Neutrophil AKT2 regulates heterotypic cell-cell interactions during vascular inflammation

Abstract

Interactions between platelets, leukocytes, and activated endothelial cells are important during microvascular occlusion; however, the regulatory mechanisms of these heterotypic cell-cell interactions remain unclear. Here, using intravital microscopy to evaluate mice lacking specific isoforms of the serine/threonine kinase AKT and bone marrow chimeras, we found that hematopoietic cell-associated AKT2 is important for neutrophil adhesion and crawling and neutrophil-platelet interactions on activated endothelial cells during TNF-α-induced venular inflammation. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet- and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under shear conditions. In particular, neutrophil AKT2 was critical for membrane translocation of αMβ2 integrin, β2-talin1 interaction, and intracellular Ca2+ mobilization. We found that the basal phosphorylation levels of AKT isoforms were markedly increased in neutrophils and platelets isolated from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow rates. Our results provide evidence that neutrophil AKT2 regulates αMβ2 integrin function and suggest that AKT2 is important for neutrophil recruitment and neutrophil-platelet interactions under thromboinflammatory conditions such as SCD.

Figure 1. AKT2 is required for neutrophil recruitment and neutrophil-platelet interactions on the activated endothelium during TNF-α–induced venular inflammation.

Vascular inflammation was induced by intrascrotal injection of TNF-α into WT and AKT isoform–specific KO mice. Intravital microscopy was performed as described in the Methods. Neutrophils and platelets were visualized by infusion of Alexa Fluor 647–conjugated anti-Gr1 and Dylight 488–conjugated anti-CD42c antibodies, respectively. (A) Representative images at various time points after monitoring. Arrows show direction of blood flow. Scale bar: 10 μm. (B) Number of adherent neutrophils. (C) Ratio of crawling/adherent neutrophils (percentage). (D) Median integrated fluorescence intensities of anti-CD42c antibodies (F platelets) were obtained from 45 to 52 venules, normalized by the number of adherent neutrophils, and plotted as a function of time. Data represent the mean ± SEM (n = 45–52 venules in 6 mice per group). *P < 0.05 versus WT mice by ANOVA and Dunnett’s test.

Figure 2. Hematopoietic cell AKT2 is important for neutrophil recruitment and neutrophil-platelet interactions on the TNF-α–inflamed endothelium.

Chimeric mice were generated by bone marrow transplantations in WT and Akt2 KO mice and used for intravital microscopy as described in Figure 1. (A) Number of adherent neutrophils. (B) Ratio of crawling/adherent neutrophils (percentage). (C) Median integrated fluorescence intensities of anti-CD42c antibodies (F platelets) were measured. Data represent the mean ± SEM (n = 60–64 venules in 8 mice per group). **P < 0.01 versus WT control mice by ANOVA and Dunnett’s test.

Figure 3. Neutrophil and platelet AKT2 regulate heterotypic neutrophil-platelet aggregation under shear conditions.

Human neutrophils (A) and platelets (B) were pretreated with 5 to 15 μM AKTi XII and stimulated with fMLF and thrombin, respectively. Lysates were immunoprecipitated with antibodies against p-AKT-Ser473, followed by immunoblotting. The band density of AKTi XII–treated groups was normalized to that of a control group (mean ± SD, n = 3). *P < 0.05 and **P < 0.01 versus control by ANOVA and Dunnett’s test. (C–F) Human neutrophils and platelets were incubated with 5 μM AKTi XII and labeled with FITC-conjugated anti–L-selectin and APC-conjugated anti-CD41a antibodies, respectively. Thrombin-activated platelets were mixed with neutrophils under a stirring condition (1,000 rpm). Cells were analyzed by flow cytometry. R1, leukocyte-platelet aggregates; R2, neutrophils; and R3, number of cell aggregates in the R1 gate. Neutrophil-platelet aggregation was measured by cell-cell aggregation (R3, E) and the fluorescence signal of anti-CD41a antibodies (F) in the R1 gate. Data represent the mean ± SD (n = 5). **P < 0.01 and ***P < 0.001 versus control by ANOVA and Dunnett’s test, and ^#P < 0.05 by Student’s t test. (D) Neutrophils and platelets were labeled with calcein red and calcein AM, respectively. After mixing both cells under a stirring condition, fluorescence microscopy was performed as described in the Methods. DAPI: blue. Scale bar: 10 μm. (G) Heterotypic aggregation assay was performed as described above using neutrophils and platelets isolated from WT and KO mice. Data represent the mean ± SD (n = 4). ^#P < 0.05 between groups by ANOVA.

Figure 4. AKT2 regulates αMβ2 integrin–mediated neutrophil recruitment and heterotypic neutrophil-platelet interactions during TNF-α–induced venular inflammation.

(A–D) Intravital microscopy of WT and αMβ2-null mice was performed as described in Figure 1. (A) Representative images at various time points after monitoring. Arrows show direction of blood flow. Scale bar: 10 μm. (B) Number of adherent neutrophils. (C) Ratio of crawling/adherent neutrophils (percentage). (D) Median integrated fluorescence intensities of anti-CD42c antibodies (F platelets). Data represent the mean ± SEM (n = 38–40 venules in 5 mice per group). **P < 0.01 versus WT mice by Student’s t test. (E–H) Akt2 KO (E and F) or αMβ2-null mice (G and H) were treated with a blocking anti-αM antibody (2 μg/g BW) or AKTi XII (10–30 μg/g BW), respectively, 3 hours after intrascrotal injection of TNF-α. The number of adherent neutrophils (E and G) and the integrated fluorescence intensities of anti-CD42c antibodies (F and H) were measured. Data represent the mean ± SEM (n = 35–38 venules in 4 mice per group). **P < 0.01 and ***P < 0.001 versus vehicle control by ANOVA and Dunnett’s test.

Figure 5. Effect of deletion and inhibition of AKT on the expression of platelet and neutrophil surface molecules.

(A and C) Flow cytometric analysis was performed to determine P-selectin exposure on WT and Akt KO platelets or on human platelets treated with 5 μM AKTi XII following thrombin (thr) stimulation as described in the Methods. *P < 0.05 and **P < 0.01 versus WT or vehicle control by ANOVA and Dunnett’s test (A) or Student’s t test (C). (B) Representative blot of P-selectin expression in lysates of WT and Akt KO platelets. (D) WT and Akt KO neutrophils were treated with or without fMLF and analyzed by flow cytometry using anti–PSGL-1 antibodies. (E) GPIbα expression was analyzed by flow cytometry using WT and Akt KO platelets treated with or without thrombin. (F) Surface expression of αMβ2 and αLβ2 integrins on unstimulated and fMLF-stimulated WT and Akt KO neutrophils. *P < 0.05 and **P < 0.01 versus unstimulated cells by Student’s t test. (G) Representative blot of αM, β2, and actin in lysates of WT and Akt KO neutrophils. (H) Human neutrophils pretreated with 5 μM AKTi XII were stimulated with fMLF. Flow cytometric analysis was performed using antibodies against total (ICRF44) and activated αMβ2 (CBRM1/5). *P < 0.05 versus vehicle by Student’s t test. All data were obtained from four independent experiments (mean ± SD).

Figure 6. AKT2 regulates β2-talin1 interaction and intracellular Ca²⁺ mobilization during neutrophil activation.

(A and B) Human neutrophils treated with 5 to 15 μM AKTi XII or neutrophils isolated from WT and Akt KO mice were incubated with or without fMLF for 1 minute. Lysates were immunoprecipitated with antibodies against β2 and immunoblotted. (C–F) Neutrophils isolated from WT and Akt KO mice or human neutrophils treated with 5 to 15 μM AKTi XII were preincubated with a Ca²⁺ dye and then treated with or without fMLF or thapsigargin in the absence of extracellular Ca²⁺. Intracellular Ca²⁺ mobilization was measured and quantified by AUC (× 10⁵ arbitrary units). All data were obtained from four independent experiments and represent the mean ± SD. *P < 0.05 and ***P < 0.001 versus WT or vehicle control by Student’s t test (B) or ANOVA and Dunnett’s test. RFU, relative fluorescence units.

Figure 7. AKT2 regulates in vitro heterotypic aggregation of neutrophils and platelets isolated from SCD patients.

Neutrophils (A) and platelets (B) of SCD patients were pretreated with or without 5 μM AKTi XII or 30 μM AKTi X and then stimulated with fMLF and thrombin, respectively. Lysates were immunoprecipitated with antibodies against p-AKT-Ser473 and immunoblotted. Band density represents the mean ± SD (n = 6 patients per group). *P < 0.05, **P < 0.01, and ***P < 0.001 versus unstimulated healthy donor by ANOVA and Dunnett’s test. (C) Neutrophils and platelets from patients were pretreated with 5 μM AKTi XII or 30 μM AKTi X and used for the in vitro aggregation assay described in Figure 3. Heterotypic aggregation was quantified by cell-cell aggregation. Data represent the mean ± SD (n = 8 patients). **P < 0.01 and ***P < 0.001 versus vehicle control by ANOVA and Dunnett’s test, and ^#P < 0.05 by ANOVA.

Figure 8. AKTi XII dose-dependently inhibits heterotypic cell-cell interactions in venules of Berkeley mice.

(A) Smears of Berkeley mouse blood were observed under a microscope (n = 4). Arrows show sickle red cells. Scale bar: 10 μm. (B–E) Berkeley mice were treated with vehicle or 10 or 30 μg/g BW AKTi XII, and intravital microscopy was performed without intrascrotal injection of TNF-α, as described in the Methods. (B) Sickle red cells attached to adherent neutrophils (Gr1-positive, red) and other leukocytes (Gr1-negative) in Berkeley mice. Green indicates platelet(s). (C) Representative images at various time points after monitoring. Arrows show direction of blood flow. Scale bar: 10 μm. Number of rolling (D) and adherent neutrophils (E). (F) Median fluorescence intensities of anti-CD42c antibodies (F platelets). (G) Blood flow rates were measured as described in the Methods. Data represent the mean ± SEM (n = 48–52 venules in 6 mice). *P < 0.05, **P < 0.01, and ***P < 0.001 versus vehicle control by ANOVA and Dunnett’s test.