Brooke Troxell

Tyler Wright

Christopher Ott

Dustin Praylow

The Effect of Interleukin 8-F on the Production of Interferons

Abstract

Interferons are cytokines that produce an immune response to pathogens.

Interleukin 8-F (IL8-F) is a primer produced by macrophages. We

hypothesized that cells treated with interferons would show the IL8-F genes.

We used PCR to try and show this but our results were inconclusive. This

experiment could be improved by instead treating cells with IL8-F and seeing

if they produce interferons.

Introduction

Interferons are a type of glycoproteins belonging to the group of

cytokines (Takaoka 2003). Interferons are released in a response to

pathogens such a bacteria, viruses, parasites, and tumors (Takaoka 2003).

They are used for communication between cells to trigger protective

defenses by activating immune cells such as macrophages (Takaoka 2003).

Interleukin 8-F is a primer (Tanaka). It is a type of chemokine produced by

macrophages (Tanaka). Interleukin induces chemotaxis in target cells and

causes them to migrate towards the site of infection (Tanaka). We wanted to

see if leukemia cells treated with Interleukin 8-F would still produce

interferons. This experiment is important because if leukemia cells treated
with IL8-F stilled produced an immune response (producing interferons) then

this could be a possible way to treat cancer. To perform the experiment, we

used HL-60 cells which are multipotent stem cells derived from the blood of a

36-year old Caucasian female with acute promyelocytic leukemia (Collins

1987). HL-60 cells are able to differentiate into granulocytes or monocytes

(Collins 1987). To perform this experiment we treated cells with Interleukin

8-F, dimethyl sulfoxide (DMSO), and phorbol 12-myristate 12-acetate (PMA).

PMA is a chemical tumor promoter that activates certain pathways in the cell

via kinase C to differentiate HL-60 cells into monocytes (Schumpert 2014).

DMSO is a chemical solvent that acts as a control for PMA treatment

conditions and therefore induces HL-60 cells to differentiate into

granulocytes (Schumpert 2014). To test for the presence of interferons we

used the polymerase chain reactions (PCR). PCR is used to amplify a single

or few copies of a piece of DNA. We hypothesized that groups treated with

interferons should show the IL8-F gene.

Materials and Methods

We had 12 treatment groups of cells, half looking for interleukin 8 and

half looking for β-actin as a control. The groups were untreated cells,

untreated and interferons, DMSO, DMSO and interferons, PMA, and PMA and

interferons. First, we calculated cell density and viability of each of the six

treatments using Trypan blue protocol. We centrifuged the remaining cells

for 1 minute at 6000 rpm. We removed the cells from the centrifuge and

discarded the cell culture medium. To disrupt and homogenize the cells we
added 350 µL of buffer RLT and then vortexed each solution for 5 seconds.

We added 350 µL of 70% ethanol to each sample and transferred the each

sample into a separate RNeasy column. We then centrifuged each sample

for 15 seconds at 12000rpm and discarded the flow through that collected at

the bottom of the collection tubes. We added 700 µL of Buffer RW1 to the

RNeasy columns, centrifuged the samples for 15 seconds at 12000 rpm, and

discarded the flow through that collected at the bottom of the collection

tubes. We added 500 µL of Buffer RPE to the RNeasy columns, centrifuged

the samples for 15 seconds at 12000 rpm, and discarded the flow through

that collected at the bottom of the collection tubes. We added 40 µL of

RNase-free water to the RNeasy columns, centrifuged the samples for one

minute at 12000 rpm. Then, we immediately placed the flow through (the

isolated RNA) on ice (Cirtain et al., 2002).

After leaving the isolated RNA samples on ice for a few minutes, we

took 2 µL of the RNA extract from each treatment and combined it with 2 µL

of random decamers and 8 µL of nuclease free water. We mixed these

combinations, spun them briefly, and heated them for three minutes at 80°C.

We then added the remaining RT components, mixed gently, spun briefly,

and incubated the treatments at 44°C for one hour. Then we incubated the

samples at 92°C to 2 composed of 2.5 µL 10X PCR Buffer, 0.5 µL dNTPs, 1.0

µL forward primer (50 pmol), 1.0 reverse primer (50 pmol), 1.0 µL Taq

polymerase, and 14.0 µL nuclease-free dH2O. We then loaded the sample
into the thermal cycler with either MMP-9 or β-actin (acts as a control) and

ran the program (Cirtain et al., 2002).

Finally, we mixed 15 µL of PCR product of each sample with the same

treatment group with 3 µL of 6X gel loading buffer. We loaded the sample

into the well of the gel. We ran the gel between 100-130 volts for 30-45

minutes and photographed the gel under an ultraviolet light (Cirtain et al.,

2002).

Results

The cell density for the untreated cell group was 2.6 x 105 and the

percent viability was 23.0%. The cell density for the untreated and

interferon was 3.8 x 105 and the percent viability was 31.6%. The cell

density for the DMSO group was 3.6 x 105 and the percent viability was

37.5%. The cell density for DMSO and interferon was 2.6 x 105 and the

percent viability was 23.0%. The cell density for PMA was 6.2 x 105 and the

percent viability was 21.1%. The cell density for PMA and interferons was 3.5

x 105 and the percent viability was 20.0%.
1 2 3 4 5 6 7

Figure 1. A photograph of the results of the agarose gel electrophoresis of

the PCR samples.

Lane 1 contained the untreated groups looking for β-actin and IL8-F. Lane 2

contained the untreated plus interferons looking for β-actin and IL8-F. Lane

3 contained the groups of DMSO-treated cells looking for β-actin and IL8-F.

Lane 4 contained the groups of DMSO-treated cells and interferons looking

for β-actin and IL8-F. Lane 5 contained the groups of PMA-treated cells

looking for β-actin and IL8-F. Lane 6 contained the groups of PMA-treated

cells and interferons looking for β-actin and IL8-F.

Discussion

The percent viability of all of our treatments groups were relatively low.

This could have been a factor in that we did not find any results of IL8-F at

the end of our agarose gel electrophoresis. The result that we cannot

determine whether IL8-F is present could be due to contaminated agarose,
power sources that may not have provided an even electrical current, or that

the loading dye had contaminants that affected our results. However, we do

know that the PCR worked because the product of β-actin is present in the

gel. Due to the lack of data, we were not able to come to a conclusion about

the validity of our hypothesis. I would improve upon this experiment by

instead of using PCR to test for the IL8-F gene, I would treat cells with IL8-F

and see if they produced interferons by SDS-PAGE and/or zymography. If the

treatment with IL8-F did produce interferons, that information could be used

to make leukemia cells targets of the immune system.

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