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

Dec 24, 2015

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

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

Page 2: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 3: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

Glutamate ReceptorsGlutamate Receptors

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

Page 4: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

Introduction to Introduction to ImmunologyImmunology

Parham, Peter. The Immune System. 2000. Garland Publishing/Elsevier Science Ltd.

Page 5: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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:

↑↑KAR → ↑ADAM10 → ↓mCD23 & ↑sCD23 → ↑IgE synthesisKAR → ↑ADAM10 → ↓mCD23 & ↑sCD23 → ↑IgE synthesis

Page 6: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

Cleavage of CD23

Page 7: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 8: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 9: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

Naive Active Spleen Naive Active Spleen

GRIK primers Actin (control)

RT-PCR results

Page 10: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

FACSFACSFluorescent Activated Cell Fluorescent Activated Cell

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

Page 11: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 12: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

Human 8866 Cells

GRIK4

1 10 100 1000

Re

lativ

e C

ell

Nu

mb

er

0

70

IsotypeGRIK4

Page 13: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 14: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

Mouse Spleen Cells

GRIK4

1 10 100 1000

Re

lativ

e C

ell N

umbe

r

0

70

IsotypeGRIK4

Page 15: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Naïve, semi-activated, activatedNaïve, semi-activated, activated T CellsT Cells MonocytesMonocytes MacrophagesMacrophages Natural Killer CellsNatural Killer Cells

Page 16: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 17: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 18: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 19: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 20: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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)

Page 21: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 22: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

Mouse Spleen Cells

Ionomycin DA

ATPAAPTA

followed by Ionomycin

Page 23: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 24: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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)

Page 25: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

*

Page 26: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 27: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 28: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

Page 29: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

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

occuroccur

Page 30: Do Glutamate Receptors Exist in the Rodent Immune System? Cate Kurkjian Dr. Conrad Lab Department of Microbiology and Immunology.

AcknowledgmentsAcknowledgments

Dr. ConradDr. ConradJamie SturgillJamie SturgillJill FordJill FordYvette OrihuelaYvette OrihuelaJoel MatthewsJoel MatthewsDavid GibbDavid GibbNicole DallyNicole Dally

Dr. Allison Johnson -- HHMI Summer Dr. Allison Johnson -- HHMI Summer Scholars ProgramScholars Program