IMMUNOHISTOCHEMICAL ANALYSIS OF WKY->F344 RAT SKIN ALLOGRAFTS AND CYTOKINE GENE EXPRESSION IN DRAINING LYMPH NODES: COMPARISON OF IMMUNOCOMPETENT ANIMALS TO ANIMALS TOLERIZED WITH DONOR-SPECIFIC LUNG ALLOGRAFTS

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158 We have previously shown that F344 animals develop a spontaneous functional tolerance of WKY lung allografts. They also show long-term retention of donor-specific skin grafts (63 vs. 12 days, without prior lung transplantation), but reject third party skin grafts (Transplantation 1995; 59:1509). To investigate immunologic mechanisms potentially responsible for the prolonged skin graft survival in animals tolerized with lung allografts, here we examine the following parameters: a) macrophage and lymphocyte infiltration in the skin grafts; b) number of Langerhans cells (LCs) in epidermal sheets of the grafts, as an indicator of LCs migration to draining lymph nodes and c) cytokine gene expression in draining lymph nodes. Methods: In the rejection group, WKY skin grafts were placed on normal F344 rats, while, in the tolerance group, the skin grafts were placed on F344 rats that had received a WKY lung transplant 35 days prior to skin grafting. Skin grafts were removed on days 1, 3, 5, and 7 and their draining lymph nodes on days 1, 3, 5, 7, and 11 post-grafting from 4 to 6 animals per time point. Immunohistochemical staining was performed on frozen sections of the grafts for CD4+ and CD8+ cells, macrophages, natural killer (NK) cells and B cells. Epidermal sheets were separated from skin grafts and stained for LCs. mRNA levels for γ-IFN, IL-2, IL-4, IL-10, and TGF-β were determined in lymph nodes by RT-PCR, followed by densitometry. Results: a) No cellular infiltration was detected in either group up to day 3. On day 5, cellular infiltration for CD8+ cells, macrophages and NK cells increased significantly in the rejection group, compared to the tolerance group (P<0.019, p<0.015, and p< 0.015, respectively) and the difference persisted on day 7 (p<0.008, p<0.04, and p<0.03, respectively). Neither CD4+ cells nor B cells were detected at any time point in either group. b) In both groups, there was a similar reduction in the number of LCs in the epidermis of the grafts, with no significant difference seen between the two groups. c) There was a significantly increased expression of γ-IFN mRNA in the rejection group on day 5 (p<0.002), day 7 (p< 0.006) and day 11 (p<0.05) and of IL-2 mRNA on day 1 (p<0.03), day 5 (p<0.02), day 7 (p<0.04) and day 11(p<0.04). No differences were detected in expression of IL-10 and TGF-β, while IL-4 was rarely detected. Conclusions: Reduced Th1 (γ-IFN and IL-2) cytokine responses in the draining lymph nodes and lower infiltration rates of CD8+ cells, macrophages and NK cells in the skin grafts of functionally tolerant animals may play a role in the prolongation of skin graft survival after lung transplantation. The equal reduction of LCs in the grafts of both groups suggests that migration of LCs to lymph nodes takes place at a similar rate, but T cell responses in the lymph nodes are muted in animals rendered functionally tolerant by lung allografts, resulting in improved skin allograft survival.