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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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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]).
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fig9: 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]).

Mentions: CD99 has been shown to be critical for leukocyte transmigration in vivo (Bixel et al., 2004; Dufour et al., 2008; Bixel et al., 2010). To test if sAC is also required for leukocyte extravasation in vivo, we used a model of dermatitis. Before stimulation with croton oil, mice were pretreated with either anti-CD99 (clone 3F11 mAb; Fig. 9 a), KH7, or carrier alone (DMSO). 3D confocal microscopy of fixed tissue revealed that although mice treated with carrier alone showed robust neutrophil influx into the perivascular space of the ear, treatment with either anti-CD99 or KH7 significantly attenuated this response (Fig. 9, b and c). To further identify the step at which anti-CD99 and KH7 were acting to block extravasation, we analyzed xz-orthogonal sections to discern where neutrophils were being arrested (Fig. 9 b, insets). Cells were scored as being in one of six positions (Fig. 9 d). Both anti-CD99 and KH7 arrested the vast majority of leukocytes partway through the endothelial junctions (Fig. 9 e), replicating the phenotype seen upon blocking CD99 or sAC function in vitro. There was no difference in mean vessel length, diameter, or the total number of PMN per field among the groups (unpublished data). Additionally, pretreatment of PMN with KH7 did not affect their ability to transmigrate across endothelial monolayers in vitro (not depicted). Therefore, we conclude that the effect of anti-CD99 and KH7 was on transmigration and not previous steps required for leukocyte requirement.


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)

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]).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig9: 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]).
Mentions: CD99 has been shown to be critical for leukocyte transmigration in vivo (Bixel et al., 2004; Dufour et al., 2008; Bixel et al., 2010). To test if sAC is also required for leukocyte extravasation in vivo, we used a model of dermatitis. Before stimulation with croton oil, mice were pretreated with either anti-CD99 (clone 3F11 mAb; Fig. 9 a), KH7, or carrier alone (DMSO). 3D confocal microscopy of fixed tissue revealed that although mice treated with carrier alone showed robust neutrophil influx into the perivascular space of the ear, treatment with either anti-CD99 or KH7 significantly attenuated this response (Fig. 9, b and c). To further identify the step at which anti-CD99 and KH7 were acting to block extravasation, we analyzed xz-orthogonal sections to discern where neutrophils were being arrested (Fig. 9 b, insets). Cells were scored as being in one of six positions (Fig. 9 d). Both anti-CD99 and KH7 arrested the vast majority of leukocytes partway through the endothelial junctions (Fig. 9 e), replicating the phenotype seen upon blocking CD99 or sAC function in vitro. There was no difference in mean vessel length, diameter, or the total number of PMN per field among the groups (unpublished data). Additionally, pretreatment of PMN with KH7 did not affect their ability to transmigrate across endothelial monolayers in vitro (not depicted). Therefore, we conclude that the effect of anti-CD99 and KH7 was on transmigration and not previous steps required for leukocyte requirement.

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