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Thomas Thornton, Danal Mayan, Carey Ray, and Luke Raymond

Yvon Woappi
Nicotine Induced Downregulation of Major Histocompatibility Complex Class I
Molecules in Monocytes and Neutrophils Derived from Induced HL-60 Cell
Differentiation
April 12, 2016

Abstract:
The MHC-class I molecules are key cells in the body for important reasons.
MHC-class I molecules are used by cells to present antigens that are created in the body.
Previous studies conducted have shown that nicotine has the properties necessary to
down regulate MHC-class I molecule presentation in cells. In our experiment, cells were
treated with PMA, DMSO, and others were treated with DMSO and nicotine and PMA
and nicotine. RNA was isolated from each of the sample cells. mRNA was then
converted into cDNA via reverse transcriptase PCR. Each of the samples was loaded into
a quantitative PCR machine to amplify genes of interest. Gel electrophoresis was
performed to see MHC expression after treatment. Our results showed that cells treated
with nicotine showed a down regulation of MHC-class I presentation.
Introduction:
The major histocompatibility complex molecules present in all cells in the body,
excluding red blood cells, are what eliminates a invading pathogen or infected cell. This
complex is proving to be one of the more important steps that detects and potentially
eradicates threats to the human body. MHC molecules are grouped into two classes: I
and II. MHC class I molecules are found in all nucleated cells in the body. They are
used by the cell to present certain proteins that are created by that cell and they are found
as a form of ID for the body. This ID is used by the body to monitor its conditions and to
detect any potential threats. If a cell has become infected with an intracellular pathogen, it
will display new proteins on its surface that are unfamiliar to the body and will therefore
elicit an immune response. The other type of MHC molecules, MHC class II molecules,
are found only in capable antigen presenting cells, primarily monocytes and their

differentiated forms: macrophages and dendritic cells. These cells are considered first
responders in the body because they arrive first to the site of infection and phagocytize
the pathogen through endocytosis. It then proceeds to digest the invader and presents a
resulting peptide on its cell surface via this complex of molecules to B cells in the lymph
nodes to create specific antibodies to target present and future attacks by invaders
containing this same antigen. However, some viruses and substances can promote the
down regulation of these MHC molecules, thus rendering them nearly invisible to the
immune system as seen in the case of tumors. This type of selective down regulation can
be exhibited in HIV-infected cell as a way of functioning unnoticed by the immune
system. Typically, nonspecific lymphocytes, primarily natural killer cells, target these
specific cells with down regulated MHC molecules. Although this appears to solve the
problem of foreign pathogenic invaders, these cells can also be down regulated, thus
leaving the invader even more impermeable to attack. The class of choice in this study is
MHC class I molecules, as it has been observed to be down regulated in the case of
cancer cells and thus allow for metastasis. As the down regulating structures of capable
viruses along with the cause of the phenomenon itself are not entirely known, this
continues to be a problem in the battle against cancer. Furthermore, nicotine acting as a
potential down regulator for this gene in cells became a topic of interest in our research
group. Although tobacco is the main variable linked to cancer in cigarette smoking, it
does not weave out the possibility of nicotine playing a role as well. Although not a
supposed carcinogen, like certain chemicals in tobacco smoke, nicotine has been
previously shown to be mutagenic and can therefore theoretically alter the genome of a
cell to silence the expression of these MHC molecules. Structurally, the MHC class I

molecules are composed of two polypeptide chains known as alpha and beta-2
microglobulin. The alpha chain is polymorphic and therefore is active in different forms.
A human leukemia antigen gene encodes this polypeptide while the beta-2 microglobulin
gene encodes the beta subunit. Based on the knowledge that the HLA system is a gene
complex and therefore has multiple forms, requiring different primers, the expression of
the beta-2 microglobulin gene will be our dependent variable and therefore our gene of
interest. Previous studies have shown nicotine induces down regulation of natural killer
cell functions by simulating the neurotransmitter acetylcholine and binding to receptors
on the natural killer cells; however, studies have not been concluded to determine its
effect on MHC molecules itself, henceforth altering its susceptibility to attack from other
immune cells. Moreover, with hundreds of millions of people being exposed to cigarettes
daily worldwide, with the direct cause of cancer focused on chemicals in tobacco smoke,
we believe a molecular look at the effects of nicotine in professional antigen presenting
cells can provide insight on its degree of assistance in causing cancer. The HL-60 cell
line will be our model of choice as differentiation into neutrophils and monocytes can be
easily induced in vitro with the addition of DMSO (neutrophils) and PMA (monocytes).
These multipotent promyelocytic leukemia stem cells have been used in prior studies
with results that show nicotine does influence the expression of certain genes during invitro differentiation. For this study, however, HL-60 cells induced to differentiate into
neutrophils and monocytes will only then be treated with nicotine; likewise, a control
group will be treated with saline. After treatment, reverse transcription PCR will then be
ran to produce cDNA, as PCR cannot be done with RNA. Primers specific for beta-2
microglobulin will be used in conjugation with PCR to amplify this gene. An

electrophoresis gel will subsequently be run in order to test for the down regulation of
this gene. Furthermore, a study has been done prior where the effects of nicotine in the
presence of HL-60 cell differentiation into neutrophils with the addition of DMSO were
tested. After 5 days of incubation, the results assert that nicotine caused for an increase in
differentiated cells during late stages of differentiation3. As we will be testing HL-60
cells when already differentiated, we are expecting dissimilar results in the aspect of cell
viability. By testing for this specific down regulating effect of nicotine in these particular
leukocytes, we will gain a better understanding of nicotines role in cancer, which could
in turn provide insight on ways to inverse its effects. In consideration of the
aforementioned material, we consequently believe that the addition of nicotine during
monocyte and neutrophil differentiation in the presence of PMA and DMSO respectively
will cause for the down regulation of MHC Class I molecules.
Hypothesis:
We believe that the addition of nicotine during monocyte and neutrophil
differentiation in the presence of PMA and DMSO respectively, will cause the down
regulation of MHC Class I molecules.
Materials and Methods:
Four culture samples of differentiated HL-60 cells are to first be obtained from
our teaching assistant, two being neutrophils (DMSO) and two being monocytes (PMA).
A Trypan blue assay will be performed initially to affirm that we have an appropriate
amount of cells. This dye exclusion test is based on the idea that dead cells with
damaged membranes will be tagged while alive cells with intact membranes will be
untouched. The process for the experiment will begin by making a one-to-one ratio of

cells to trypan blue dye; 50 l of each differentiated HL-60 cell culture samples will be
added to 50 l of 0.4% trypan blue dye. This ratio yields a dilution factor of one-to-two
(50 l cell suspension: 100 l solution), which is accounted for in the calculations. 10 ul
of both dye mixture and suspension culture will be added to a slide with a
hemacytometer. The hemacytometer will allow us to determine the estimated amount of
viable cells per mL. 1.6 x 10-2 l of nicotine will be added to a set of previously obtained
neutrophils and monocytes. A previously performed experiments used 10^-6 M of
nicotine and we arrived at our number by multiplying this concentration by the molecular
weight of nicotine, 162.23, and then converting it to microliters. Saline will be used as
our control for nicotine, and will therefore be added at the same increment of 100 l to
the other sets of previously obtained neutrophils and monocytes. Another trypan blue
assay will then be performed to determine cell viability following the addition of
nicotine/saline. RNA will then be extracted from each differentiated HL-60 cell sample
and reverse transcription PCR will be conducted in order to synthesize a complimentary
DNA strand of the RNA known as cDNA. This is done because PCR cannot be run with
RNA and therefore has to have this alternate engineered form. Furthermore, primers
specific for MHC class I molecules will be used to amplify genes responsible for the
production of these proteins. As the complex itself is composed of 2 polypeptide chains,
primers for either of the genes encoding these molecules can be used. However, we will
reasonably use primers for the Beta-2 microglobulin gene since it is not polymorphic and
is thus likely easier to detect. An agarose gel will then be prepared, loaded with each of
our samples as well as ethidium bromide, which will bind to the peptide bonds and allow
us to view them. The electrophoresis gel will be run at 130 volts for the duration of 45

minutes and following completion will be observed under an ultraviolet light to detect the
expression of Beta-2 microglobulin and therefore determine down regulation.
Results:

MHC Class 1 HL-60 Gel Electrophoresis:

Expected amplicon size: Beta-actin: ~300 bp
2.

Forward Primer: 5-TGACCCAGATCATGTTTGAGA-3

Reverse Primer: 5-AGTCCATCACGATGCCAGT-3

Expected amplicon size: MHC-Class1: ~500 bp

2.

Forward Primer: 5- CTCGCGTGTCTTTTCCTCG-3

3.

Reverse Primer: 5- GCCGACATGACGAAGTTGG-3

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The primers used in both sets of samples were used to amplify gene

expression. Primers allow for new strands of DNA to be synthesized that help us
reach our ideal product of cDNA.
Following gel electrophoresis, the DNA MHC samples were examined under an
ultraviolet light to reveal how far they traveled. The PMA in lane four traveled roughly
to the 500 bp mark. The fifth lane, which was treated with PMA, traveled to the 475 bp
mark. The eighth lane, which was treated with DMSO, traveled roughly to the 450 bp
mark.

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The results of gel electrophoresis for the DNA Beta-actin samples are listed here.

For the second well, treated with PMA and nicotine, the sample reached the 275 bp mark.
For the third well, also treated with PMA and nicotine, the sample reached 290 bp. The
fourth well, treated with PMA only, reached 300 bp. The fifth well, treated with PMA,
also reached the 300 bp mark. The sixth well, which contained sample treated with
DMSO and nicotine, reached the 320 bp mark. The seventh well, treated with DMSO
and nicotine as well, reached 325 bp. The eighth and final well, treated with solely
DMSO, reached the 330 bp mark.
Conclusions:
The expected amplicon size for both the Beta-actin and MHC-Class I were around
their expected 300 bp and 500 bp marks respectively. The wells containing the samples
that were only PMA and DMSO in the MHC-Class I test all seemed to hit their mark of
500 bp while the other test samples did not. In the wells containing the samples that were
treated with beta-actin almost all samples reached near the 300 bp mark.
The total RNA was isolated from HL-60 cells treated with different
concentrations of PMA/DMSO and nicotine combinations. The mRNA collected was
converted into cDNA through the process of reverse transcriptase. Our primers for the
experiment were added and our total mixture was loaded to a PCR machine that
amplified our genes of interest. After the gel electrophoresis had ran for 45 minutes at
130 volts, it was analyzed under UV light so that the ethidium bromide fluorescence
could be observed. This ethidium bromide fluorescence helped to determine amplicon
base pair length and quantity. In Table 1, the ladder showed us that both of our PMA and
our one DMSO sample reached the 500 bp mark. The other samples that were treated

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with PMA/DMSO and nicotine did not reach that mark. Their results did not come out as
clearly as hoped, but since there was no definitive line as to where they ended up, one can
assume that there was a down regulation of the MHC-class I histocompatibility complex.
The addition of nicotine resulted in the MHC-class I molecules not clearly presenting
themselves. This result is important because MHC-class I molecules are how cells
present antigens for phagocytosis. Nicotine acting as a down regulator for MHC-class I
molecules goes to show why cancer cells are so effective at never being eliminated from
the body. With nicotine down regulating MHC-class I molecules, the cancer cells have
free roam to colonize with no fear of being broken down.
Future studies involving this type of experiment can help to narrow down exactly
how and why nicotine down regulates MHC-class I molecules. A study examining what
exactly is in nicotine and its chemical properties can show how it inhibits presentation.
Running samples from a cigarette containing nicotine and just nicotine, one could
compare and contrast the results of down regulation.
Another experiment involving this study could be to test the viability of cells that
were covered in a puff of smoke. The smoke inhaled from cigarettes is a huge factor in
why smoking is so bad for your lungs and it only makes sense to have a test study that.
The actual smoke inhaled from puffing a cigarette is the vessel that contains all the
possible carcinogens. Studies further documenting the smoke and how potential threats
travel in the smoke could yield interesting results.

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References:
Hewitt, Eric W. "The MHC Class I Antigen Presentation Pathway: Strategies for Viral
Immune Evasion." Immunology. Blackwell Science Inc. Web. 13 Apr. 2016.
Fust, George, Judith Kramer, Bi Zhou, and Carol Blanchog. "International Immunology."
Genetic Basis of Tobacco Smoking: Strong Association of a Specific Major
Histocompatibility Complex Haplotype on Chromosome 6 with Smoking
Behavior. Web. 13 Apr. 2016.