Roles Of NF-κB Decoy Oligodeoxyribonucleotides In The Allograft Rejection After Penetrating Keratoplasty

PurposeTo study the roles of NF-kappa B decoy oligodeoxynucleotides (ODNs) in corneal transplant rejection by injecting into the anterior chamber, and to explore the underlying mechanism in it.Methods1. Mouse model of penetrating keratoplasty. Three BALB/C mice animal models of penetrating keratoplasty were established, grafts from C57BL/6 mice. Group A: keratoplasty without any treats. Group B: NF-κB scrambled ODNs. Group C: NF-κB decoy ODNs. Group B and C were injected 1μg/μl scrambledof NF-κB or decoy-ODNs 2μl in anterior chamber respectivly on intraoperative, postoperative three days and five days. Changes in the corneal grafts were recorded by regular observation.2. Immunology and pathology detection after corneal transplantation. On postoperative 3, 7, 14 day and/or the time of rejection, to test the iris/ciliary body and cornea graft of mice in each group: (1) immunohistochemistry for detecting the expression of CD11c, CD80, CD86. (2) eyeball frozen sections for detecting CD4+T cell infiltration; (3) paraffin-embedded for detecting inflammatory cell infiltration of graft.3. RT-PCR analysis for mRNA expression of the chemokine receptor (CCR5, CCR7) and cytokine (IFN-γIL-4, IL-12p40) in the iris/ciliary body tissues on the 3, 7, 14 days postoperatively.Results1. The mean time for allograft rejection in each group. The mean time for allograft rejection in each group A, B, C was 15.5±2.4 days, 13.8±2.6 days, 22.9±4.8 days respectivly. The difference among the three groups was statistically significant (P=19.691, P =0.000). The time for allograft rejection in group C was later than the other two groups. (P=0.000).2. The expression of CD11c, CD80, CD86 in the iris/ciliary body of each group postoperative. The expression of CD11c, CD80, CD86 in normal iris/ciliary body expressed weakly in iris/ciliary body by whole-mount iris, and positive staining cells located in the margin of iris nearly pupil. The expression of CD11c, CD80, CD86 were gradually increasing after keratoplasty in group A and B with positive staining cells distributing in the whole iris and ciliary, up to the peak when rejection, whille the expression of these were lower in group C at the same time.3. Infiltration of inflammatory cells and the mRNA expression of chemokine receptors of each group after keratoplasty. The graft and iris/ciliary body had a large number of CD4+ T cells, and inflammatory cells infiltration in group A and B postoperatively, reaching peak when rejection, and the mRNA expression of the chemokine receptor such as CCR5, CCR7 correspondingly increased. In group C, the local infiltration of inflammatory cells and the mRNA expression of CCR5, CCR7 were lower than those in group A and B.4. The mRNA expression of IFN-γ,IL-12p40 and IL-4 in the iris/ciliary body of each group postoperative. Anterior chamber injection of NF-κB decoy ODNs could reduce the mRNA transcription of Thl-type cytokine such as IFN-γand IL-12 p40, and increase of Th2-type such as IL-4 in the iris/ciliary body. In group A and B postoperatively, the mRNA transcription of IFN-γand IL-12p40 increased dramaticly, while no mRNA expression of IL-4 was detected in iris/ciliary body.ConclusionsAnterior chamber injection of NF-κB decoy oligonucleotide could inhibit the corneal allograft immunorejection in mice. The roles in inhibiting dendritic cells activation, reducing the secretion of Th1-type cytokines and increasing Th2-type in the iris/ciliary body, may be the anti-rejection mechanism inside the anterior chamber. Dendritic cells activation of the iris/ciliary body may be intimatedly associated with occurrence of corneal graft rejection.