Endothelial histamine H1 receptor signaling reduces blood-brain barrier permeability and susceptibility to autoimmune encephalomyelitis

Abstract

Disruption of the blood-brain barrier (BBB) underlies the development of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis. Environmental factors, such as Bordetella pertussis, are thought to sensitize central endothelium to biogenic amines like histamine, thereby leading to increased BBB permeability. B. pertussis-induced histamine sensitization (Bphs) is a monogenic intermediate phenotype of EAE controlled by histamine H(1) receptor (Hrh1/H(1)R). Here, we transgenically overexpressed H(1)R in endothelial cells of Hrh1-KO (H(1)RKO) mice to test the role of endothelial H(1)R directly in Bphs and EAE. Unexpectedly, transgenic H(1)RKO mice expressing endothelial H(1)R under control of the von Willebrand factor promoter (H(1)RKO-vWF(H1R) Tg) were Bphs-resistant. Moreover, H(1)RKO-vWF(H1R) Tg mice exhibited decreased BBB permeability and enhanced protection from EAE compared with H(1)RKO mice. Thus, contrary to prevailing assumptions, our results show that endothelial H(1)R expression reduces BBB permeability, suggesting that endothelial H(1)R signaling may be important in the maintenance of cerebrovascular integrity.

Fig. 1.

Endothelial-specific H₁R activation in vivo. (A) Brain endothelial cells from H₁RKO and H₁RKO-vWF^H1R Tg mice were stained with anti-HA mAb, and HA-H₁R expression (red) was visualized by confocal microscopy. Nuclei were stained with DAPI (blue). (B) Isolated brain endothelial cells were loaded with the Ca²⁺-sensing dye Fluo-4, and the change in Fluo-4 fractional fluorescence after stimulation with the H₁R agonist 2-[(3-trifluoromethyl)phenyl] histamine dimaleate was measured by real-time confocal microscopy.

Fig. 2.

Decreased BBB permeability mediated by endothelial H₁R expression. (A) BBB permeability on day 10 after PTX injection of WT C57BL/6J (n = 9), H₁RKO (n = 9), and H₁RKO-vWF^H1R Tg (n = 6) mice was determined as described in Materials and Methods. Values were normalized to H₁RKO, and a one-sample t test was used to assess significance compared with H₁RKO mice (***P < 0.0001, H₁RKO vs. WT and H₁RKO vs. H₁RKO-vWF^H1R Tg). WT and H₁RKO-vWF^H1R Tg mice were also significantly different from each other (*P < 0.05) as assessed by the Student's t test. (B) Brain endothelial cells from WT, H₁RKO, and H₁RKO-vWF^H1R Tg mice (n = 4 per strain) were stimulated with PBS (no histamine) or with 10 μM histamine for 30 min, and intracellular cAMP was determined by enzyme immunoassay (*P < 0.05; ***P < 0.0001, as assessed by the Student's t test).

Fig. 3.

Endothelial H₁R activation is protective in EAE. EAE was elicited in WT, H₁RKO, and H₁RKO-vWF^H1R Tg mice using a 1× MOG_35–55 + CFA + PTX (A) or 2× MOG_35–55 + CFA (B) immunization protocol. Number in parentheses indicates number of animals per group. Regression analysis revealed that the disease course in mouse strains fit a variable slope sigmoidal curve (shown as solid lines) that was significantly different (P < 0.0001) in all strains as assessed by the extra sum-of-squares F test.

Fig. 4.

Endothelial H₁R expression did not alter the antigen-specific cytokine response in 1× MOG_35–55 + CFA immunized mice. Spleen and draining lymph node cells were isolated from WT, H₁RKO, and H₁RKO-vWF^H1R Tg (n = 15–17 per strain) mice that were immunized 10 d previously with the 1× MOG_35–55 + CFA + PTX protocol. (A) Cells were restimulated ex vivo with the indicated doses of MOG_35–55 for 72 h, and proliferation was determined by [H³]-thymidine incorporation. The mean cpm ± SD were calculated from triplicate wells, and the results shown are representative of two experiments. (B–D) Cells were isolated as in A and restimulated with 50 μg/mL MOG_35–55 for 72 h. Supernatants were harvested and IFN-γ (B), IL-4 (C), and IL-17 (D) levels were determined by ELISA (*P < 0.05; **P < 0.01, as determined using one-way ANOVA with Kruskal–Wallis and Dunn's multiple comparison tests).