An antigen system expressed on human endothelial cells and monocytes, which is distinct from major histocompatibility encoded antigens HLA- class I and II, is thought to play an important role in renal and cardiac allograft rejection and certain peripheral vascular autoimmune diseases. In spite of this clinical significance, very little is currently known with respect to the biochemistry and structure of these antigens as well as the genes encoding for them. Using monoclonal antibodies (mAb) (SK2H10,SK2F11) specific for human endothelial-monocyte (EM) antigens developed in our laboratories, we have determined the cellular expression and biochemistry of two of these EM antigens. Furthermore, we have obtained strong evidence that the mAb to EM antigens and EM specific alloantibodies present in allograft recipients share antigenic determinants. Thus, studies proposed in this application are designed to further characterize EM antigens utilizing the mAb as a tool in order to define the biology and immunopathogenesis of this new antigenic system. Towards this objective, we will employ a unique cDNA clone (KA13) identified by immunoscreening of an umbilical vein endothelial expression library using the SK2H10 mAb. We will determine the clinical significance of this unique EM antigen encoded by cDNA clone KA13 by testing serial sera from allograft recipients on KA13 transfected cell lines and quantitating the message in rejected allograft biopsies using semiquantitative polymerase chain reaction and northern blot analysis. To understand the regulation of the KA13 gene we will study the levels of KA13 mRNA induction and its stability in endothelial cells and monocytes subsequent to incubating with agents which are shown to augment the cell surface expression of these antigens. The involvement of transcriptional and posttranscriptional regulation will also be established. Polymorphism of the KA13 gene will be determined using restriction fragment linked polymorphism and direct sequencing. The chromosomal location of the gene will also be determined utilizing bioblots of chromosome specific DNA. The hypothesis that EM antigens can transduce intracellular signals which may have important bearing in the immunopathology of both allograft rejection and vascular diseases will be tested by determining whether alloantibodies, autoantibodies, and mAb specific for these EM antigens can activate intracellular Ca++. Measurement of second messengers, e.g., inositol phosphates and diacylglycerol, determination of the involvement of G proteins in the signalling events and activation of protein kinases will be done towards defining the mechanism of intracellular signalling pathways. The overall goal of this proposal is to define the functional and molecular nature of the new endothelial-monocyte antigenic system in order to gain further understanding of its possible role in clinical transplantation and peripheral vascular diseases.
