Do Glutamate Do Glutamate Receptors Exist in Receptors Exist in the Rodent Immune the Rodent Immune System? System? Cate Kurkjian Cate Kurkjian Dr. Conrad Lab Dr. Conrad Lab Department of Microbiology and Department of Microbiology and Immunology Immunology
30
Embed
Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Do Glutamate Do Glutamate Receptors Exist in Receptors Exist in
the Rodent the Rodent Immune System?Immune System?
Cate KurkjianCate Kurkjian
Dr. Conrad LabDr. Conrad Lab
Department of Microbiology and Department of Microbiology and ImmunologyImmunology
The Purpose of my The Purpose of my ProjectProject
Studies show that glutamate receptors are a part of Studies show that glutamate receptors are a part of the human nervous system and can be localized the human nervous system and can be localized within the brainwithin the brain
Research within our lab suggests the presence of Research within our lab suggests the presence of kainate receptors (a specific type of glutamate kainate receptors (a specific type of glutamate receptors) within the human immune systemreceptors) within the human immune system
We want to show that kainate receptors can also be We want to show that kainate receptors can also be found within the rodent immune systemfound within the rodent immune system
To date, the presence of kainate receptors has never To date, the presence of kainate receptors has never been shown in the mouse immune systembeen shown in the mouse immune system
If present, the mouse system can used to help expand If present, the mouse system can used to help expand upon studies on the human immune systemupon studies on the human immune system
Such findings will better link epilepsy and allergies in Such findings will better link epilepsy and allergies in humanshumans Studies in humans show that people who have epilepsy are Studies in humans show that people who have epilepsy are
more prone to allergic diseasemore prone to allergic disease
Introduction to Introduction to ImmunologyImmunology
Parham, Peter. The Immune System. 2000. Garland Publishing/Elsevier Science Ltd.
Why is it important?Why is it important? Kainate receptors are shown to influence Kainate receptors are shown to influence
ADAM10ADAM10 ADAM10 is responsible for the cleavage of CD23 ADAM10 is responsible for the cleavage of CD23
(a low affinity IgE receptor and a protein known (a low affinity IgE receptor and a protein known to regulate IgE synthesis)to regulate IgE synthesis)
Cleavage of CD23 influences IgE production, Cleavage of CD23 influences IgE production, which leads to increased allergies in humanswhich leads to increased allergies in humans
This model has been worked out in the human This model has been worked out in the human system:system:
DataData RT-PCR and FACS were methods RT-PCR and FACS were methods
used to determine the presence of used to determine the presence of kainate receptors within mouse kainate receptors within mouse lymphocyteslymphocytes
Calcium Assays and Sodium Assays Calcium Assays and Sodium Assays were performed to determine if KA were performed to determine if KA influx in the immune system is influx in the immune system is similar to that of the nervous systemsimilar to that of the nervous system The nervous system will primarily flux The nervous system will primarily flux
sodium into cells, but a smaller amount sodium into cells, but a smaller amount of Calcium will also flux into the cellof Calcium will also flux into the cell
RT-PCR ResultsRT-PCR Results Naïve B cells, activated B cells, and whole Naïve B cells, activated B cells, and whole
spleen cells were tested with GRIK primersspleen cells were tested with GRIK primers Actin ( a house keeping primer) was used as Actin ( a house keeping primer) was used as
the controlthe control It is a good control to use because it can be It is a good control to use because it can be
localized in almost all cell typeslocalized in almost all cell types Results:Results:
Using GRIK primers, the appropriate location for Using GRIK primers, the appropriate location for kainate receptors was present, but faintkainate receptors was present, but faint
A non-specific band was present below the desired A non-specific band was present below the desired bandband
Actin was present at the appropriate bandActin was present at the appropriate band Conclusion: results were not significant to Conclusion: results were not significant to
confirm or reject the presence of kainate confirm or reject the presence of kainate receptors on mouse cellsreceptors on mouse cells
SorterSorter Used to analyze the distribution of Used to analyze the distribution of
surface molecules on cellssurface molecules on cells In this case, it is used to detected the In this case, it is used to detected the
distribution of kainate receptors on distribution of kainate receptors on mouse lymphocytesmouse lymphocytes
The data is analyzed and graphed The data is analyzed and graphed using a program on the computer, using a program on the computer, CXPCXP
FACS results – Proof of FACS results – Proof of PrinciplePrinciple
FACS was run on human 8866 cells to FACS was run on human 8866 cells to determine if kainate receptors could determine if kainate receptors could be localized on the cells using this be localized on the cells using this method of experimentationmethod of experimentation Human 8866 cells were used since we Human 8866 cells were used since we
have already observed the presence of have already observed the presence of the receptors on these cellsthe receptors on these cells
Results:Results: A significant shift in the graph confirmed A significant shift in the graph confirmed
the presence of kainate receptors on the the presence of kainate receptors on the human 8866 cellshuman 8866 cells
Human 8866 Cells
GRIK4
1 10 100 1000
Re
lativ
e C
ell
Nu
mb
er
0
70
IsotypeGRIK4
FACS ResultsFACS Results
FACS was run on mouse spleen cells FACS was run on mouse spleen cells to determine the presence of kainate to determine the presence of kainate receptors on mouse lymphocytesreceptors on mouse lymphocytes
Results:Results: a slight shift in the graph suggested a slight shift in the graph suggested
that kainate receptors can be located on that kainate receptors can be located on mouse spleen cellsmouse spleen cells
Mouse Spleen Cells
GRIK4
1 10 100 1000
Re
lativ
e C
ell N
umbe
r
0
70
IsotypeGRIK4
FACS dataFACS data FACS was run on all the different cell FACS was run on all the different cell
types within the mouse spleen to types within the mouse spleen to determine the exact cells in which determine the exact cells in which kainate receptors can be localizedkainate receptors can be localized B CellsB Cells
Presence of Kainate Receptors Presence of Kainate Receptors on B Cellson B Cells
Naive B cells
GRIK4
1 10 100 1000
Rel
ativ
e C
ell N
umbe
r
0
70
GRIK4Isotype
Semi-Activated B Cells
GRIK4
1 10 100 1000
Rel
ativ
e C
ell N
umbe
r
0
70
GRIK4 + B220Isotype
Activated B cells
GRIK4
1 10 100 1000
Re
lativ
e C
ell
Nu
mb
er
0
70
IsotypeGRIK4 + B220
Presence of Kainate Receptors Presence of Kainate Receptors on T Cellson T Cells
T Cells
GRIK4
1 10 100 1000
Rel
ativ
e C
ell N
umb
er
0
Grik4
Presence of Kainate Presence of Kainate Receptors on NK Cells and Receptors on NK Cells and
MonocytesMonocytes
Activated Monocytes
GRIK4
1 10 100 1000
Re
lativ
e C
ell
Nu
mb
er
0
70
140
210
280
350
420
490
560
630
700
770
IsotypeGRIK4 + Gr1
Monocytes
GRIK4
1 10 100 1000
Re
lativ
e C
ell
Nu
mb
er
0
70
140
210
280
350
420
490
560
630
700
IsotypeGRIK4 + CD11b
Natural Killer Cells
GRIK4
1 10 100 1000
Rel
ativ
e C
ell N
umbe
r
0
70
140
210
280
350
420
490
560
630
700
770
IsotypeGRIK4 + NK1.1
Comparison of GRIK4 Levels on Spleen Cells
GRIK41 10 100 1000
Rel
ativ
e C
ell N
umbe
r
0
20
40
60
80
100
120
Isotype NK Mac T cell B cell
Summary of FACS DataSummary of FACS Data B Cells (Naïve, Semi-Activated, and Activated)B Cells (Naïve, Semi-Activated, and Activated)
Positive for receptors on all B Cell subsetsPositive for receptors on all B Cell subsets T CellsT Cells
Positive for receptors on all T Cells, but moderate Positive for receptors on all T Cells, but moderate compared to other cell typescompared to other cell types
Natural Killer CellsNatural Killer Cells Positive for receptors (showed the highest Positive for receptors (showed the highest
fluorescence)fluorescence) MonocytesMonocytes
Positive for receptors (showed the highest Positive for receptors (showed the highest fluorescence) fluorescence)
Activated Monocytes (Macrophages)Activated Monocytes (Macrophages) Positive for receptors (showed the highest Positive for receptors (showed the highest
fluorescence)fluorescence)
Calcium AssayCalcium Assay
The Calcium Assay was performed The Calcium Assay was performed using ionomycin as a control (to show using ionomycin as a control (to show high influx of calcium into the cells)high influx of calcium into the cells)
ATPA and DA (agonists of KA) were ATPA and DA (agonists of KA) were tested to see if their presence would tested to see if their presence would increase calcium flux into the cellsincrease calcium flux into the cells
No calcium flux was observed within a No calcium flux was observed within a 300 second time frame with ATPA or 300 second time frame with ATPA or DADA
Mouse Spleen Cells
Ionomycin DA
ATPAAPTA
followed by Ionomycin
Sodium AssaySodium Assay
The Sodium Assay was performed to The Sodium Assay was performed to observe a possible influx of sodium observe a possible influx of sodium into the cells since there was so into the cells since there was so significant influx of calcium significant influx of calcium observedobserved
However, we were unable to get However, we were unable to get sodium conditions worked out, and sodium conditions worked out, and therefore were not able to collect therefore were not able to collect useable datauseable data
Stimulation of naïve B cells Stimulation of naïve B cells in the presence of Kainic in the presence of Kainic
AcidAcid The purpose of the stimulation was to The purpose of the stimulation was to
determine if KA affect mouse naïve B cellsdetermine if KA affect mouse naïve B cells KA has shown to increase cell proliferation KA has shown to increase cell proliferation
and IgE production in human cellsand IgE production in human cells We tested this to observe the levels of cell We tested this to observe the levels of cell
proliferation, as well as IgE productionproliferation, as well as IgE production Cell proliferation and IgE production was Cell proliferation and IgE production was
observed under different parameters set observed under different parameters set in the stimulation, including in the stimulation, including the amounts of IL-4 (varying units of IL-4 per the amounts of IL-4 (varying units of IL-4 per
well)well) the number of cells plated (10,000 cells per the number of cells plated (10,000 cells per
well vs. 50,000 cells per well)well vs. 50,000 cells per well)
Murine B cell Proliferation in the Presence of Varying doses of KA
0.0
20000.0
40000.0
60000.0
80000.0
100000.0
120000.0
140000.0
160000.0
180000.0
(-) 5mM 2.5mM 1.25mM
Dose of KA
CPM
Mouse B cells
*
* Represents a P-Value < .05
*
IgE Synthesis in the IgE Synthesis in the Presence of KAPresence of KACell Dose
Cell No
1e+3 1e+4 1e+5
IgE
(ng
/ml)
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
AloneKA
Disappointing Results?Disappointing Results?
Although we wanted an increase of Although we wanted an increase of IgE, just as in humans, our data IgE, just as in humans, our data corresponds to other phenomena corresponds to other phenomena observed in the mouseobserved in the mouse IL-21 is a cytokine that increases cell IL-21 is a cytokine that increases cell
proliferation and IgE synthesis in the proliferation and IgE synthesis in the human, HOWEVER IL-21 increases cell human, HOWEVER IL-21 increases cell proliferation but decreases IgE proliferation but decreases IgE synthesis in the mousesynthesis in the mouse
ConclusionsConclusions We have identified the kainate receptors on all We have identified the kainate receptors on all
cell types in the mouse immune systemcell types in the mouse immune system We have shown that KA increases proliferation We have shown that KA increases proliferation
on naïve B cellson naïve B cells A modest, but significant, increase (similar as to A modest, but significant, increase (similar as to
humans)humans) We have also shown that KA decreases IgE We have also shown that KA decreases IgE
productionproduction Studies further emphasize that you must be Studies further emphasize that you must be
careful when comparing the mouse and the careful when comparing the mouse and the humanhuman The human system and the mouse system can be The human system and the mouse system can be
drastically differentdrastically different
Future StudiesFuture Studies
We want to determine mechanismWe want to determine mechanism Understand more thoroughly the Understand more thoroughly the
decrease of IgEdecrease of IgE Does it act like IL-21?Does it act like IL-21?
We want to see what would happen We want to see what would happen with in vivo studywith in vivo study Add KA to a mouse to see what would Add KA to a mouse to see what would