Download Finding a probable origin for the secretion of

December 10, 2012
Finding a probable origin for the secretion of
Interleukin-4 cytokine in the immune response
Michael Weissenberger
I. Introduction
Our bodies are under constant attack from foreign invaders, and we have our immune systems to
thank for keeping us healthy and productive every day. One of the many important pieces to our
immune response is Interleukin-4, which has many essential functions. It stimulates and signals
the division and differentiation of activated B cells1 and T cells2, and help B cells turn into
Plasma Cells3, which will allow for more antibodies to be produced and more foreign invaders to
be destroyed as a result. IL-4 also switches antibody production over to the Immunoglobin E and
Immunoglobin G4 classes4,5, to help better fight certain kinds of infections. It up regulates the
production of Major Histocompatibility Complex Class II6.
Needless to say, it is a key component of our immune response, and as such is made by quite a
few different cells. It is currently known to be made by Th2 T cells2, B cells3, NK1.1 T7 cells,
Basophils8,9,10, and Mast cells11. But we don’t have a definite answer as to which cell starts
making IL-4 first, nor do we know at what point IL-4 is first produced. My experiment hopefully
will shed some light as to when it is produced and the order in which cells produce it.
II. Experiment
My experiment will be provoking an immune response in mice, harvesting them at
predetermined intervals, and analyzing the harvested cells for IL-4 production. By analyzing
different cells at each interval, we can establish an order in which IL-4 is hopefully produced.
To start with, I would inject a batch of mice with a solution of ovum proteins and an Aluminum
Hydroxide adjunct to provoke an immune response. Over the course of 10 days, I would
administer a boost of the ovum protein and Aluminum Hydroxide solution on days 3 and 7, and
harvest a portion of the mice on the remaining days.
When the mice are harvested, I will collect splenocytes, lymph node cells, and periteneal cells as
these places will contain the particular cells I am looking for. I will then incubate the cells with
the ova in a cell culture with 5µM Monensin for 5 hours. The Monensin will block the secretion
of any IL-4 made from exiting the cell and will allow us to detect the amount of IL-4 present.
Next, the cell membranes would be fixed in place by adding a solution of 4% paraformaldehyde
to the culture. This would prevent the cell from lysing from osmotic pressure when the next step
is preformed, which is to permeabilize and stain the membrane with a Saponin detergent buffer
and antibodies. The antibodies would bind to specific molecules and IL-4 depending on the cell.
The antibodies are dyed so that they could be analyzed using flow cytometry, and will fluoresce
a different color depending on the molecule it was bound to (Table 1)
December 10, 2012
Cells harvested
FITC Dye
PE Dye
Alexafluor Dye
Control
IgG
IgG
IgG
Th2 T Cells
CD4
T1/ST2
IL-4
NK1.1 T Cells
CD4
NK1.1
IL-4
Basophils
FcERI
Kit
IL-4
Mast Cells
FcERI
Kit
IL-4
B Cell
B220
IgE
IL-4
Table 1: Upon harvesting, antibodies with the dyes listed with enter the fixed, permiablized
membrane and bind to a specific molecule based on the cell type.
III. Discussion
After flow cytometry is done, I would analyze the data and counting the amount of IL-4
produced by each cell type. After repeating this procedure 8 times of the course of 10 days, I
should be able to graph each cell’s production of IL-4 as well find an origin point for production.
That would be the ideal result, having only one cell begin IL-4 production and in enough
quantities to be picked up by the flow cytometer. Another possible result would be that two or
more cell types begin production of IL-4 at the same time, in which case I would have to begin
looking for a common element or trigger that would initiate production of IL-4.
Some possible errors this experiment could face is that the ovum solution would not produce a
strong enough immune response to warrant IL-4 production, and if so a stronger infection agent
would be required. Another obstacle would be that IL-4 would already be present in the
background in sufficient quantity to mask the cell’s production, but the possibility of this is low
and most likely the result of a genetically defective mouse that run counter to the experiment’s
design, and would be replaced.
The known limitations would be that the time intervals may not be short enough to catch the first
cell producing IL-4, and that it would not be definitive proof of the origin of IL-4.
December 10, 2012
References
1) Jelinek, D.F. and Lipsky, P.E., 1988, Inhibitory Influence of IL-4 on Human B Cell
Responsiveness, Journal of Immunology, Volume 14(1), pages 164-173
2) Khan et al., 2011, CD4+ T-cell Derived Novel Peptide Thp5 Induces Interleukin-4 Production
in CD4+ T-cells to Direct T Helper 2 Cell Differentiation., Journal of Biological Chemistry,
2011, Volume 287(4), pages 2830-2835
3) Neuberger, M. S.; Honjo, T.; Alt, Frederick W. , 2004, Molecular biology of B cells.
Amsterdam: Elsevier., pages 189–191.
4) Fujieda et al., 1998, Immunodeficiency and other Clinical Immunology: CD-1 Restricted TCells Influence IgG Subclass and IgE Production, Journal of Allergy and Clinical Immunology,
Volume 101(4), pages 545-551
5) Bacharier and Geha, 2000, Molecular Mechanisms of IgE Regulation, Journal of Allergy and
Clinical Immunology, Volume 105(2) pages S547-S558
6) Zhou, X., Jiang, Y., Lu, L., Ding, Q., Jiao, Z., Zhou, Y., Xin, L. and Chou, K.-Y. (2007),
MHC class II transactivator represses human IL-4 gene transcription by interruption of promoter
binding with CBP/p300, STAT6 and NFAT1 via histone hypoacetylation. Journal of
Immunology, Volume 122, pages 476–485.
7) Enomoto, Nishimura, and Yoshikai, 1997, Predominant appearance of NK1.1+ T cells
producing IL-4 may be involved in the increased susceptibility of mice with the beige mutation
during Salmonella infection, Journal of Immunology, Volume 158(5), pages 2268-2277.
8) Devouassoux et al, 2002, Chemical Constituents of Diesel Exhaust Particles Induce IL-4
Production and Histamine Release by Human Basophils, Journal of Allergy and Clinical
Immunology, Volume 109(5), pages 847-853.
9) Heinzmann, 2001, Production of IL-4 by Lung Basophils, Respiratory Research, Volume 2,
page 68469
10) Van Punhuys et al, 2011, Basophils are the major producers of IL-4 during primary helminth
infection., Journal of immunology, volume 186(5), pages 2719-2728.
11) Toru et al, 1998, Human mast cells produce IL-13 by high-affinity IgE receptor crosslinking: Enhanced IL-13 production by IL-4 primed human mast cells., The Journal of Allergy
and Clinical Immunoloy, Vol 102(3), page 491-502.