiments. Error bars represent SEM (***, P < 0.001; ****, P < 0.0001; Student’s t test [a] and ANOVA [b, d, and f]).
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fig5: Inhibiting sAC or PKA blocks leukocyte transmigration. (a) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin or anti-CD99, as well as DMSO, PKI, KH7, or ddAdo. Before fixation, either GαM IgG (cross-linking antibody, XL) or GαRb IgG (control) was added to samples for 10 min (b and c) PBMCs were added to HUVECs pretreated with DMSO, anti-CD99, PKI, KH7, or ddAdo. Samples were stained with anti–VE-cadherin (EC) and anti-CD18 (leukocyte). Confocal images were taken to assess the site of blockade. Leukocytes were scored as being above the endothelium, blocked partway through, or migrated below HUVEC monolayers. (d and e) HUVECs were pretreated with anti–VE-cadherin, anti-PECAM, or anti-CD99. PBMCs were allowed to transmigrate for 1 h. Cells were then fixed, stained, imaged, and analyzed (as described above). (f) Eluate control TEM assays were performed as previously described (Mamdouh et al., 2009). In brief, HUVECs were pretreated with anti–VE-cadherin, anti-CD99, PKI, KH7, ddAdo, or DMSO for the duration of the incubation in blocking experiments. Cells were then washed and fresh media was added to samples. HUVECs were incubated at 37°C for 1 h (the duration of the normal blocking experiments). The media was collected from each well. The eluate media contains all of the inhibitor that would have eluted out of the cultures over the duration of the blocking experiment. PBMCs were resuspended in the eluate media, added to untreated HUVECs, and incubated at 37°C for 1 h. Cells were subsequently fixed and analyzed. Bars, 10 µm. Images are representative of two (e) or three (c) independent experiments. Numerical values are the average of two (d and f) or three (a and b) independent experiments. Error bars represent SEM (***, P < 0.001; ****, P < 0.0001; Student’s t test [a] and ANOVA [b, d, and f]).

Mentions: Having demonstrated that CD99 associates with and signals through sAC to activate PKA, we examined if sAC is required for transmigration. We performed TEM assays in HUVECs pretreated with either anti-CD99 (mouse IgG1 mAb) or anti–VE-cadherin (mouse IgG2a mAb), as well as PKI, KH7, ddAdo, or DMSO. As seen above, inhibiting endothelial CD99 with function-blocking mAb substantially ablated TEM (Fig. 5 a). As expected (Fig. 1), this blockade could be overcome if GαM cross-linking antibody was briefly (10 min) added to polyvalently cluster the function-blocking CD99 antibody, thus providing the necessary activation to propagate EC CD99 signaling cascade. As a control, adding goat anti–rabbit antibody had no effect on anti-CD99 blockade of TEM. Inhibiting sAC or PKA not only prevented CD99 cross-linking-mediated restoration of TEM, but also blocked TEM in HUVECs treated with control antibody (Fig. 5 a); this is expected if sAC and PKA function sequentially downstream of CD99. Conversely, blocking tmACs did not have any effect on leukocyte TEM.


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)

Inhibiting sAC or PKA blocks leukocyte transmigration. (a) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin or anti-CD99, as well as DMSO, PKI, KH7, or ddAdo. Before fixation, either GαM IgG (cross-linking antibody, XL) or GαRb IgG (control) was added to samples for 10 min (b and c) PBMCs were added to HUVECs pretreated with DMSO, anti-CD99, PKI, KH7, or ddAdo. Samples were stained with anti–VE-cadherin (EC) and anti-CD18 (leukocyte). Confocal images were taken to assess the site of blockade. Leukocytes were scored as being above the endothelium, blocked partway through, or migrated below HUVEC monolayers. (d and e) HUVECs were pretreated with anti–VE-cadherin, anti-PECAM, or anti-CD99. PBMCs were allowed to transmigrate for 1 h. Cells were then fixed, stained, imaged, and analyzed (as described above). (f) Eluate control TEM assays were performed as previously described (Mamdouh et al., 2009). In brief, HUVECs were pretreated with anti–VE-cadherin, anti-CD99, PKI, KH7, ddAdo, or DMSO for the duration of the incubation in blocking experiments. Cells were then washed and fresh media was added to samples. HUVECs were incubated at 37°C for 1 h (the duration of the normal blocking experiments). The media was collected from each well. The eluate media contains all of the inhibitor that would have eluted out of the cultures over the duration of the blocking experiment. PBMCs were resuspended in the eluate media, added to untreated HUVECs, and incubated at 37°C for 1 h. Cells were subsequently fixed and analyzed. Bars, 10 µm. Images are representative of two (e) or three (c) independent experiments. Numerical values are the average of two (d and f) or three (a and b) independent experiments. Error bars represent SEM (***, P < 0.001; ****, P < 0.0001; Student’s t test [a] and ANOVA [b, d, and f]).
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fig5: Inhibiting sAC or PKA blocks leukocyte transmigration. (a) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin or anti-CD99, as well as DMSO, PKI, KH7, or ddAdo. Before fixation, either GαM IgG (cross-linking antibody, XL) or GαRb IgG (control) was added to samples for 10 min (b and c) PBMCs were added to HUVECs pretreated with DMSO, anti-CD99, PKI, KH7, or ddAdo. Samples were stained with anti–VE-cadherin (EC) and anti-CD18 (leukocyte). Confocal images were taken to assess the site of blockade. Leukocytes were scored as being above the endothelium, blocked partway through, or migrated below HUVEC monolayers. (d and e) HUVECs were pretreated with anti–VE-cadherin, anti-PECAM, or anti-CD99. PBMCs were allowed to transmigrate for 1 h. Cells were then fixed, stained, imaged, and analyzed (as described above). (f) Eluate control TEM assays were performed as previously described (Mamdouh et al., 2009). In brief, HUVECs were pretreated with anti–VE-cadherin, anti-CD99, PKI, KH7, ddAdo, or DMSO for the duration of the incubation in blocking experiments. Cells were then washed and fresh media was added to samples. HUVECs were incubated at 37°C for 1 h (the duration of the normal blocking experiments). The media was collected from each well. The eluate media contains all of the inhibitor that would have eluted out of the cultures over the duration of the blocking experiment. PBMCs were resuspended in the eluate media, added to untreated HUVECs, and incubated at 37°C for 1 h. Cells were subsequently fixed and analyzed. Bars, 10 µm. Images are representative of two (e) or three (c) independent experiments. Numerical values are the average of two (d and f) or three (a and b) independent experiments. Error bars represent SEM (***, P < 0.001; ****, P < 0.0001; Student’s t test [a] and ANOVA [b, d, and f]).
Mentions: Having demonstrated that CD99 associates with and signals through sAC to activate PKA, we examined if sAC is required for transmigration. We performed TEM assays in HUVECs pretreated with either anti-CD99 (mouse IgG1 mAb) or anti–VE-cadherin (mouse IgG2a mAb), as well as PKI, KH7, ddAdo, or DMSO. As seen above, inhibiting endothelial CD99 with function-blocking mAb substantially ablated TEM (Fig. 5 a). As expected (Fig. 1), this blockade could be overcome if GαM cross-linking antibody was briefly (10 min) added to polyvalently cluster the function-blocking CD99 antibody, thus providing the necessary activation to propagate EC CD99 signaling cascade. As a control, adding goat anti–rabbit antibody had no effect on anti-CD99 blockade of TEM. Inhibiting sAC or PKA not only prevented CD99 cross-linking-mediated restoration of TEM, but also blocked TEM in HUVECs treated with control antibody (Fig. 5 a); this is expected if sAC and PKA function sequentially downstream of CD99. Conversely, blocking tmACs did not have any effect on leukocyte TEM.

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