Recent research has begun to elucidate the immune function of platelets. A few clinical studies have reported platelet accumulation in renal transplants during acute rejection. However, the role of platelets in transplant rejection has not been fully examined.

We hypothesized that, in antibody-mediated rejection (AMR), circulating donor specific antibodies activate platelets by inducing elevation of platelet-activating molecules such as Von Willebrand factor (vWf) on stimulated endothelial cells. Activated platelets may enhance inflammatory responses in the grafts.

B10.A kidneys were orthotopically transplanted to B6 rag-/- mice. After postoperative inflammation subsided, a mixture of IgG1, 2a and 2b monoclonal antibodies to H-2a (or isotype controls) were transferred i.p. in single or multiple doses. By 1 hr after a single dose of alloantibodies, C4d and C3d deposited on peritubular and glomerular capillaries in a strong, diffuse pattern. Extensive aggregates of P-selectin and vWf positive platelets co-localized with C4d and C3d. Capillaries also contained platelet-monocyte conjugates. When 4 doses of alloantibodies were transferred every other day for one week to replicate a more sustained alloantibody response and recipients were sacrificed an hour after the last dose, increased Mac2 positive macrophages were localized to the glomeruli in a focal and segmental pattern. Macrophage counts on 100 glomeruli per allograft established that 28% of the glomeruli in alloantibody treated recipients had ≥3 macrophages per glomerulus compared to 7% in controls. Glomeruli with increased macrophages also contained platelet aggregates. Many macrophages had recently proliferated as indicated by Ki67 staining.

Platelet depletion treatment by injecting anti-CD42b antibody 1 hr prior to alloantibody transfer, decreased percentage of glomeruli with ≥4 macrophages per glomerulus from 27% to 12%. The platelet depleted group also had lower levels of pro-inflammatory cytokines, including IL-1b, CCL5, MCP-1 and MIP-1a measured by ELISA and qRT-PCR.

In summary, our model demonstrated that alloantibodies cause pathologic features of transplant glomerulitis. It also established that antibodies initiate rapid localization of platelets in glomeruli followed by macrophages. Platelet depletion treatment decreased numbers of infiltrating macrophages and levels of inflammatory cytokines in kidney transplants. Effects of platelets depletion on glomeruli microvasculature is being further examined by laser capture microdissection.

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