Immunization with Cry1Ac from Bacillus Thuringiensis Increases Intestinal IgG Response and Induces the Expression of FcRn in the Intestinal Epithelium of Adult Mice

Abstract

We have shown that Cry1Ac protoxin from Bacillus thuringiensis is a potent mucosal and systemic immunogen with adjuvant properties. Interestingly, we have observed that Cry1Ac preferentially induces high specific IgG responses in intestinal fluid when it is intraperitoneally administered to mice; therefore, in the present study, we used this protocol, as a model to address the influence of systemic immunization on the induction of the intestinal IgG response. The data shown indicate that upon intraperitoneal immunization with Cry1Ac, significant intestinal specific IgG cell responses were produced in the lamina propria, accompanied by an increased frequency of intestinal IgG+ lymphocytes and epithelial cells containing IgG. Considering that FcRn is the receptor responsible for the transport of IgG in neonatal intestinal epithelia, but it is developmentally downregulated in the rodent intestine, we analysed whether upon intestinal IgG induction, FcRn mRNA expression was induced in intestinal epithelial cells, of adult mice. Whereas in intestinal epithelia of unimmunized adult mice FcRn mRNA was not detected, in Cry1Ac immunized mice it was expressed, although the level was lower in comparison with that found in neonatal epithelia. Then using flow cytometry and immunofluorescence we confirmed that the expression of the protein FcRn was induced in the intestines of adult immunized mice especially in the large intestine. Finally, we found that Cry1Ac also increased FcRn expression in isolated intestinal epithelial cells stimulated in vitro. The outcomes suggest that the expression of FcRn in intestinal epithelium might be reactivated upon immunization, and possibly facilitate IgG transport.

Intraperitoneal Immunization with Cry1Ac Protoxin from Bacillus thuringiensis Provokes Upregulation of Fc‐Gamma‐II/and Fc‐Gamma‐III Receptors Associated with IgG in the Intestinal Epithelium of Mice

“…immunization of mice with pCry1Ac is achieved an increased proportion of lymphocytes IgG+ located in the lamina propria accompanied with increased proportion of IgG+ epithelial cells [12], we used this protocol as a model of intestinal IgG induction to determine whether Fcc-R such as Fcc-RII and Fcc-RIII were expressed in the intestinal epithelia of mice. Firstly, we examined by immunohistochemistry in intestinal sections whether CD16/32 was expressed in the intestinal epithelia of mice.…”

“…Intestinal epithelial cells were isolated as described previously [12]. Briefly, for intestinal epithelial cell isolation, discontinues 20%, 40% and 70% Percoll gradients were used, and intestinal cells recovered after EDTA treatment were centrifuged at 453 g for 30 min at room temperature.…”

“…Although, in a previous study, we have observed that the expression of FcRn at the intestinal epithelium of adult mice can be fairly expressed upon intestinal IgG induction, this was too slight to account for the substantial epithelial IgG recorded [12]. As a model of intestinal IgG induction, we have used intraperitoneal immunization of mice with Cry1Ac protoxin (pCry1Ac) from Bacillus thuringiensis, which is a potent mucosal and systemic immunogen with adjuvant properties [13][14][15], because this treatment preferentially induces high specific IgG responses in intestinal fluids produced by intestinal lamina propria lymphocytes and an increased proportion of intestinal lymphocytes expressing IgG.…”

“…Tissue slices 5-6 mm thick were fixed in a freshly prepared ZSF solution (0Á1 M Tris base buffer with Ca acetate 0Á05% [pH 7-7Á4], containing Zn acetate 0Á5% and Zn chloride 0.5%) [17]. For CD16/32 detection in intestinal 5-lmthick tissue sections, a standard three-stage indirect immunoperoxidase technique (in which endogenous peroxidase, avidin or biotin were eliminated) was performed as described previously [12]. Blocked slides were then incubated with biotin-conjugated anti-mouse CD16/32 mAb diluted 1:1000 in the blocking buffer for 2 h at room temperature.…”

“…As a model of intestinal IgG induction, we have used intraperitoneal immunization of mice with Cry1Ac protoxin (pCry1Ac) from Bacillus thuringiensis, which is a potent mucosal and systemic immunogen with adjuvant properties [13][14][15], because this treatment preferentially induces high specific IgG responses in intestinal fluids produced by intestinal lamina propria lymphocytes and an increased proportion of intestinal lymphocytes expressing IgG. Interestingly, the high intestinal IgG responses elicited with pCry1Ac were accompanied by a marked increase in frequency of epithelial cells bearing IgG, suggesting that IgG might be uptaken or transported through the epithelia [12]. So, the slight upregulation of FcRn in the epithelia in comparison with the marked increase of epithelial cells bearing IgG observed upon immunization with pCry1Ac led us to propose that additional Fcc receptors (Fcc-R) may be participating in the uptake of IgG by epithelial cells.…”