Celiac disease IgA modulates vascular permeability in vitro through the activity of transglutaminase 2 and RhoA

Abstract

Celiac disease is characterized by the presence of specific autoantibodies targeted against transglutaminase 2 (TG2) in untreated patients' serum and at their production site in the small-bowel mucosa below the basement membrane and around the blood vessels. As these autoantibodies have biological activity in vitro, such as inhibition of angiogenesis, we studied if they might also modulate the endothelial barrier function. Our results show that celiac disease patient autoantibodies increase endothelial permeability for macromolecules, and enhance the binding of lymphocytes to the endothelium and their transendothelial migration when compared to control antibodies in an endothelial cell-based in vitro model. We also demonstrate that these effects are mediated by increased activities of TG2 and RhoA. Since the small bowel mucosal endothelium serves as a "gatekeeper" in inflammatory processes, the disease-specific autoantibodies targeted against TG2 could thus contribute to the pathogenic cascade of celiac disease by increasing blood vessel permeability.

Fig. 1

Endothelial paracellular permeability to macromolecules, a 4-kD and b 70-kD FITC-dextran, in the presence of celiac disease (CD) and control patient (non-CD)-derived antibodies as well as commercially available anti-transglutaminase 2 (TG2) antibody, CUB7402. Both types of celiac-patient derived antibodies, CD total IgA and CD anti-TG2-specific IgG, significantly increased macromolecular permeability (a, b) when compared to relevant control antibodies, while CUB7402 had no significant effect. Experiments were performed after 24 h incubation with the antibodies and the measurements were carried out 3 h after administration of the macromolecules. Experiments were performed in duplicate and repeated at least three times. Bars represent mean values and error bars standard error of means. Only significant differences (P < .05) between the relevant antibody groups are depicted

Fig. 2

Lymphocyte adhesion on endothelial cells. a Lymphocytes adhered more prominently to endothelial monolayer treated with celiac disease (CD) patient-derived antibodies [CD total IgA and CD anti-transglutaminase 2 (TG2)-specific IgG] when compared to control (non-CD) antibodies. Commercially available anti-TG2 antibody CUB7402 had no effect. b CD IgA increased the expression of the endothelial adhesion molecule E-selectin significantly when compared to non-CD IgA (b) analysed by flow cytometry (b, c) and by immunofluorescent labelling (d, in red). Experiments were performed in duplicate and repeated at least three times. Bars represent mean values and error bars standard error of means. Only significant differences (P < .05) between the relevant antibody groups are depicted

Fig. 3

Lymphocyte transendothelial migration was significantly increased in the presence of celiac disease (CD)-derived antibodies [CD total IgA and CD anti-transglutaminase 2 (TG2)-specific IgG] when compared to relevant control (non-CD) antibodies. Commercially available anti-TG2 antibody CUB7402 had no effect. Experiments were performed in duplicate and repeated at least three times. Bars represent mean values and error bars standard error of means. Only significant differences (P < .05) between the relevant antibody groups are depicted in figures

Fig. 4

a Celiac disease patient IgA (CD IgA) significantly increased the extracellular enzymatic activity of TG2 in endothelial cells when compared to control patient antibodies (non-CD IgA) whereas commercial transglutaminase 2 (TG2) antibody, CUB7402 had no effect. The co-administration of site-directed TG2 inhibitor, R281 prevented the enhancement of the TG2 activity. Bars represent mean TG2 activity in arbitrary units (AU). b Western blot analysis of TG2 expression in endothelial cells without any treatment (basal) and after administration of non-CD and CD IgA. Data are representative of three independent experiments. c Celiac disease (CD) patient IgA (n = 3) and CD patient serum samples (n = 20) bind to purified TG2 both in the presence and absence of R281 in enzyme-linked immunoassay

Fig. 5

Effects of site-directed transglutaminase 2 inhibitor R281 on a 4-kD macromolecule permeability, b lymphocyte adhesion, and c lymphocyte transendothelial migration in the presence of celiac (CD)- and control patient (non-CD)-derived total IgA. TG2 inhibition blocked the effects of CD IgA on macromolecular permeability (a) and lymphocyte transendothelial migration (c), and attenuated their effect on lymphocyte adhesion (b). Bars represent mean values of experiments performed in duplicate and repeated at least three times. Error bars indicate standard error of means. P < .05 was considered significant

Fig. 6

Role of Rho activity in response to celiac disease (CD) patient IgA. a RhoA activity in endothelial cells is significantly increased after 24 h administration with CD but not with control (non-CD) patient IgA. Co-administration of the Rho inhibitor C3 endotoxin together with CD IgA abolished the effect of celiac antibodies on the paracellular permeability of 4 kD FITC-dextran (b) and lymphocyte transendothelial migration (d), and diminished lymphocyte adhesion to endothelial cells (c). Bars represent mean values of experiments performed in duplicate and repeated at least three times. Error bars indicate standard error of means. P < .05 was considered significant