Anti-inflammatory functions of protein C require RAGE and ICAM-1 in a stimulus-dependent manner

Abstract

By binding β 2-integrins both ICAM-1 and the receptor for advanced glycation end products (RAGE) mediate leukocyte recruitment in a stimulus-dependent manner. Using different inflammatory mouse models we investigated how RAGE and ICAM-1 are involved in anti-inflammatory functions of protein C (PC; Ceprotin, 100 U/kg). We found that, depending on the stimulus, RAGE and ICAM-1 are cooperatively involved in PC-induced inhibition of leukocyte recruitment in cremaster models of inflammation. During short-term proinflammatory stimulation (trauma, fMLP, and CXCL1), ICAM-1 is more important for mediation of anti-inflammatory effects of PC, whereas RAGE plays a major role after longer proinflammatory stimulation (TNF α ). In contrast to WT and Icam-1(-/-) mice, PC had no effect on bronchoalveolar neutrophil emigration in RAGE(-/-) mice during LPS-induced acute lung injury, suggesting that RAGE critically mediates PC effects during acute lung inflammation. In parallel, PC treatment effectively blocked leukocyte recruitment and improved survival of WT mice and Icam-1-deficient mice in LPS-induced endotoxemia, but failed to do so in RAGE-deficient mice. Exploring underlying mechanisms, we found that PC is capable of downregulating intracellular RAGE and extracellular ICAM-1 in endothelial cells. Taken together, our data show that RAGE and ICAM-1 are required for the anti-inflammatory functions of PC.

Figure 1

Effect of PC on leukocyte recruitment in wild-type, RAGE ^−/−, and Icam-1 ^−/− mice during trauma-induced inflammation. Leukocyte adhesion (number of adherent cells per mm² of surface area) in cremaster muscle venules of wild-type (WT) control mice, RAGE ^−/− mice, and Icam-1 ^−/− mice was recorded with and without treatment with PC (100 U/kg, 3 hours) during trauma-induced inflammation (a). Leukocyte adhesion in the same genotypes and treatment groups was shown after additional stimulation with fMLP (superfusion at 1 μM, 15 min) (b). Leukocyte transmigration (per mm² surface area) was analyzed in Giemsa-stained cremaster muscle whole mounts after 15 min fMLP superfusion (1 μM) in the trauma model in WT, Icam-1 ^−/−, and RAGE ^−/− mice with and without PC treatment (100 U/kg, 3 hours) (c). All values are presented as mean ± SEM from three or more mice per group. Significant differences (P < 0.05) to WT and RAGE ^−/− control mice are indicated by the asterisks and pound key, respectively.

Figure 2

Effect of PC on leukocyte recruitment in wild-type, RAGE ^−/−, and Icam-1 ^−/− mice after TNFα stimulation. Leukocyte adhesion (number of adherent cells per mm² of surface area) in 3 h TNFα-stimulated (500 ng/mouse) cremaster muscle venules of wild-type (WT) control mice and Icam-1 ^−/− and RAGE ^−/− mice was documented with and without treatment with PC (100 U/kg, 3 hours) (a). Leukocyte adhesion in the same genotypes and treatment groups was depicted after additional stimulation with fMLP (superfusion at 1 μM, 5 min) (b). Leukocyte transmigration (per mm² surface area) was analyzed in Giemsa-stained cremaster muscle whole mounts after 5 min fMLP superfusion (1 μM) in the TNFα-model in WT, Icam-1 ^−/−, and RAGE ^−/− mice with and without PC treatment (100 U/kg, 3 hours) (c). All values are presented as mean ± SEM from three or more mice per group. Significant differences (P < 0.05) to WT and Icam-1 ^−/− control mice are indicated by the asterisks and pound key, respectively.

Figure 3

Leukocyte transmigration in PC-treated wild-type, RAGE ^−/−, and Icam-1 ^−/− mice after TNFα stimulation. Leukocyte transmigration is illustrated by representative micrographs of Giemsa-stained cremaster muscle whole mounts after 5 min fMLP superfusion (1 μM) in the TNFα-model in WT, Icam-1 ^−/−, and RAGE ^−/− mice with and without PC treatment (100 U/kg, 3 hours) (a–f). Reference bar is shown in (c). Arrows point to extravasated leukocytes.

Figure 4

Effect of PC treatment on bronchoalveolar neutrophil emigration during LPS-induced ALI in wild-type, RAGE ^−/−, and Icam-1 ^−/− mice. In a 6-hour model of LPS-induced ALI, the number of intra-alveolar neutrophils obtained by bronchoalveolar lavage was analyzed in wild-type, Icam-1 ^−/−, and RAGE ^−/− mice treated with 100 U/kg PC 30 min after intratracheal LPS instillation (Escherichia coli 0111:B4 (10 μg LPS/50 μL PBS)). All values are presented as mean ± SEM from at least three mice per group. Significant differences (P < 0.05) to WT control mice are indicated by the asterisks.

Figure 5

Effect of PC treatment on survival during LPS-induced endotoxemia in wild-type, RAGE ^−/−, and Icam-1 ^−/− mice. Lethal endotoxemia was induced by Escherichia coli LPS (serotype 055:B5, 40 mg/kg i.p.) and treated with PC (100 U/kg, i.p.) or human albumin (8 mg/mL) as controls at 0.5, 8, and 24 h after LPS challenge. Survival is shown in Kaplan-Meier plots for the respective treatments in Icam-1 ^−/− mice (a) and RAGE ^−/− mice (b) and compared to WT mice. Significant differences by log-rank test were set at P < 0.05 and indicated by asterisks.

Figure 6

Effect of PC treatment on lung inflammation during LPS-induced endotoxemia in wild-type, RAGE ^−/−, and Icam-1 ^−/− mice. Lethal endotoxemia was induced by Escherichia coli LPS (serotype 055:B5, 40 mg/kg i.p.) and treated with PC (100 U/kg, i.p.) or human albumin (8 mg/mL) as controls at 0.5, 8, and 24 h after LPS challenge. Lungs of WT mice (a and b), Icam-1 ^−/− mice (c and d), and RAGE ^−/− mice (e and f) with and without PC treatment were harvested 24 h after LPS challenge and prepared as 3 μm paraffin-embedded sections for H&E staining. Representative micrographs are shown for at least three mice per group. Arrows indicate infiltrating neutrophils. Reference bar for (a)–(f) is shown in (a) and represents 50 μm.

Figure 7

Effect of PC on intracellular RAGE expression in endothelial cells. Intracellular RAGE expression of TNFα-stimulated (25 ng TNFα/mL, 4 h) endothelial cells was measured by flow cytometry after permeabilization with saponin in response to PC (5 U/10⁶ leukocytes, 3 h) and compared to respective isotype controls. Mean fluorescence intensities are presented as mean ± SEM from at least three mice per group. Significant differences (P < 0.05) to isotype and anti-RAGE + PC are indicated by the asterisks.

Figure 8

Effect of PC on endothelial ICAM-1 expression. Surface ICAM-1 expression was measured on freshly prepared (a) and TNFα-stimulated (25 ng TNFα/mL, 4 h) (b) endothelial cells with or without PC preincubation (5 U/10⁶ leukocytes, 3 h) and compared to respective isotype controls. Mean fluorescence intensities are presented as mean ± SEM from at least three mice per group. Significant differences (P < 0.05) to isotype and anti-ICAM-1 + PC are indicated by the asterisks.