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Ischemia-reperfusion injury induces the expression of an immune response gene, MHC II-DR-[beta], in the host peripheral T lymphocytes via an alloantigen-independent pathway : (a swine model of single lung transplantation)

University of British Columbia

The complex and multifactorial pathogenesis of ischemia-reperfusion injury (IRI) has made IRI one of the major challenges faced by organ transplantation. Recently, many attempts have been made to establish a relationship between IRI and organ rejection. IRI is now believed to increase the chance of organ rejection via the release of inflammatory mediators such as platelet-activating factor (PAF) which leads to activation of inflammatory cells, causing tissue damage and increased immunogenecity of the graft. To our knowledge, all studies have focused on the effect of IRI on the transplanted donor organ. However, whether IRI can induce an immune response in the host has not been investigated. The objective of this thesis was to investigate whether ischemia-reperfusion injury, independent of tissue incompatibility, could induce MHC H-up-regulation in host peripheral T lymphocytes in a swine model of single-lung transplantation and whether PAF played a role in the mechanism of MHC II up-regulation due to IRI. Single lung transplantation was performed on three groups of domestic swine. Group A/E (n=7) and group B (n=6) had ex vivo preservation times of 4 hr and 15 hr respectively at 4°C hypothermia. To eliminate the allogenecity effect, group C (n=6) underwent 2 hours of warm ischemia via dissection and isolation of the left lung with ligation of its bronchial artery and cross-clamping of the left pulmonary artery, vein, and bronchus without explantation. PAFantagonist, TCV-309, in combination with prostaglandin Et (PGEj) was administered before and during surgery in group E. The sham-operated group (group D, n=6) was used as the control group. Blood samples were collected at pre, one, two, three day post-reperfusion for two-color flow cytometry analysis using swine anti-CD3 and anti-MHC II-DR-β antibodies. Blood samples were also collected for semi-quantitative and competitive reverse transcription polymerase chain reaction (RT-PCR) analysis at pre, two hr, one, two, and three day postreperfusion. Tissue culture experiments were performed using purified resting/proliferating T lymphocytes and T lymphocytes in the presence of accessory cells [peripheral blood lymphocytes (PBL)] treated with PAF only and PAF + TCV-309. The cells were incubated for 4 days at 37°C. Samples were removed every day, and the level of MHC II intensity in T lymphocytes was measured. The results indicated that the level of MHC II-DR-β intensity on host's peripheral T lymphocytes increased significantly (p<0.05) on day 2 post-reperfusion for group A, B, and C. Comparison between groups suggested that the level of MHC II intensity increased as the ex vivo preservation time was extended from 4 hr to 15 hr. The intensity of MHC II on peripheral T cells in group C appeared to be higher than that in group A and B. Results from group D indicated that administration of TCV-309 + PGEj inhibited the up-regulation of MHC II. RTPCR analysis indicated that the steady state level of MHC II mRNA in T cells significantly increased by 2 hr post-reperfusion (p<0.01) for group B and C. The results of tissue culture experiments indicated that PAF at 10⁻¹ M caused a small but significant increase (p<0.05) in MHC II surface expression in T cells only in the presence of accessory cells by day 2 posttreatment. The results of this study indicated that IRI by itself, independent of tissue incompatibility, induced an immune response in the host manifested by MHC II up-regulation in host peripheral T cells. The result also suggested that MHC II up-regulation increased further as the ex vivo preservation time increased. Furthermore, PAF appeared to play a role in the mechanism of MHC II up-regulation in T cells. However, this effect appeared to be an indirect one via intervention of accessory cells. TCV-309 in combination with PGE₁ appeared to have preventive effects against IRI-induced immune response and a potential for clinical application.

Thesis/Dissertation

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2009-06-02

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http://hdl.handle.net/2429/8584

Pathology

University of British Columbia

UBCV

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