Experimental Study On Suppressive Effect Of Chemokine Receptor Antisense Peptide Nucleic Acids On Murine Skin Allograft Rejection

Purpose: We tested the ability of chemokine receptor CCR5 antisense peptide nucleic acids (PNA) and chemokine receptor CXCR3 antisense PNA to suppress rejection of skin allografts in mice, and explored the potential mechanisms of action of CCR5 antisense PNA and CXCR3 antisense PNA. Methods: At the first stage, to detect the role of CCR5 antisense PNA and CXCR3 antisense PNA in the rejection of C57BL/6 allogeneic skin grafts, BALB/c recipients mice were randomly divided into 6 groups and received normal saline (NS), CCR5 antisense oligo, CXCR3 antisense oligo, CCR5 antisense PNA, CXCR3 antisense PNA or CsA respectively. At the second stage, allografts and spleen T lymphocytes were retrieved on day 7 posttransplanting from the groups that were treated with NS, CCR5 antisense PNA, CXCR3 antisense PNA or CsA. mRNA expression of chemokine (MCP-1, MlP-la, eotaxin, RANTES, Mig and IP-10) and chemokine receptor (CCR5 and CXCR3) in skin grafts were measured by real-time quantitative RT-PCR technique. Histologic evaluation was undertaken using hematoxylin and eosin-stained paraffin sections. CCR5+ cells and CXCR3"l~cells in skin grafts were detected by immunohistochemistry. Spleen T lymphocytes chemotactic activity was performed using a 48-well microchamber technique. CCR5 or CXCR3 expression on spleen T lymphocytes was detected by flow cytometry. Results: In comparison with negative controls (8.12+0.64 days), treatment of allograft recipients with CCR5 antisense PNA (11.75 + 1.66 days) and CXCR3 antisense PNA (12?.58 days) resulted in significant prolongation of skin allografts survival, but CCR5 antisense oligo (8.28+0.95 days) and CXCR3 antisense oligo (8.38?.76 days) had no effectson allografts survival. Infiltrating mononuclear cells, CCR5 mRNA and CXCR5+ cells, but not CXCR3 mRNA, CXCR3 + cells or chemokine mRNA, were significantly reduced in allografts from recipients treated with CCR5 antisense PNA compared with negative controls. Spleen T lymphocytes from the same recipients displayed reduction in chemotaxis toward CCR5 ligands (RNATES, MIP-1a) and surface expression of CCR5. In allografts from recipients treated with CXCR3 antisense PNA, infiltrating mononuclear cells, CXCR3 mRNA and CXCR3+cells, but not CCR5 mRNA, CCR5+cells or chemokine mRNA, were significantly reduced compared with negtive controls. Spleen T cells from the same recipients displayed reduction in chemotaxis towards CXCR3 ligand (Mig, IP-10) and surface expression of CXCR3. In comparison, treatment with CsA did not significantly alter the above parametes except for the reduction of infiltrating mononuclear cells. Conclusions: Both CCR5 antisense PNA and CXCR3 antisense PNA are effective agents in countering transplant rejection. It appears that the mechanism of action of them stems from their ability to inhibit the migration of activated immune cells, particularly activated T lymphocytes, to intragaraft. This may be because of selective inhibition of CCR5 and CXCR3 expression. These data strongly suggest that CCR5 and CXCR3 play important roles in allograft rejection, and therapies to interference with the interaction of CCR5/ligand or CXCR3 /ligand will have a positive effect on preventing transplantation rejection.