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

Abstract

CD99 is a critical regulator of leukocyte transendothelial migration (TEM). 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. Pharmacologic or genetic inhibition of EC sAC or PKA, like CD99 blockade, arrests neutrophils and monocytes partway through EC junctions, in vitro and in vivo, without affecting leukocyte adhesion or the expression of relevant cellular adhesion molecules. This is the first description of the CD99 signaling pathway in TEM as well as the first demonstration of a role for sAC in leukocyte TEM.

Figure 1.

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 IgG_2a (550-GαM IgG_2a, first antibody) labeled LBRC membrane (labeled with nonblocking mouse anti-PECAM IgG_2a antibody, clone P1.1) that trafficked before the CD99-dependent step of TEM. DyLight488 GαM IgG_2a (GαM IgG_2a, 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-GαM_2a. (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 IgG_2a 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).

Figure 2.

Engagement of EC CD99 activates PKA. (a) Quantitative TEM assays were performed using HUVECs pretreated with nonblocking anti–VE-cadherin, anti-PECAM, or anti-CD99 mAb. After 50 min, histamine (10 µM) was added to samples for 10 min at 37°C. (b) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin (nonblocking control) or anti-CD99 mAb. Additionally, HUVECs were pretreated with diphenhydramine (H1-R antagonist, 10 µM), ranitidine (H2-R antagonist, 10 µM), or JNJ-10191584 (H4-R antagonist, 10 µM) or DMSO (carrier). After 50 min, histamine (10 µM) or dimaprit (H2-R agonist, 10 µM) was added to samples for 10 min. (c) Immunoblot analysis of phospho-VASP S157 and phospho-CREB S133 activity after anti-CD99 or anti-PECAM mAb (control) cross-linking (XL) in resting HUVECs. (d and e) Quantification of results in c, pVASP and pCREB signals were normalized to total VASP and total CREB, respectively. Values were then normalized to CD99 XL. (f) Antibody-coated polystyrene bead recruitment of CD99 and activation of phospho-PKA. Beads precoupled with mIgG₁, anti-CD99 mAb, or anti-PECAM mAb were added to HUVEC monolayers expressing hCD99-GFP. Monolayers were subsequently stained with anti–VE-cadherin and anti-pPKA T197 PKA antibodies. Arrows indicate beads bound to HUVECs. (g and h) Quantification of data; percent of beads in the field of view with either hCD99-GFP or pPKA T197 enrichment. Bars, 10 µm. Images are representative of three (f) or four (c) independent experiments. Numerical values are the mean of three (a, b, e, g, and h) or four (d) independent experiments. Error bars represent SD (d and e) or SEM (a, b, g, and h; ***, P < 0.001; ****, P < 0.0001; Student’s t test [a, b, d, and e] and ANOVA [g and h]).

Figure 3.

Raising intracellular cAMP reverses anti-CD99 blockade of transmigration and restores TR of the LBRC. (a and b) Quantitative TEM assays were performed using HUVECs pretreated with anti–VE-cadherin (control) or anti-CD99 mAb (IgG₁). After 50 min, 8-CPT (30 µM), Forskolin (30 µM), or DMSO (control) was added to the cells for 10 min. (c) Two-color TR assays were performed (as previously described). In brief, before warming monolayers to 37°C, 488-GαM IgG_2a and 8-CPT, Forskolin, or DMSO were added. Cells were then incubated at 37°C for either 0 or 5 min and subsequently washed, fixed, and stained. Arrows denote LBRC enrichment around anti-CD99–arrested monocytes. (d and e) LBRC enrichment was quantified for both 550- or 488-GαM antibodies. (f) Quantitative TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin (control) or anti-CD99 mAb. PBMCs were then added and allowed to transmigrate at 37°C for 50 min. 10 min before fixation, either 8-CPT (general cAMP analogue) or 007-AM (selective-Epac activator) was added to cells. (g) HUVECs were treated in parallel with either 8-CPT or 007-AM for 10 min and then lysed. Immunoblot analysis of pVASP-S157 normalized to total VASP was used to assess PKA activity induced by the drugs. (h) Quantification of results above. Bars, 10 µm. Images are representative of two (c) or three (g) independent experiments. Numerical values are the average of two (d and e) or three (a, b, f, and h) independent experiments. Error bars represent SD (h) or SEM (a, b, and d–f; **, P < 0.01; ***, P < 0.001; Student’s t test [a, b, and d–f] and ANOVA [h]).

Figure 4.

Inhibition of sAC prevents CD99 activation of PKA. (a) HUVECs were pretreated with 50 µM KH7 (sAC inhibitor), 25 µM ddAdo (tmAC inhibitor), or DMSO (control). HUVECs were then cross-linked with mIgG₁ control or anti-CD99 and lysed. Immunoblot analysis was performed for pVASP-S157 and total VASP. (b) Quantification of immunoblots. Values denote pVASP-S157 signal normalized to total VASP signal. Values were then normalized to the CD99/DMSO condition. (c) Amine-modified polystyrene latex beads were precoupled with mIgG₁, anti-CD99, or anti-PECAM. HUVECs expressing hCD99-GFP were pretreated with DMSO, PKI, KH7, or ddAdo. Beads were added to HUVECs for 20 min at 37°C. Samples were then fixed and stained for VE-cadherin (not depicted) and pPKA-T197. Arrows indicate where polystyrene beads bound to HUVECs. (d and e) Data were quantified for percent of beads in the field of view with either hCD99-GFP or pPKA-T197 enrichment. (f) HUVECs were pretreated with DMSO, KH7, or ddAdo. PGE₂ (100 ng/ml) was added to HUVECs for 10 min. Cells were then lysed and immunoblot analysis was performed for pCREB-S133 and total CREB. (g) Quantification of immunoblots. Values denote pCREB-S133 signal normalized to total CREB signal. Values for all conditions were then normalized to DMSO/PGE₂ condition. Bars, 10 µm. Images are representative of two (c), three (f), or four (a) independent experiments. Numerical values are the average of two (d and e), three (g) four (b) independent experiments. Error bars represent SD (b) or SEM (d, e, and g; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test [b] and ANOVA [d, e, and g]).

Figure 5.

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]).

Figure 6.

Genetic ablation of endothelial sAC inhibits leukocyte transmigration. (a) Quantitative PBMC TEM assays were performed on HUVECs expressing either scrambled (SCR) or sAC shRNA. Where indicated, the rescue construct (sAC_t) was also expressed. (b) Immunoblot analysis for sAC and β-actin was performed on samples treated in parallel. (c) Quantification of immunoblots. Total sAC expression was normalized to β-actin for each sample and then normalized to SCR control. (d and e) TEM-IF assays were performed on HUVECs expressing SCR, sAC, or CD99 shRNA. (f) Immunoblot analysis for CD99, sAC, and β-actin was performed on samples treated in parallel. (g) Quantification of immunoblots. Total sAC or CD99 expression was normalized to β-actin for each sample and then normalized to SCR control. (h) Quantitative PMN TEM-IF assays were performed on TNF-activated (4 h) HUVECs expressing SCR, sAC, or CD99 shRNA. (i) Quantitative PMBC TEM-IF assays were performed on TNF-activated (4 h) HUVECs expressing SCR, sAC, or CD99 shRNA. (j) Immunoblot analysis of samples treated in parallel were performed for sAC, ICAM-1, PECAM, JAM-A, and CD99 expression. Bars, 10 µm. Images are representative of three (b, e, and j) independent experiments. Numerical values are the mean of two (h and i) or three (a, c, d, and g) independent experiments. Error bars represent SD (c and g) or SEM (a, d, h, and i; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test [a and c] and ANOVA [d, g, h, and i]).

Figure 7.

CD99, sAC, PKA, and ezrin form a signaling complex to regulate transmigration. (a) HUVECs were infected with sAC_t-GFP encoding adenovirus. Cells were then fixed and stained for CD99. Arrows denote junctional localization. (b and c) CD99 and sAC were immunoprecipitated from HUVEC whole-cell lysate. Immunoblot analysis was performed for CD99, sAC, PKA-RIIα, ezrin, and AKAP-13. Cell lysates were probed in parallel for loading controls. (d) Endogenous CD99 was immunoprecipitated from iHUVECs expressing either SCR or sAC shRNA. Immunoblot analysis was performed for ezrin, sAC, and CD99 for both cell lysates and immunoprecipitation samples. (e) Quantification of lysates. Efficacy of sAC knockdown was quantified by normalizing sAC expression to CD99. Values were then normalized to the SCR shRNA/CD99 IP condition. (f and g) Quantification of coimmunoprecipitation samples. The amount of sAC (f) and ezrin (g) that coimmunoprecipitated with CD99 was calculated and normalized to the SCR shRNA CD99 IP condition. (h) Endogenous CD99 was immunoprecipitated from iHUVECs expressing either SCR or ezrin shRNA. Immunoblot analysis was performed for ezrin, sAC, and CD99 for both cell lysates and immunoprecipitation samples. (i) Quantification of lysates. Efficacy of ezrin knockdown was quantified by normalizing ezrin expression to CD99. Values were then normalized to the SCR shRNA CD99 IP condition. (j and k) Quantification of coimmunoprecipitations. The amount of sAC (j) and ezrin (k) that coimmunoprecipitated with CD99 was calculated and normalized to the SCR shRNA CD99 IP condition. (l) Quantitative TEM assays were performed using HUVECs pretreated with CD99, ezrin, or SCR shRNA. (m) HUVECs were treated in parallel and used for immunoblot analysis of ezrin, CD99, and β-actin. (n) Quantification of results above. Ezrin and CD99 expression was normalized to β-actin for each condition. Values were then normalized to SCR shRNA conditions. Bars, 10 µm. Images are representative of three (a–d, h, and m) independent experiments. Numerical values are the mean of three (e–g, i–l, and n) independent experiments. Error bars represent SD **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test [e–g and i–l] and ANOVA [n]).

Figure 8.

CD99 cytoplasmic tail mediates TEM through a positively charged juxtamembrane region. (a) Diagram of CD99 cytoplasmic tail and CD99-GFP constructs. (b) Endogenous CD99 in HUVECs was knocked down using CD99 shRNA. CD99-GFP constructs were then reexpressed. Samples treated in parallel were lysed and used for immunoblot analysis of CD99 to assess degree of reexpression and knockdown. (c) Quantitative TEM assays were then performed on knockdown samples. (d) CD99-GFP constructs were overexpressed in iHUVECs. CD99 was then immunoprecipitated from the surface of cells (ensuring only fully processed CD99 was being analyzed). Immunoblot analysis for ezrin, sAC, and CD99 was then performed. (e and f) Quantification of results above. Amount of ezrin (e) and sAC (f) in each sample was normalized to the amount of CD99 immunoprecipitated (to account for any variability in pull-down efficiency between samples). Values were then normalized to wild-type CD99-GFP condition. Images are representative three (b and d) independent experiments. Numerical values are the mean of three (c, e, and f) independent experiments. Error bars represent SD (e and f) or SEM (b; ***, P < 0.001; ****, P < 0.0001; ANOVA).

Figure 9.

SAC is critical for leukocyte transmigration in vivo. (a) Validation of rat anti–mouse CD99 monoclonal antibody, clone 3F11. Flow cytometry analysis of control (shaded) or anti–mouse CD99 (3F11, thick line) mAb binding to parental or mouse CD99-transfected Ba/F3 cells. (b) The ears of wild-type FVB/n mice (age and sex matched littermates) pretreated with DMSO, anti-CD99 (3F11 mAb), or KH7 (5 µmol/kg) were stimulated with croton oil (1%, 20 µl/ear) or carrier (10% olive oil/90% acetone). After 5 h, mice were sacrificed, their ears were harvested, and immunohistochemical staining was performed using anti-PECAM (ECs), anti-MRP14 (neutrophils), and anti–collagen-IV (basement membrane). 3D confocal images were acquired for each sample. (c) Quantification of results above. Percent of leukocytes extravasated within 50 µm of venules per field of view. (d) Model for quantification of site of arrest. Neutrophils were scored as being in one of six positions: luminal (1), apically arrested (2), arrested partway through the endothelium (3), arrested on the basement membrane (4), migrating through the basement membrane (5), or fully extravasated (6). (e) Quantification of the site of arrest for anti-CD99 and KH7-treated animals. (f) The ears of wild-type FVB/n mice (age- and sex-matched littermates) pretreated with anti-CD99 or rat IgG (control) were stimulated with croton oil. After 3 h, mice received dimaprit (10 mg drug/kg animal) or carrier (H₂O). Mice were sacrificed 2 h later, their tissue stained, and analyzed as described in panel b. (g) Quantification of results. Percent of leukocytes extravasated within 50 µm of venules per field of view. (h) Additional mice pretreated with anti-CD99 mAb were sacrificed at the time dimaprit was given (3 h) to ensure that the anti-CD99 blockade was present throughout the experiments (5 h total). (i) Quantification of the site of arrest for anti-CD99/carrier and anti-CD99/dimaprit-treated animals. 100–200 cells were analyzed per ear. Total PMN per field of view, vessel length, and vessel diameter were equivalent for all conditions tested (not depicted). Images were acquired with a 40× objective (n = 1.00). Insets show xz-orthogonal view (where yellow bar dissects the vessel) to demonstrate site of neutrophil arrest. Bars, 25 µm. Two to three mice per condition were used for each experiment. Images are representative of two (f) or three (a and b) independent experiments. Data represent the average value of two (g-i) or three (c and e) independent experiments. Error bars denote SEM ***, P < 0.001; ****, P < 0.0001; Student’s t test [c and g] and ANOVA [e and i]).

Figure 10.

Endothelial-specific knockout of sAC blocks leukocyte transmigration in vivo. (a) Heart tissue and peripheral blood was collected from sAC-C2^flox/flox and sAC-C2^flox/flox/VE-Cre⁺ mice used in the following experiments (see Materials and methods). FACS was used to sort MHEC (CD31⁺/CD45⁻) and leukocyte (Ly6G⁺/CD45⁺) cell populations from the heart tissue and peripheral blood, respectively. MHEC and leukocyte DNA was isolated from each mouse. PCR was performed to assess the expression of either sAC WT allele (top band) or sAC C2-KO allele (bottom band). (b) The ears of sAC-C2^flox/flox or sAC-c2^flox/flox/VE-Cre⁺ mice (mixed background, see Materials and methods; Chen et al., 2013) were stimulated for 5 h with croton oil. Tissue was then harvested, stained, and analyzed. (c) Quantification of results above. (d) Quantification of site of arrest for sAC-C2^flox/flox/VE-Cre⁺ mice. Percent of leukocytes extravasated within 50 µm of venule per field of view. (e) Our current model of how CD99 signals during TEM. Under resting conditions, CD99, sAC, PKA, and ezrin form a signaling complex at endothelial junctions. During TEM, homophilic engagement of endothelial CD99 with leukocyte CD99 (#1) signals through sAC (#2) to elevate cAMP (#3), to activate PKA, which works through a yet to be defined mechanism (#4) to induce LBRC membrane trafficking to sites of leukocyte–endothelial contact (#5). (f) Flow diagram of model detailed above. 100–200 cells were analyzed per ear. PMN per field of view, vessel length and vessel diameter were equivalent for all conditions tested (not depicted). Images were acquired with a 40× objective (n = 1.00). Insets show xz-orthogonal view (where yellow bar dissects the vessel) to demonstrate site of neutrophil arrest. Bars, 25 µm. Three mice per condition were used for each experiment. Images are representative of three (a and b) independent experiments. Data represent the average value of three (c and d) independent experiments. Error bars denote SEM (****, P < 0.0001; Student’s t test [c] and ANOVA [d]).