Methylene blue modulates transendothelial migration of peripheral blood cells

Abstract

Vasoplegia is a severe complication after cardiac surgery. Within the last years the administration of nitric oxide synthase inhibitor methylene blue (MB) became a new therapeutic strategy. Our aim was to investigate the role of MB on transendothelial migration of circulating blood cells, the potential role of cyclic cGMP, eNOS and iNOS in this process, and the influence of MB on endothelial cell apoptosis. Human vascular endothelial cells (HuMEC-1) were treated for 30 minutes or 2 hours with different concentrations of MB. Inflammation was mimicked by LPS stimulation prior and after MB. Transmigration of PBMCs and T-Lymphocytes through the treated endothelial cells was investigated. The influence of MB upon the different subsets of PBMCs (Granulocytes, T- and B-Lymphocytes, and Monocytes) was assessed after transmigration by means of flow-cytometry. The effect of MB on cell apoptosis was evaluated using Annexin-V and Propidium Iodide stainings. Analyses of the expression of cyclic cGMP, eNOS and iNOS were performed by means of RT-PCR and Western Blot. Results were analyzed using unpaired Students T-test. Analysis of endothelial cell apoptosis by MB indicated a dose-dependent increase of apoptotic cells. We observed time- and dose-dependent effects of MB on transendothelial migration of PBMCs. The prophylactic administration of MB led to an increase of transendothelial migration of PBMCs but not Jurkat cells. Furthermore, HuMEC-1 secretion of cGMP correlated with iNOS expression after MB administration but not with eNOS expression. Expression of these molecules was reduced after MB administration at protein level. This study clearly reveals that endothelial response to MB is dose- and especially time-dependent. MB shows different effects on circulating blood cell-subtypes, and modifies the release patterns of eNOS, iNOS, and cGMP. The transendothelial migration is modulated after treatment with MB. Furthermore, MB provokes apoptosis of endothelial cells in a dose/time-dependent manner.

Figure 1. Endothelial cells apoptosis after MB treatment.

The number of APC-positive/PI-negative HuMEC-1 after MB increased with higher MB dosages. (A) Unstained and untreated controls served as initial point for the gating strategy. (B) Untreated control. (C–D) HuMEC-1 treated with 10 µM MB for 30 min and 120 min. (E–F) HuMEC-1 treated with 30 µM MB for 30 min and 120 min. (G-H) HuMEC-1 treated with 60 µM MB for 30 min and 120 min. (each experiment n = 3)

Figure 2. Influence of MB on transendothelial migration of PBMCs.

Transmigration of PBMCs through an endothelial layer after treatment with (A) 10 µM MB for 30 min. (B) 10 µM MB for 2 h. (C) 30 µM MB for 30 min. (D) 30 µM MB for 2 h. (E) 60 µM MB for 30 min. (F) 60 µM MB for 2 h (each experiment n = 5–8) (*p<0.05).

Figure 3. Influence of MB on transendothelial migration of Jurkat cells.

Transmigration of Jurkat cells through an endothelial layer after treatment with (A) 10 µM MB for 30 min. (B) 10 µM MB for 2 h. (C) 30 µM MB for 30 min. (D) 30 µM MB for 2 h. (E) 60 µM MB for 30 min. (F) 60 µM MB for 2 h (each experiment n = 3–6) (*p<0.05).

Figure 4. Change of migrated PBMC subtypes after MB and LPSMB treatment.

(A1–E1) MB treatment of HuMEC-1 previous to transmigration assay. (A2–E2) LPSMB treatment of HuMEC-1 previous to transmigration assay. (A1, A2) Migrated granulocytes, (B1, B2) Migrated B-lymphocytes. (C1,C2) Migrated T-lymphocytes. (D1, D2) Migrated CD14++ monocytes. (E1,E2) Migrated CD14++CD16++ monocytes. (each experiment n = 3) (§ p<0.05 vs. control; # p<0.05 vs. LPS, *p<0.05).

Figure 5. Prophylactic MB treatment previous to inflammation modulates transendothelial migration.

(A) Transmigration of PBMCs through HuMEC-1 after prophylactic treatment with MB followed by LPS (n = 9). (B) Transmigration of Jurkat cells through HuMEC-1 after prophylactic treatment with MB followed by LPS (each experiment n = 4–8) (*p<0.05).

Figure 6. L-NMMA inhibits transendothelial migration after MB treatment.

(A) Treatment of HuMEC-1 with either LPS or 10 µM MB for 30 min previous to transendothelial migration. L-NMMA was added during transmigration to LPS and MB group. (B) Treatment of HuMEC-1 with either LPS or 60 µM MB for 30 min previous to transendothelial migration. L-NMMA was added during transmigration to LPS and MB group. (each experiment n = 3–6) (*p<0.05)

Figure 7. Modulation of eNOS and iNOS expression and secretion of cGMP of HuMEC-1 after MB and LPSMB administration.

(A) iNOS expression is dose dependent elevated by MB administration compared to control. (B) Stimulation of HuMEC-1 with LPS followed by MB treatment increases iNOS expression after 2 h MB. (C) MB treatment of HuMEC-1 reduces eNOS expression. (D) LPS stimulation prior to MB administration reduces eNOS expression. (E) cGMP content in HuMEC-1 supernatants after different MB administrations. (F) Blocked cGMP released from the endothelial cells after LPSMB treatment. (each experiment n = 4) (*p<0.05).

Figure 8. Protein expression levels of eNOS vs. phosphor-eNOS and iNOS vs. phosphor-iNOS.

(A) Relative amount of iNOS protein expression after MB treatment. (B) Relative amount of iNOS protein expression after LPSMB treatment. (C) Relative amount of phosphorylated iNOS protein expression after MB treatment. (D) Relative amount of phosphorylated iNOS protein expression after LPSMB treatment. (E) Relative amount of eNOS protein expression after MB treatment. (F) Relative amount of eNOS protein expression after LPSMB treatment. (G) Relative amount of phosphorylated eNOS protein expression after MB treatment. (H) Relative amount of phosphorylated eNOS protein expression after LPSMB treatment. (each experiment n = 2-3) ((§ p<0.05 vs. control; *p<0.05).