1. Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology

2. The Purpose of my Project Studies show that glutamate receptors are a part of the human nervous system and can be localized within the brain Studies show that glutamate receptors are a part of the human nervous system and can be localized within the brain Research within our lab suggests the presence of kainate receptors (a specific type of glutamate receptors) within the human immune system Research within our lab suggests the presence of kainate receptors (a specific type of glutamate receptors) within the human immune system We want to show that kainate receptors can also be found within the rodent immune system We want to show that kainate receptors can also be found within the rodent immune system To date, the presence of kainate receptors has never been shown in the mouse immune system To date, the presence of kainate receptors has never been shown in the mouse immune system If present, the mouse system can used to help expand upon studies on the human immune system If present, the mouse system can used to help expand upon studies on the human immune system Such findings will better link epilepsy and allergies in humans Such findings will better link epilepsy and allergies in humans Studies in humans show that people who have epilepsy are more prone to allergic disease Studies in humans show that people who have epilepsy are more prone to allergic disease

3. Glutamate Receptors http://www.giccs.georgetown.edu/cnsi/research/glur_files/Slide3.JPG

4. Introduction to Immunology Parham, Peter. The Immune System. 2000. Garland Publishing/Elsevier Science Ltd.

5. Why is it important? Kainate receptors are shown to influence ADAM10 ADAM10 is responsible for the cleavage of CD23 (a low affinity IgE receptor and a protein known to regulate IgE synthesis) Cleavage of CD23 influences IgE production, which leads to increased allergies in humans This model has been worked out in the human system: ↑KAR → ↑ADAM10 → ↓mCD23 & ↑sCD23 → ↑IgE synthesis

6. Cleavage of CD23

7. Data RT-PCR and FACS were methods used to determine the presence of kainate receptors within mouse lymphocytes RT-PCR and FACS were methods used to determine the presence of kainate receptors within mouse lymphocytes Calcium Assays and Sodium Assays were performed to determine if KA influx in the immune system is similar to that of the nervous system Calcium Assays and Sodium Assays were performed to determine if KA influx in the immune system is similar to that of the nervous system The nervous system will primarily flux sodium into cells, but a smaller amount of Calcium will also flux into the cell The nervous system will primarily flux sodium into cells, but a smaller amount of Calcium will also flux into the cell

8. RT-PCR Results Naïve B cells, activated B cells, and whole spleen cells were tested with GRIK primers Naïve B cells, activated B cells, and whole spleen cells were tested with GRIK primers Actin ( a house keeping primer) was used as the control Actin ( a house keeping primer) was used as the control It is a good control to use because it can be localized in almost all cell types It is a good control to use because it can be localized in almost all cell types Results: Results: Using GRIK primers, the appropriate location for kainate receptors was present, but faint Using GRIK primers, the appropriate location for kainate receptors was present, but faint A non-specific band was present below the desired band A non-specific band was present below the desired band Actin was present at the appropriate band Actin was present at the appropriate band Conclusion: results were not significant to confirm or reject the presence of kainate receptors on mouse cells Conclusion: results were not significant to confirm or reject the presence of kainate receptors on mouse cells

9. NaiveActiveSpleenNaiveActiveSpleen GRIK primersActin (control) RT-PCR results

10. FACS Fluorescent Activated Cell Sorter Used to analyze the distribution of surface molecules on cells Used to analyze the distribution of surface molecules on cells In this case, it is used to detected the distribution of kainate receptors on mouse lymphocytes In this case, it is used to detected the distribution of kainate receptors on mouse lymphocytes The data is analyzed and graphed using a program on the computer, CXP The data is analyzed and graphed using a program on the computer, CXP

11. FACS results – Proof of Principle FACS was run on human 8866 cells to determine if kainate receptors could be localized on the cells using this method of experimentation FACS was run on human 8866 cells to determine if kainate receptors could be localized on the cells using this method of experimentation Human 8866 cells were used since we have already observed the presence of the receptors on these cells Human 8866 cells were used since we have already observed the presence of the receptors on these cells Results: Results: A significant shift in the graph confirmed the presence of kainate receptors on the human 8866 cells A significant shift in the graph confirmed the presence of kainate receptors on the human 8866 cells

13. FACS Results FACS was run on mouse spleen cells to determine the presence of kainate receptors on mouse lymphocytes FACS was run on mouse spleen cells to determine the presence of kainate receptors on mouse lymphocytes Results: Results: a slight shift in the graph suggested that kainate receptors can be located on mouse spleen cells a slight shift in the graph suggested that kainate receptors can be located on mouse spleen cells

15. FACS data FACS was run on all the different cell types within the mouse spleen to determine the exact cells in which kainate receptors can be localized FACS was run on all the different cell types within the mouse spleen to determine the exact cells in which kainate receptors can be localized B Cells B Cells Naïve, semi-activated, activated Naïve, semi-activated, activated T Cells T Cells Monocytes Monocytes Macrophages Macrophages Natural Killer Cells Natural Killer Cells

16. Presence of Kainate Receptors on B Cells

17. Presence of Kainate Receptors on T Cells

18. Presence of Kainate Receptors on NK Cells and Monocytes

20. Summary of FACS Data B Cells (Naïve, Semi-Activated, and Activated) B Cells (Naïve, Semi-Activated, and Activated) Positive for receptors on all B Cell subsets Positive for receptors on all B Cell subsets T Cells T Cells Positive for receptors on all T Cells, but moderate compared to other cell types Positive for receptors on all T Cells, but moderate compared to other cell types Natural Killer Cells Natural Killer Cells Positive for receptors (showed the highest fluorescence) Positive for receptors (showed the highest fluorescence) Monocytes Monocytes Positive for receptors (showed the highest fluorescence) Positive for receptors (showed the highest fluorescence) Activated Monocytes (Macrophages) Activated Monocytes (Macrophages) Positive for receptors (showed the highest fluorescence) Positive for receptors (showed the highest fluorescence)

21. Calcium Assay The Calcium Assay was performed using ionomycin as a control (to show high influx of calcium into the cells) The Calcium Assay was performed using ionomycin as a control (to show high influx of calcium into the cells) ATPA and DA (agonists of KA) were tested to see if their presence would increase calcium flux into the cells ATPA and DA (agonists of KA) were tested to see if their presence would increase calcium flux into the cells No calcium flux was observed within a 300 second time frame with ATPA or DA No calcium flux was observed within a 300 second time frame with ATPA or DA

22. Mouse Spleen Cells IonomycinDA ATPA APTA followed by Ionomycin

23. Sodium Assay The Sodium Assay was performed to observe a possible influx of sodium into the cells since there was so significant influx of calcium observed The Sodium Assay was performed to observe a possible influx of sodium into the cells since there was so significant influx of calcium observed However, we were unable to get sodium conditions worked out, and therefore were not able to collect useable data However, we were unable to get sodium conditions worked out, and therefore were not able to collect useable data

24. Stimulation of naïve B cells in the presence of Kainic Acid The purpose of the stimulation was to determine if KA affect mouse naïve B cells The purpose of the stimulation was to determine if KA affect mouse naïve B cells KA has shown to increase cell proliferation and IgE production in human cells KA has shown to increase cell proliferation and IgE production in human cells We tested this to observe the levels of cell proliferation, as well as IgE production We tested this to observe the levels of cell proliferation, as well as IgE production Cell proliferation and IgE production was observed under different parameters set in the stimulation, including Cell proliferation and IgE production was observed under different parameters set in the stimulation, including the amounts of IL-4 (varying units of IL-4 per well) the amounts of IL-4 (varying units of IL-4 per well) the number of cells plated (10,000 cells per well vs. 50,000 cells per well) the number of cells plated (10,000 cells per well vs. 50,000 cells per well)

25. * * Represents a P-Value <.05 *

26. IgE Synthesis in the Presence of KA

27. Disappointing Results? Although we wanted an increase of IgE, just as in humans, our data corresponds to other phenomena observed in the mouse Although we wanted an increase of IgE, just as in humans, our data corresponds to other phenomena observed in the mouse IL-21 is a cytokine that increases cell proliferation and IgE synthesis in the human, HOWEVER IL-21 increases cell proliferation but decreases IgE synthesis in the mouse IL-21 is a cytokine that increases cell proliferation and IgE synthesis in the human, HOWEVER IL-21 increases cell proliferation but decreases IgE synthesis in the mouse

28. Conclusions We have identified the kainate receptors on all cell types in the mouse immune system We have identified the kainate receptors on all cell types in the mouse immune system We have shown that KA increases proliferation on naïve B cells We have shown that KA increases proliferation on naïve B cells A modest, but significant, increase (similar as to humans) A modest, but significant, increase (similar as to humans) We have also shown that KA decreases IgE production We have also shown that KA decreases IgE production Studies further emphasize that you must be careful when comparing the mouse and the human Studies further emphasize that you must be careful when comparing the mouse and the human The human system and the mouse system can be drastically different The human system and the mouse system can be drastically different

29. Future Studies We want to determine mechanism We want to determine mechanism Understand more thoroughly the decrease of IgE Understand more thoroughly the decrease of IgE Does it act like IL-21? Does it act like IL-21? We want to see what would happen with in vivo study We want to see what would happen with in vivo study Add KA to a mouse to see what would occur Add KA to a mouse to see what would occur

30. Acknowledgments Dr. Conrad Jamie Sturgill Jill Ford Yvette Orihuela Joel Matthews David Gibb Nicole Dally Dr. Allison Johnson -- HHMI Summer Scholars Program