Breaking Down the Mechanisms behind CD43 Shedding on T Lymphocytes and Neutrophils Carl Earl Lambert, Jr1, Jonathan M. Green2, Biology Department, Washington University, St. Louis, MO1, Departments of Medicine and Pathology and Immunology, Washington University School of Medicine, St. Louis, MO2 AbstractT cells and neutrophils are major regulatory cells and mediators of the immune system. T cells are able to destroy pathogens after their fragmentation and presentation of pathogenic peptides by MHC molecules. However, T cell directed immune responses involve many surface receptors, called accessory receptors, which lead to the activation of intracellular signaling cascades. This whole process is known as the formation of the immunologic synapse. In such a process, strict regulation of adhesion molecule expression is necessary, or else unwanted adhesive action, or the lack thereof, can lead to disease. The focus of our laboratory is to understand the role of these accessory proteins in the activation of T cells in the presence of a specific antigen. CD43 (also known as leukosialin or sialophorin) is one of the most abundant accessory proteins on the surface of a T cell, covering approximately 28 percent of a T cell’s surface. However, CD43 remains to be one of the least understood surface proteins in T cell activation. It has been hypothesized that the large, rigid, and sterically hindered extracellular domain CD43 is a crucial regulator of interactions between the corresponding receptors on T cells and on the APC surface. Supposedly, CD43 sterically hinders the ability of cell surface receptors to bind to ligands located on the APC. However, this hypothesis has not been rigorously tested, and recent findings suggest that steric hindrance may not be the central mechanism in successful T cell activation. Direct visualizations of the immunologic synapse show that the extracellular domain of CD43 is not even involved in the formation of the immunologic synapse. However, it is considered that once the immune response has begun, the ectodomain of CD43 “cleaves” or “sheds” in order to remove any steric hindrance and its large negative charge in order for better T cell receptor and APC ligand binding to occur. Recently, galactoglycoprotein (Galgp), a protein with high homology to the ectodomain of CD43, has been detected in human serum. While the origin of Galgp is unknown, it was suggested that its presence in human serum is due to the ectodomain of CD43 being cleaved or shed during the commencement of the immune response. Through the usage of flow cytometry, it is possible to rigorously test the hypothesis that the CD43 ectodomain is shed during T cell activation. We used phorbol myristate acetate (PMA) to activate neutrophils, which causes CD43 shedding over a relatively short period of time. Through incubating untreated and PMA-treated neutrophils extracted from wild type mice and staining them with fluorescent antibodies directed against CD43 for 1 hour, one can monitor the relative fluorescent intensity of these cells over this period of time. Essentially, these intensities indicate the amount of antibody bound to CD43 and thus allow analysis of the amount of CD43 on the surface of over time. Though it was evident that fluorescence on these neutrophils, and thus the amount of CD43, had greatly decreased, this occurrence could have arisen from two different situations. For, either CD43 could have indeed been shedding or actually have gone through a process known as internalization, in which the CD43 ectodomain reverts back into the neutrophil. In order to test which situation took place, one would use the ELISA (Enzyme-Linked Immunosorbent Assay) technique in order to determine whether the CD43 ectodomain is located in the incubated PMA-activated mouse serum. A sample of this serum is added to a polystyrene well that is coated with antibodies directed against CD43, and time is allowed for all of the CD43 tails present in the serum to bind to the antibodies inside of the well. Next, antibodies that are specific for CD43 and marked with an enzymatic tag are added to the wells. If CD43 is present, the antibody will bind to it. Washing with phosphate buffer solution (PBS) removes any unbound antibodies. Finally, a substrate that will change color from reaction with the enzyme is added to each well. A color change shows the presence of CD43, and hence proves that CD43 was indeed shed during PMA activation over time. Unfortunately, due to time constraints, we were not able to execute this part of the experiment. As there remains much to know and understand about the mechanisms behind CD43 shedding and its relationship to T cell activation, future experiments will continue towards the discovery of more of this protein’s functions. For instance, we will define the target site on CD43 cleavage and mutate the DNA sequence of CD43 to inhibit cleavage. Such a mutation will shed more light upon how this protein functions Scholar Index Next   Natural Sciences Learning Center Washington University - Biology All contents copyright © 2003 Email comments to nslc webmanager