An Investigation into the Effects of the Addition of Synthetic Receptor on Chemokine Induced Jurkat T-Cell Migration Jessica Jurado, Jianfang Hu, Avery August, PhD PSU Undergraduate Animal Bioscience July 2004 Abstract Extravasation is the process in which leukocytes enter irritated or injured tissue. One aspect of extravasation is T-cell migration in which the cells are drawn to damaged tissue from circulation in the blood stream. Chemokines are responsible for alerting T- cells to an area of damaged tissue to illicit an immune response. Recently novel Synthetic Receptors have been synthesized and the effects of the insertion of such Synthetic Receptors on chemokine induced T-cell migration were investigated in this experiment. It was hypothesized that the insertion of Synthetic Receptor into the cell membrane affects cell signaling such that chemokine induced migration will be enhanced. The results show that the percent migration of chemokine induced T-cells with the added Synthetic Receptor doubled compared to % migration of chemokine induced T-cells with no Synthetic Receptor. The % migration of the cells with the Synthetic Receptor increased almost ten fold compared to the control which consisted of cells that had no Synthetic Receptor and no chemokine available. The purpose of this experiment was to determine if chemokine induced migration of T cells would be effected by the addition of Synthetic Receptor and the results show that the percent migration of T-cells with Synthetic Receptor is increased compared to cells with no Synthetic Receptor with chemokine available. Introduction Lymphocytes are cells of the immune system that help our bodies combat foreign antigens, or compounds that can trigger an immune response. Two major types of lymphocytes include B lymphocytes ( B-cells ) and T lymphocytes ( T-cells ). All lymphocytes originate in bone marrow; however T-cells travel to the thymus where they mature. Lymphocytes can be found in various tissues including the lymph nodes and spleen. In order for T-cells to migrate to a site of tissue injury, they must be informed that there is some sort of tissue damage. Chemokines are such informants because as they seep through the vascular endothelium, they can attract T-cells to the site of injury (1). SDF-1α is a chemokine and its receptor, CXCR4, is located in what are called lipid rafts in the cell membrane of T-cells. These lipid rafts are areas in the cell membrane where signaling molecules aggregate (2). Once the cells have migrated through the bloodstream to the location of the chemokines, the cell is activated by chemokines and is able to adhere onto the cells of the vascular endothelium. Examples of such endothelium include high-endothelial venules (HEVs), which are located in the postcapillary venules of some lymphoid organs (1).
Once the chemokine binds to its receptor on the cell membrane of T-cell, the T- cells migrate to an area of increasingly higher chemokine concentration (1). The binding of the chomokine to its receptor on the T-cell induces cell signaling, leading to an increase ability of cell-adhesion molecules (CAMs) to bind to their receptors on the HEVs, this strengthening the interaction between the T-cell and vascular endothelium. Essentially the T-cell is able to stick onto the vasculature. Like a marble on a table, the T-cell begins to roll over the vascular endothelium. Once the cell is firmly adhered to the cells of the endothelium it is then able to migrate through the vessel into the site of tissue damage. It should be noted that the terms migration and adhesion apply to the process of extravasation. Extravasation is the process in which leukocytes (including lymphocytes) enter injured or irritated tissue (1). Human white blood cells naturally contain fragment crystallizable receptors (FcRs) which bind to the constant region (Fc) of Immunoglobulins so that they can be cleared from the body. This results in the Immunoglobulins being swallowed up by the leukocyte and destroyed (1). Novel Synthetic Receptors for the Fc region of Immunoglobulins have recently been synthesized. The Synthetic Receptors mimic FcRs and consist of a plasma membrane anchor, linker, and immunoglobulin-g binding motif. The plasma membrane anchor has a structure similar to cholesterol and is therefore able to stay firmly inside the lipid soluble environment of the plasma membrane. This is unlike the FcR s which must be recruited to the lipid raft before activation. The rest of the molecule is extracellular (3). The purpose of this experiment is to see if the migration rate of Jurkat T-cells towards the chemokine SDF-1α, a ligand of the receptor CXCR4 located on the Jurkat T cell membrane is affected by the addition of Synthetic Receptor. It is hypothesized that the insertion of Synthetic Receptor into the cell membrane affects cell signaling such that chemokine induced migration will be enhanced. Methodology Cells Jurkat T cells were grown in the media detailed below: 5.0 ml of sodium pyruvate 2.5 ml hepes 5.0 ml of non-essential amino acids 25.0 ml fetal bovine serum 5.0 ml penicillin/streptomycin, 1 µl β-mercaptoethanol The above were combined under sterile conditions and adjusted for a final volume of 500 ml. Cells were grown in this media in an incubator set at 37 C with 5% CO 2. Migration Assay About 7 X 10 6 cells were removed from the incubated mixture and centrifuged at 400 x g s for 10 minutes. The supernatant was then removed and the pellet resuspended with 2.8 ml of and placed in ice for further use. The Synthetic Receptor was prepared by mixing 90 µl of sterile dimethyl sulphoxide, 900 µl of media and 10 µl of 1mM Synthetic Receptor DMSO stock. The final 2
concentration of this solution was 1 µm, 1% DMSO. A master mixture of 995 µl of and 5 µl of a 1x10 4 ng/ml stock solution of SDF-1α was prepared and stored in ice until further use. Seven eppendorf tubes were prepared with the following contents: Tube Contents SDF/No Synthetic Receptor 360 µl & resuspended pellet mixture, 40 µl SDF/No Synthetic Receptor 360 µl & resuspended pellet mixture, 40 µl SDF & Synthetic Receptor 360 µl & resuspended pellet mixture, 40 µl Synthetic Receptor SDF & Synthetic Receptor 360 µl & resuspended pellet mixture, 40 µl Synthetic Receptor No SDF/No Synthetic 360 µl & resuspended pellet mixture, 40 µl No SDF/No Synthetic 360 µl & resuspended pellet mixture, 40 µl Next 6 migration chambers were prepared and were labeled in correspondence to the eppendorf tubes. A migration chamber, cap, and filter are pictured below. Figure 1: Migration assay equipment. Above is a picture of the migration chamber, cap, and polycarbonate filter used. The bottom of the migration chambers were filled as follows: Chamber CONTENTS SDF/No Synthetic Receptor 200 ul SDF/ mixture SDF/No Synthetic Receptor 200 ul SDF/ mixture SDF & Synthetic Receptor 200 ul SDF/ mixture SDF & Synthetic Receptor 200 ul SDF/ mixture No SDF/No Synthetic 200 ul No SDF/No Synthetic 200 ul 3
The chambers containing no SDF or Synthetic Receptor served as negative controls. The chamber containing SDF but no synthetic receptor served as the positive control and the chambers containing SDF and Synthetic Receptor will test whether the addition of Synthetic Receptor enhances the migration rate of T-cells. A 13 mm wide, 5-micron thick polycarbonate filter was placed on top of the above contents and the migration chamber cap was tightly fastened to the bottom piece of the chamber. The contents of each eppendorf tube were then added to the corresponding migration chamber cap. The chambers were incubated at 37 C for 3 hours and 5% CO 2. After this time the cell mixture in the migration caps was removed as well as the cap and filter paper. The cells that migrated into the bottom portion of the chamber were then diluted 3 fold with sterilized phosphate buffered saline (PBS) and counted using the Advia TM 120 Hematology System. Results The purpose of this experiment was to see if chemokine induced migration is enhanced by the addition of Synthetic Receptor to the cell membrane of Jurkat T-cells. It was hypothesized that the Synthetic Receptor would enhance migration. To test this hypothesis a migration assay was performed and the % migration of cells under various chemical environments through the filter of a migration chamber was calculated (4). The chambers that served as negative controls consisted of cells with no SDF-1α or cells with Synthetic Receptor. The chambers containing SDF-1α but no Synthetic Receptor served as the positive control. The test chambers contained cells with Synthetic Receptor and SDF-1α available. The migration assay was performed 13 times. After each assay the Advia TM 120 Hematology System was used to calculate the number of cells that migrated per µl. The total number of migrated cells was calculated by multiplying this value by 200 µl (the volume in the bottom portion of the migration chamber). This calculation was carried out for each migration chamber. Cells/µL x 200uL = Number of Migrated Cells The number of migrated cells for each duplicate was averaged. The percent of migrated cells was calculated by dividing the average number of migrated cells by the original amount of cells loaded into the chamber and then multiplying by 100. (Average Number of Migrated Cells/Original Number Cells Loaded per Chamber) x 100 = Percent of Migrated Cells All of the data for the % Migration of each assay was compiled into one figure as shown below: 4
Compiled Data of % Chemokine Induced Migration of Jurkat T Cells SDF SDF & SR Negative Control SDF SDF&SR Control 1 2.3 22.5 44.92 0 10 20 30 40 50 % Migration of Jurkat T Cells Figure 2: Compiled Data of % Chemokine Induced Migration of Jurkat T Cells As expected the negative control which consisted of no Synthetic Receptor or chemokines had the lowest % migration compared to the positive control and the test. Only 2.3 % of the cells loaded into the chambers migrated. By contrast and as expected the chambers containing cells with the Synthetic Receptor and chemokines (positive control) had almost 10 times more cells migrate as compared to the negative control. The percent of cells that migrated was 22.5%. Also as expected the test chambers containing chemokines and cells with Synthetic Receptor had the highest % migration as compared to the negative and positive controls. The % migration of cells loaded into the test chambers was almost double that of the % migration of the cells in the positive control. The test chambers also had about 20 times the % migration of cells as compared to the negative control. This data suggests that the Synthetic Receptor does enhance chemokine induced Jurkat T- cell migration. Discussion The results support the hypothesis that Jurkat T-cell migration would be enhanced by the insertion of Synthetic Receptor into the cell membrane. The % migration of cells loaded into the test chambers was almost double that of the % migration of the cells in the positive control chambers. The test chambers also had about 20 times the % migration of cells as compared to the negative control. It is proposed that this enhanced chemokine induced migration is a result of the interaction between the CXCR4 in the lipid raft of the Jurkat T-cell and the Synthetic Receptor. Lipid rafts are areas in the cell membrane of high levels of cholesterol and glycosphingolipids. The rafts serve to concentrate signaling molecules such as receptors and substrates. The close proximity of these molecules allows for a faster interaction than 5
if the signaling molecules were dispersed in different areas of the cell membrane. Cell signaling is activated when a signaling molecule normally present elsewhere in the cell membrane is recruited to the lipid raft. That way incomplete signaling pathways (or inactivated) become complete (2). Once a Jurkat T-cell encounters the chemokine SDF-1α, the receptor CXCR4 is recruited to the lipid raft of the cell membrane. Once the receptor is in the lipid raft the SDF- α/cxcr4 complex is able to form. Perhaps the reason why there is an increase in chemokine induced cell migration when the Synthetic Receptor is added to Jurkat T-cells is that the Synthetic Receptor aids in stabilizing the CXCR4/SDF-1α complex; therefore leading to enhanced cell signaling. Also the presence of the Synthetic Receptor in the lipid raft may also stabilize other signaling molecules involved in the immune response initiated by the formation of the chemokine/synthetic receptor complex. The results of this experiment could have a clinical application to medicine. For example if the Synthetic Receptor and chemokine SDF-1α could be injected into a site tissue injury, the percent migration of T-cells to the area of tissue injury may increase which would illicit a faster immune response. This project investigated only one aspect of extravasation which is migration. Another aspect is adhesion of T-cells to the vascular endothelium. Future experiments will continue to test this hypothesis by examining the ability of the Synthetic Receptor to affect adhesion. This will be done by performing adhesion assays which will give some insight into the effect of adding the Synthetic Receptor to Jurkat T-cells on the adhesiveness of Jurkat T cells to the cells of the endothelium. The purpose of this experiment was to see if chemokine induced migration is enhanced by the addition of Synthetic Receptor to the cell membrane of Jurkat T-cells. It was hypothesized that the Synthetic Receptor would enhance chemokine induced Jurkat T- cell migration and indeed the results supported this hypothesis. The % migration of cells loaded into the test chambers was almost double that of the % migration of the cells in the positive control chambers. References 1. Goldsby, Richard A. Immunology. 5th ed. New York: W.H. Freeman and Company, 2003. 338-348. 2. Pike, Linda J. "Lipid Rafts: Bringing Order to Chaos." Journal of Lipid Research 44 (2003): 665-667. 3. Scott E. Martin and Blake R. Peterson, A Synthetic Mimic of Human Immunolglobulin Fc Receptors, Submitted for publication 4. Fischer, Angela M., Mercer, J.C., Iyer, A., Ragin, M.J., August, A., et al. "Regulation of CXC Chemokine Receptor 4-mediated Migration by the Tec 6
Family Tyrosine Kinase ITK." Journal of Biological Chemistry 279 (2004): 29816-29820. Acknowledgements Dr. Avery August, Professor of Immunology: Acknowledged for the idea of the experiment, for teaching me new lab techniques, for answering any question I had, for his encouragement, patience, and most importantly for being a mentor. Cindy Mueller, Research Technician: Acknowledged for insightful discussions and answering any technical and conceptual problem I might have had. Jianfang Hu, Graduate Student: Acknowledged for answering any question I asked and for teaching me new lab techniques. Jason Mercer, Graduate Student: Acknowledged for answering any question I asked and for teaching me new lab techniques. Archana Iyer, Graduate Student: Acknowledged for insightful discussions concerning the application of this project. Melanie Ragin, Graduate Student: Acknowledged for insightful discussions concerning the application of this project. Summer Research Opportunity Program Staff: Acknowledged for providing the opportunity and resources to complete this project. 7