The use of allogeneic bone to repair osseous defects arising from various causes, including trauma and malignancy, is a widely accepted treatment modality. However, as many as 50% of these procedures fail. Although attributed to a variety of issues, the precise biologic reasons for these failures remains poorly understood. One of the major presumed causes of graft failure is rejection mediated by the host's immune system. Bone is unique as a transplanted tissue because it presents both a specialized matrix as well as calcified mineral. Bone graft rejection, by definition, requires loss of calcified mineral as a direct consequence of a specific immune response. Appropriately coupled bone resorption and formation or both are compromised. It is the investigator's hypothesis that allogeneic bone activates immune cells, the result of which is excessive resorptive activity that leads to the biologic and clinical failure associated with """"""""rejection"""""""". Bone resorption is mediated by osteoclasts. The pathways by which osteoclasts are activated during allograft rejection or incorporation are unknown. The immune system determines whether a particular graft is incorporated or rejected. However, little information exists on the role played by the immune system in bone allograft biology, particularly with regard to the cell mediated response. Therefore, the long term goal of this proposal is to identify and characterize the immune response to bone allografts using functional, serological,biochemical and molecular techniques. Three distinct but interrelated Specific Aims will be pursued. The applicants will: (1) determine the role Class I and Class II antigens play in bone allograft reactivity; (2) identify the immune cells responding to allogeneic bone; and (3) identify the cytokines secreted by bone-activated immune cells. Murine long bone (with and without marrow or fresh frozen) will be used as a source of antigen to stimulate T cells and macrophages in vitro. The resulting alloreactive T cells will be cloned and analyzed for their antigen recognition (Specific Aim I). This analysis will be accomplished by genetic mapping using recombinant inbred strains and by blocking mixed lymphocyte cultures (MLC; responding T cells and macrophages + stimulating allogeneic bone) using monoclonal antibodies directed at specific antigenic epitopes. The types of responding T cells (Specific Aim II) will be determined by FACS analysis, inhibition of MLC response by monoclonal antibody and Northern blot analysis (Specific Aim III). A better understanding of the immune response to allogeneic bone should advance the basic understanding of this process, allow investigators to compare this response to that of soft tissue allografts, and permit the development of modalities which could significantly increase the predictable success of bone grafting.
