Park
BSCI Annie 330
Natural Killer Cells in Obesity
The researchers are investigating the question of whether or not obese individuals should
expect a decrease in life expectancy, and if smoking increases their chances of a shorter life
expectancy (OShea 2010). The authors cited information about how smokers had increased
maglinancies and other health complications, as well as obese individuals susceptibility to various
health complications leading to reduced life expectancy. The researchers also cited that NK cells
and their capacity to kill tumor cells, and how the NK cell activity is decreased in smokers in
comparison to non-smokers as well as a smokers greater proneness to infections. The authors also
cited information about NK cells having a decreased cytotoxicity in obese individuals in
comparison to lean individuals (OShea, 2010). The biological systems they used in their
experiments were the blood cultures of both obese and lean individuals (OShea, 2010). Some
words in the introduction that were crucial to ones understanding of the
paper included; malignancy which can be defined as an unwanted
proliferation of cells that are indicative of a cancer harmful to the body. In
vivo
is another term which can be defined as in a culture, in this case the cells
were grown and observed in culture. Metastasis can be defined as the
spread of cancerous cells throughout ones body. Cytotoxicity is the toxicity or how effective cells
may be in killing one another utilizing chemical toxins. Adipokines are a class of proteins or
cytokines that includes adiponectin and leptin which helps regulate ones metabolism and are
secreted by fats cells. Phenotypes are the manifestation of an organisms physical or mechanical
attributes derived from its genotype.
Fluorescence is used throughout the scientific community as an easy way to establish the
presence, count, and cell sorting techniques. By making a cell fluoresce you are literally treating
that cell with a glowing dye giving the cell the capability to emit photons thereby emitting light .
Flow Cytometry is a method by which one may be able to separate and analyze the definitive
Annie Park
BSCI 330
Natural Killer Cells in Obesity
numbers of cells based on their fluorescence using lasers and various light sensors . The cells are
first put through a central core which decreases in diameter, as they are pushed down by a sheath
flow, which eventually leads to a single cell by cell stream (see figure 1) (Nolla, 2009). The cells
are then subject to a variety of lasers which can give one qualitative and quantitative information
based on the distinct light produced by the cells with fluorescent cytokines or DNA based on their
response to the laser (Rahman, 2006). The responses to the lasers can include the wavelength at
which the photon is emitted and etc. There are also usually a series of filters which can block out
certain wavelengths while transmitting others, which are arranged in a line so that each distinct
wavelength can be measured to categorize the cell being analyzed (Rahman, 2006). Monoclonal
Antibodies are antibodies that are synthesized in the laboratory that can attach to any specific
mutagenized cells in the body in order to help ones immune system recognize the defective cell
better (Kimball, 2011). Cytotoxicity can be defined as the properties of a certain medium or
chemical by which a cell is subject to death but not through to routine apoptotic pathways or by
any established mechanism (Rode, 2008). Cytotoxic assays are used to determine the efficacy of
the cytotoxin available as well as the count of the cells affected by the cytotoxic medium/chemical
(Rode, 2008). These assays are generally performed by staining a cell strain, and incubating it with
the cytotoxin, and then investigating the quantitative effects of the toxin through assays such as
flow cytometry by measuring the fluorescence of the target cells (Rode, 2008).
The assays utilized in the study include; Flow Cytrometry, and Cytotoxicity Assays . The
cells were prepared in a suspension of 1x10^6 /mL and were measured for viability through the
ethidium bromide test (to assure that the cells were able to use). The cells were stained with
monoclonal antibodies (mAbs) recognizable by the immune system. For preparation of Flow
Cytrometric Analysis, the leukocytes were stained with mAbs; anti-C45, and for the identification
of the NK cells and NKR +T cells, PE CD56, and PerCP labeled anti-CD3 (SK7) mAb were also
Annie Park
BSCI 330
Natural Killer Cells in Obesity
used. After the cells were stained they were incubated and fixed with paraformaldehyde . For the
cells that were used to determine the effect of the CSE; there were various dilutions of CSE
prepared which were then incubated with the NK cells and the K562. The NK cells were then
stained with mAbs for flow cytometric analysis with the NK cells after incubation with FITC
labeled anti CD158a (HP-3E4), CD158b, anti-NKB1(DX9), and activatory marker antiCD69(L78). Cytokine production was also determined by exposing the NK and K562 cells to
saponin which permeabilized the membranes so that they could be labeled with anti-IFN-, antiTNF-, and anti-IL-10 to bind to their respective cytokines if they are produced.
In the flow cytometry assay the CD45 cells were isolated by lymphogate processes and
then they attained a >95% peripheral lymphocyte count which they then proceeded to perform
flow cytometry on giving a percentage of population of CD45+ lymphocytes to assess the efficacy
of the NK cells in the population between lean and obese individuals. The test was then repeated
with four individuals after 8-24 weeks and the results were duplicated.
In the cytotoxicity assays performed, the NK cells were first gathered by using anti-CD56
mAb coated magnetic beads to separate the NK cells. Then the K562 cancerous cells were added
to the solution of NK cells and the ethidium bromide/acrinide orange staining was performed to
determine their viability. Then K562 cells were isolated and stained with CFSE and added to the
NK cell population (in various ratios), which were then analyzed through flow cytometry. Another
set of cell cultures were incubated with or without the presence of CSE (which was available in
various dilutions) and the killing of CFSE-labeled K562 cells were measured by staining through
7-AAD after incubation and by analysis flow cytometry (OShea 2010). The role of adipokines;
adiponectin and leptin were tested with or without CSE using the Total Cytotoxicity and
Apoptosis Detection Kit.
In Fig. 1, the researchers were trying to answer the question of whether or not there was a
Annie Park
BSCI 330
Natural Killer Cells in Obesity
difference between the basal NK cell function between obese and lean individuals. The researchers
specifically wanted to measure the NK cell count by percentage, and the percentage of cells killed
between lean and obese individuals, they were able to measure this through flow cytometry assay .
The results showed in Fig 1.A; lean subjects had a much higher NK count overall, and in Fig 1.B
the cytotoxicity of lean subjects were also significantly higher meaning that the NK cell efficacy
was much lower in obese individuals leading to a compromised immune system. In Fig. 2, the
researchers were answering the question of effect of the CSE and IL2 on the NK cells. The
researchers wanted to measure the NK cell efficacy through percentage of tumor cells killed using
flow cytometry as well as the cytotoxic assay. The results showed in Fig. 2A that the NK cells
were more effective when treated with IL2, and had a significant decrease in efficacy when treated
with the CSE. In Fig 2B. the data was presented that the greater the concentration of smoke
derived constituents, the greater the percentage of tumor cells killed by the NK cells, meaning that
smoking would definitely compromise the immune system through reduced efficacy of NK cells .
In Figure 3, the researchers were answering the question of whether CSE affected lean and obese
subjects, and whether or not adiponectin or leptin affected the efficacy of the NK cells. The
researchers wanted to measure the NK efficacy through decrease in cytotoxicity as well as the %
of tumor cells killed between the various cell solutions, which were all measured by the cytometry
assay as well as the cytotoxicity assay. The results showed the there was a decrease in both obese
and lean individuals in the % tumor cells killed as well as a % decrease in cytotoxity for obese
individuals compared to lean ones. The results also showed that in lean individuals there was a
significant increase in % tumor cells killed in presence of adiponectin, whereas in obese
individuals adiponectin did not show an increase in tumor cells killed, and leptin did not have any
significant data. In Fig 4A the researchers were answering the query of how the NK cells efficacy
was affected by CSE, CSE+adiponectin, and CSE+leptin in both obese and lean individuals . The
Annie Park
BSCI 330
Natural Killer Cells in Obesity
researchers measured the % tumor cells killed in each of the cellular solutions and were able to
quantify the data through cytotoxic assays as well as flow cytometry. The data showed that CSE
greatly inhibited the NK cells cytotoxicity, and that adiponectin (in both lean and obese subjects)
restored the function of NK cells, and leptin showed no significant data . In Figure 5 the
researchers were responding to the question of whether or not the production of
cytokines/markers; CD107, CD69, IFN-, or IL-10 in NK cells were affected by the presence or
absence of CSE and adiponectin. The researchers were measuring the %of NK cells to determine
the presence or absence of the cytokines/markers. The data in Fig 5A showed that production of
CD107 and CD69 were significantly inhibited by the presence of smoke, but with adiponectin
CD107 and 69 function could be restored in the presence of smoke to normal. In Fig 5C the data
showed that cigarette smoke didnt alter the production of IFN- or IL-10 by NK cells after
exposure to tumor cells, however when adiponectin was introduced along with smoke, IFN-
increased, but there was no significant data from IL-10.
Overall, this experiment was able to deduce that there was a significant link between
immune deficiencies and obese individuals and smoking individuals . The researchers concluded
that there was a significant compromise of NK cell function and cytotoxicity due to the smoke
extract. Adiponectin was found to have a positive effect on NK cell efficacy and cytotoxicity in
lean individuals in the presence of smoke. However, in obese individuals, adiponectin was shown
to have no significant response in restoring NK cell efficacy. The findings however found that
smoking was not a significant factor in decreasing the NK cell efficacy, cytotoxicity, tumor death,
or surface inhibitory receptor production. Thus, adiponectin may be significant in blocking the bad
effects of smoke on NK cell activity, or adiponectin may enhance cell function cancelling out the
smoke. I believe that future studies should be conducted to investigate fully the pathways that the
adiponectin may utilize to enhance NK cell activity and cytotoxicity. There would also be some
Annie Park
BSCI 330
Natural Killer Cells in Obesity
great utility in investigating the effects of adiponectin on other types of immune cells and
measuring its effect on the other cells against other toxins or carcinogens such as UV light .
Yes, I agree with the conclusions of the researchers drew from their results. They seemed
to pass good judgment based on the results that they were given. However, I do not believe that
the results were presented in the best most easily understood way. I think that the researchers
should have provided a chart that includes which cells were treated with which molecules,
reagents and media. But, I believe that the results were presented in an acceptable manner.
Annie Park
BSCI 330
Natural Killer Cells in Obesity
Bibliography
Nolla, Hector (2009). Flow Cytometry Principles. Retrieved from
http://biology.berkeley.edu/crl/flow_cytometry_basic.html
OShea, Donal (January, 2010). Natural Killer Cells in Obesity: Impaired Function and Increased
Susceptibility to the Effects of Cigarette Smoke. PloS ONE, Volume 5, Issue 1, e8660.
Kimball, John (Jan 2011). Monoclonal Antibodies. Retrieved from
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mono
clonals.html#Monoclonal_Antibodies
Rahman, Misha Ph.D (2006). Introduction to Flow Cytometry. Retrieved from
http://www.abdserotec.com/uploads/Flow-Cytometry.pdf
Rode , Hans-Jurgen Ph.D. (2008). Relationship between Cytotoxicity, Apoptosis, and Necrosis.
j_Cytotox_CelProl_4th_edition.pdf