REJECTION-MEDIATED INCREASE IN SMALL BOWEL (SB) EPITHELIAL TIGHT JUNCTION PERMEABILITY REQUIRES GRAFT EPITHELIAL NF-KAPPAB ACTIVATION

Abstract

O389 Introduction: Sepsis is the major cause of morbidity and mortality in SB transplant recipients and enteric organisms are often responsible. Increased SB epithelial permeability has been shown to correlate with small bowel (SB) allograft rejection in humans and in animal models of SB transplantation. This compromise in SB allograft epithelial barrier function may exacerbate rejection and contribute to sepsis. The mechanism of rejection-mediated SB epithelial barrier dysfunction has not been defined. Tight junction proteins play a major role in regulating permeability in the intact SB epithelium. We hypothesize that alterations in graft epithelial tight junction function results in epithelial barrier dysfunction through NF-kappaB dependent mechanisms. Methods: SB tissues from C57BL6 → C57BL6 SB isografts and C57BL6 → BALB/c SB allografts were examined during early allograft rejection (POD 5). Mucosa permeability to different sized molecular probes (Alexa 350 hydrazide, FITC-Dextran 3000, and Texas Red-Dextran 10,000) were measured by Ussing Chamber. Tight junction-associated protein expression was examined by fluorescent staining with antibodies to occludin, claudin, ZO-1, and p-MLC. To determine the role of NF-kappaB activation in regulating tight junction protein expression following SB transplantation, SBs from double transgenic mice selectively expressing a mutated form of IkappaBalpha (mIkappaBalpha, a super-repressor of NF-kappaB) gene in their intestinal epithelial cells (mIkappaBalpha) were transplanted to BALB/c recipients. Results: 1) Ussing chamber studies identified a significant increase in mucosal to serosal flux of larger probes (i.e. Dextrans 3000 and 10,000) in the segments of the SB allograft compared to SB isograft (p<0.05) on POD 5. Flux of the smaller probe (Alexa 350 hydrazide) was not different from the isograft. 2) In SB isografts there were no differences in the epithelial distribution of these proteins compared to normal non-transplanted C57BL6 SB tissues. Conversely, the epithelial distribution of ZO-1, occludin, and p-MLC was altered in SB allografts compared to isografts and normal SBs. Specifically, while in the isograft and normal SB epithelium there was a smooth, linear, sub-membrane distribution of ZO-1, in SB allografts the distribution was beaded and irregular especially when viewed in tangential sections. More dramatic differences were seen with the epithelial distribution of occludin and p-MLC. While the epithelia of isografts and normal SBs had normal expression of occludin and p-MLC at the tight junctions, the SB allograft epithelia had a redistribution of these components from the tight junctions to intracytoplasmic inclusions. 3) Normal epithelial distribution of ZO-1, occludin, and p-MLC was seen in mIkappaBalpha allografts, suggesting a role of NF-kappaB activation in regulating tight junction functions during SB allograft rejection. Conclusions: These results indicate that early SB allograft rejection is associated with a defect in graft epithelial barrier function, which may be attributed to the changes in epithelial tight junction associated proteins. Furthermore, this rejection-mediated epithelial tight junction dysfunction is NF-kappaB–dependent.