No 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.

In humans, intestinal epithelial FcRn is expressed throughout life and mediates the bidirectional transport of IgG, but in mice, it is markedly expressed in neonatal intestine. In adults, its expression is only faintly upregulated after intestinal IgG induction such as that elicited by i.p. immunization with Cry1Ac protoxin (pCry1Ac) Bacillus thuringiensis. This led us to suggest that additional Fcγ receptors (Fcγ‐R) may be participating in epithelial IgG uptake. So, first we determined whether CD16/32 [an epitope shared by Fcγ‐RII (CD32) and Fcγ‐RIII (CD16)] was expressed in the intestinal epithelia of mice. Using confocal microscopy and flow cytometry, we detected co‐localization of IgG and CD16/32 in epithelial cells, whose frequency was increased by immunization with pCry1Ac. Western blot and cross‐immunoprecipitation results with anti‐CD16/32 and IgG antibodies in epithelial cell extracts suggested that epithelial cells bear both Fcγ‐RII and Fcγ‐RIII and contained IgG associated with Fcγ‐RII/RIII. Using anti‐CD32 and anti‐CD16 antibodies, we confirmed by Western blot, confocal microscopy and flow cytometry that both Fcγ‐RII and Fcγ‐RIII were expressed and suggested that upregulation occurred upon immunization in intestinal epithelia. Finally, we examined the in vitro effect of anti‐CD16/32, anti‐CD16 and anti‐CD32 antibodies on IgG uptake and transport by intestinal epithelial cells and found that it was partially reduced. Although further studies are still required, our results suggest that Fcγ‐RII and Fcγ‐RIII might participate in the uptake and/or transport of IgG through the intestinal epithelia of adult mice.