Early effects of gliadin on enterocyte intracellular signalling involved in intestinal barrier function

Abstract

Background and aims: Despite the progress made in understanding the immunological aspects of the pathogenesis of coeliac disease (CD), the early steps that allow gliadin to cross the intestinal barrier are still largely unknown. The aim of this study was to establish whether gliadin activates a zonulin dependent enterocyte intracellular signalling pathway(s) leading to increased intestinal permeability.

Methods: The effect of gliadin on the enterocyte actin cytoskeleton was studied on rat intestinal epithelial (IEC-6) cell cultures by fluorescence microscopy and spectrofluorimetry. Zonulin concentration was measured on cell culture supernatants by enzyme linked immunosorbent assay. Transepithelial intestinal resistance (Rt) was measured on ex vivo intestinal tissues mounted in Ussing chambers.

Results: Incubation of cells with gliadin led to a reversible protein kinase C (PKC) mediated actin polymerisation temporarily coincident with zonulin release. A significant reduction in Rt was observed after gliadin addition on rabbit intestinal mucosa mounted in Ussing chambers. Pretreatment with the zonulin inhibitor FZI/0 abolished the gliadin induced actin polymerisation and Rt reduction but not zonulin release.

Conclusions: Gliadin induces zonulin release in intestinal epithelial cells in vitro. Activation of the zonulin pathway by PKC mediated cytoskeleton reorganisation and tight junction opening leads to a rapid increase in intestinal permeability.

Figures

Figure 1

Effect of gliadin (0.1 mg/ml) on IEC-6 cell cytoskeleton. (A) Fluorescence microscopy of gliadin exposed IEC-6 cells. Incubation for 15 minutes of cultured cells with gliadin caused a reorganisation of actin filaments characterised by redistribution to the cell subcortical compartment and subsequent cell rounding. A normal F-actin fluorescence pattern was observed when cells were exposed to similar concentrations of either zein, a protein from maize (B), or bovine serum albumin negative control (C). The gliadin effect on the actin cytoskeleton was reversible as two hours after withdrawal of gliadin from the culture medium the actin cytoskeleton returned to its basal state (D). Magnification: 40×.

Figure 2

F-actin quantitation by spectrofluorimetry in IEC-6 cells. (A) Gliadin (0.1 mg/ml) induced a time dependent increase in the cellular content of actin filaments, beginning as early as 15 minutes after exposure to the protein. Fluorescence was measured as relative fluorescence intensity units. (B) IEC-6 cells were exposed to gliadin 0.1 mg/ml at increasing time intervals, NBD-phallacidin extracted at the indicated time interval, and measured by spectrofluorimetry. The time profile of actin polymerisation showed a peak at 60 minutes. Actin polymerisation was expressed as per cent of control. n=4 for each time point.

Figure 3

Effect of cycloheximide and CGP41251 on gliadin induced cytoskeleton rearrangement. IEC-6 cells exposed to gliadin were pretreated 30 minutes before and throughout gliadin exposure with either the protein synthesis inhibitor cycloheximide or the protein kinase C (PKC) inhibitor CGP41251. Gliadin exposed cells without pretreatment served as a positive control, while bovine serum albumin (BSA) exposed cells served as negative controls. Pretreatment with cycloheximide did not affect gliadin induced actin polymerisation, suggesting that this phenomenon is independent of new protein synthesis. In contrast, pretreatment with CGP41251 completely blocked gliadin induced actin polymerisation, suggesting that the effect of gliadin on the cell cytoskeleton is PKC mediated. The results were expressed as percentage of actin polymerisation obtained in BSA exposed cells. *p

Figure 4

Effect of gliadin on zonulin release from IEC-6 cells. Gliadin 0.1 mg/ml induced release of zonulin in the cell medium that peaked at 30 minutes post-gliadin incubation and returned to baseline when cells were incubated with gliadin for 60 minutes. BSA, bovine serum albumin. Each point represents the average of four determinations.

Figure 5

Effect of the synthetic peptide FZI/0 or the protein kinase C inhibitor CGP41251 on gliadin induced zonulin release from IEC-6 cells. When added to IEC-6 cells, neither FZI/0 nor CGP41251 induced zonulin secretion. Both molecules did not affect gliadin mediated zonulin secretion, suggesting that their inhibitory effect on gliadin mediated actin polymerisation occurs downstream of secretion of zonulin from IEC-6 cells. The effects of gliadin and bovine serum albumin (BSA) on zonulin release are shown as positive and negative controls, respectively.

Figure 6

Effect of gliadin on tissue epithelial electrical resistance (Rt) in rabbit intestinal mucosa mounted in Ussing chambers. (A) Addition of the α-gliadin peptide led to a significant reduction in Rt which was detected after a few minutes of incubation. The same effect was observed with the gliadin toxic peptide 31–55 but not with the gliadin non-toxic peptide 22–39. No change in Rt was observed in the absence of gliadin (no gliadin). (B) Treatment with the zonulin inhibitor FZI/0 did not affect Rt. The effect of gliadin on Rt decrement was significantly inhibited when the tissue was pretreated with FZI/0.