Mucosal tolerance to E-selectin provides cell-mediated protection against ischemic brain injury

Abstract

We have demonstrated that induction of mucosal tolerance to E-selectin, a cytokine-inducible adhesion molecule restricted to activating blood vessels, prevents ischemic and hemorrhagic stroke in spontaneously hypertensive, genetically stroke-prone (SHR-SP) rats. We now examine whether mucosal tolerance to E-selectin has protective effects in ischemic brain damage after permanent middle cerebral artery occlusion (MCAO) in SHR-SP rats and whether these effects are related to generation of regulatory T cells. Rats were exposed to intranasal administration of E-selectin every other day for 10 days (single tolerization group) or on two tolerization schedules separated by 11 days (booster tolerization group). Control groups received PBS on corresponding schedules. MCAO was performed 48 h after the last dose of E-selectin or PBS. There were 45.8% and 37.9% (P < 0.05) decreases of infarction volume in the E-selectin booster group compared with the PBS group at 6 and 48 h, respectively. Single tolerization with E-selectin had only a slight trend toward a decrease in infarction volume (6.3%). CD8-positive cells were decreased in brains of E-selectin booster animals (46.6%, P < 0.01) compared with controls; splenocyte-culture supernatant levels of IL-10 were increased (59.3%, P < 0.05) in E-selectin booster animals. A decrease of infarction volume (34%, P < 0.05) was also observed in SHR-SP rats subjected to MCAO after adoptive transfer of splenocytes from E-selectin-tolerized compared with PBS-tolerized donors. The results indicate that, in addition to preventing stroke, mucosal tolerance to E-selectin is cytoprotective. Thus, immunomodulation targeted to activated blood vessel segments can both reduce stroke occurrence and attenuate brain damage if a stroke supervenes.

Fig. 1.

Relative blood flow in the contralateral and ipsilateral hemispheres. Data are presented as percentage of baseline blood-flow value (n = 5 per group). Line A, the corresponding core site in the contralateral hemisphere; lines B and C, the ischemic perifocal and core region, respectively, in the ischemic hemisphere.

Fig. 2.

DTH reaction in E-selectin-tolerized compared with PBS-tolerized SHR-SP rats. Ear thickness determination was as described in Materials and Methods; rat treatment consisted of tolerization to PBS or E-selectin (E) after immunization and sensitization with E. An unpaired t test revealed a significant difference between the two groups (n = 3; ^*, P < 0.05). Data are presented as mean ± SEM.

Fig. 3.

Infarct volumes (corrected for edema) of ischemic hemisphere in PBS- and E-selectin-tolerized animals. (A) Coronal brain sections from the 48-h ischemia group stained with cresyl violet at levels of bregma 2.70, 1.6, -2.12, and -2.56 mm. Pale areas represent infarction. Summary data for these representative sections are displayed in C. Infarct volumes (mean mm³ ± SEM) for single-tolerization SHR-SP rats at 48 h (D) and booster-tolerization SHR-SP rats at 6 h (B) and 48 h (C) after MCAO are compared with simultaneously prepared PBS-treated controls. An unpaired t test revealed a significant difference between booster-tolerization groups (n = 5-10; ^*, P < 0.05; ^**, P < 0.01).

Fig. 4.

The level of IL-10 in the supernatants of splenocyte cultures. A Wilcoxon signed-rank test revealed a significant difference between the two groups (n = 5; ^*, P < 0.05). Data are presented as mean ± SEM.

Fig. 5.

Infarct volumes (corrected for edema) of ischemic hemisphere in adoptive transfer experiments. Infarct volume is presented (mean mm³ ± SEM) as measured 6 h after MCAO. An unpaired t test revealed a significant difference between the two groups (n = 6-7; ^*, P < 0.05).