Neuregulin1-β decreases IL-1β-induced neutrophil adhesion to human brain microvascular endothelial cells

Abstract

Neuroinflammation contributes to the pathophysiology of diverse diseases including stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, and multiple sclerosis, resulting in neurodegeneration and loss of neurological function. The response of the microvascular endothelium often contributes to neuroinflammation. One such response is the upregulation of endothelial adhesion molecules which facilitate neutrophil adhesion to the endothelium and their migration from blood to tissue. Neuregulin-1 (NRG1) is an endogenous growth factor which has been reported to have anti-inflammatory effects in experimental stroke models. We hypothesized that NRG1 would decrease the endothelial response to inflammation and result in a decrease in neutrophil adhesion to endothelial cells. We tested this hypothesis in an in vitro model of cytokine-induced endothelial injury, in which human brain microvascular endothelial cells (BMECs) were treated with IL-1β, along with co-incubation with vehicle or NRG1-β. Outcome measures included protein levels of endothelial ICAM-1, VCAM-1, and E-selectin, as well as the number of neutrophils that adhere to the endothelial monolayer. Our data show that NRG1-β decreased the levels of VCAM-1, E-selectin, and neutrophil adhesion to brain microvascular endothelial cells activated by IL1-β. These findings open new possibilities for investigating NRG1 in neuroprotective strategies in brain injury.

Fig.1. Incubation of BMEC with IL-1β does not result in an increase in cell death or a decrease in metabolic activity

After incubation with IL-1β (100ng/ml) for 18h in the presence of vehicle or NRG1-β (100ng/ml or 300ng/ml), changes in metabolic activity and cell death of BMECs were evaluated by LDH (right axis) or MTT assay (left axis). For MTT results, the data is represented as the percent of metabolic activity compared to the vehicle-treated control (shown as the mean + SEM). For LDH, the results are expressed as the percentage of LDH release compared to vehicle-treated control(shown as the mean + SEM). n=3.

Fig. 2. NRG1-β decreases IL-1β induced increases in protein levels of endothelial intercellular adhesion molecules

BMECs were treated with IL-1β (50ng/ml) and co-incubated addition vehicle (PBS) or NRG1-β (12.5nM and/or 37.5nM). Western blots of cell lysates were analyzed for protein levels of adhesion molecules. n=3. 2a. 6h incubation with IL-1β resulted in an increase in a 5-fold (5.1 +/- 1.0, p<0.05) increase in VCAM-1. Co-incubation with 100ng/ml (12.5nM) NRG1-β did not result in a statistically significant difference from IL-1β - treated cells; however, addition of 300ng/ml (37.5nM) NRG1-β resulted in a decrease in VCAM-1 levels to 3.5 (3.5 +/- 1.2) times that of IL-1β - treated cells that were co-incubated with vehicle, which is a statistically significant reduction of 32% (p<0.05).n=3. 2b. 6h incubation with IL-1β resulted in a 5-fold (4.9 +/-1.5, p<0.05) increase in endothelial ICAM-1 levels compared to vehicle-treated controls. BMECs that were co-incubated with NRG1 (300ng/ml or 37.5nM) had ICAM-1 protein levels which were 4.2 x that of IL-1β –treated cells, which does not represent a statistically significant difference. 2c. 6h incubation with IL-1β resulted in an increase in endothelial E-selectin expression, averaging 3.3-fold (3.3 +/- 0.6; p<0.05) increase compared to baseline (*p < .0.01). Co-incubation with 300ng/ml (37.5nM) NRG1-β resulted in a decrease in E-selectin levels to 2.4 (2.4 +/- 0.8) times that of IL-1β treated cells that were co-incubated with vehicle, a statistically significant decrease of 27% (p<0.05) (Fig. 2c). n=3.

Fig. 3. Representative images of neutrophils and endothelial cells in the neutrophil adhesion assay

3a. Cytopsin of PFA-fixed aliquot of mouse peritoneal neutrophils, Wright-Giemsa stained, photographed at 400x. 3b. High magnification view of neutrophils with fluorescent Mitotracker signal, photographed at 400x. 3c. Neutrophils and endothelial cells in neutrophil adhesion assay, photographed at 200x. 3d. mouse peritoneal neutrophils, Wright-Giemsa stained, photographed at 1000x.

Fig. 4. IL-1β dose-finding pilot experiment for neutrophil adhesion assay

At 10ng/ml (n=2), IL-1β increased the neutrophil adhesion 1.8 fold that of the baseline level; at 50ng/ml (n=3): 2.5 fold; at 100ng/ml (n=1): 4 fold. Co-incubation of NRG1- β (300ng/ml or 37.5nM) reduced neutrophil adhesion at each dose of IL-1β. a-g. Representative images of the neutrophil adhesion assay with the conditons listed. 4h. Graphical representation of the results of the dose-finding pilot experiment.

Fig. 4. IL-1β dose-finding pilot experiment for neutrophil adhesion assay

At 10ng/ml (n=2), IL-1β increased the neutrophil adhesion 1.8 fold that of the baseline level; at 50ng/ml (n=3): 2.5 fold; at 100ng/ml (n=1): 4 fold. Co-incubation of NRG1- β (300ng/ml or 37.5nM) reduced neutrophil adhesion at each dose of IL-1β. a-g. Representative images of the neutrophil adhesion assay with the conditons listed. 4h. Graphical representation of the results of the dose-finding pilot experiment.

Fig. 5. NRG1-β reduces the IL-1β induced increase in neutrophil adhesion to human brain endothelial cells

In PBS-treated BMECs, an average of 14 (14 +/- 6.5) neutrophils were adherent to the endothelial monolayer in each high-powered (10x) field. In IL-1β treated endothelial cells, the number of adherent neutrophils increased to 35 (35+/- 12.9), a 2.5 fold increase (p<0.05). In BMECs treated with IL-1β and co-incubated with NRG1-β (100ng/ml or 12.5nM) NRG1-β, the number of adherent neutrophils decreased to the baseline level of 14 (14.6 +/- 4.7; p<0.05compared to BMECs treated with IL-1β and co-incubated with PBS). In endothelial cells treated with IL-1β and 300ng/ml (37.5nM) NRG1-β, the number of adherent neutrophils was 9.9 (9.9 +/-4.0), again a significant decrease from that in cells treated with IL-1β without NRG1-β (p<0.05).