Leptin is Immunosuppressive in both Concanavalin A and Lipopolysaccharide Mitogenesis
of Murine Splenocytes In Vitro
An Honors Thesis (HONRS 499)
by
Eric L. Hensley
--
Dr. Nancy Behforouz
Ball State University
Muncie, Indiana
April 1999
May 1999
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Abstract
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The aim of this study was to determine the effect of recombinant murine
;...
leptin on the mitogen response mouse lymphocytes in ritm. Mouse splenocytes were
cultured from young mice and lymphocyte proliferation was measured in the
presence of media, Con A, or LPS either in the presence or absence of leptin. This
proliferation was measured using MTT uptake or 3H-thymidine uptake.
Proliferation was then measured when leptin is present in the cell culture.
We first established the optimum concentration of lymphocytes and the two
mitogens in
(~ell
culture. We then added leptin at various concentrations under
these optimized conditions and examined the effect which this hormone had on the
proliferation of murine T or B cells. Our findings indicate that leptin in ritm is
immunosuppressive and has a dose-dependent negative effect on proliferation of
both T and B cells in response to the specific T cell mitogen Con A and the B cell
mitogen LPS.
Introduction
Leptin was identified in 1994 as the product of a gene that is defective in an obese
strain of mice, ob/ob. Injection ofleptin into these mice led to a return to their normal
body weight and as a result, made news headlines as a cure to obesity. However, it was
found that many obese humans actually have an excess of leptin rather than a deficiency;
actually their problem was frequently a lack of the leptin receptor or a faulty leptin
receptor. After an initial interest in leptin as a potential weight management hormone, it
was discovered that weight loss in humans is a much more complex process (1).
Leptin is produced by the adipose tissue, and the level of leptin that is circulating
in the body is directly proportional to the total amount offat in the body. Cortisol and
insulin are known to be potent stimulators ofleptin expression. In mice, it has been
discovered that leptin acts on receptors in the hypothalamus where it inhibits food intake.
Leptin also counteracts the effects of neuropeptide Y, a feeding stimulant that is secreted
by cells in the gut and is also found in the hypothalamus (2). It is thought that leptints
effects on food intake and energy expenditure may be centrally mediated through this and
other neurotransmitters (3).
In humans, obesity is associated with an increased incidence of infection, diabetes,
and cardiovascular disease, which together account for most obesity-related deaths.
Decreased leptin or leptin receptor expression results in a decrease in energy expenditure,
and obesity. 1t is not yet clear if leptin-dependent signal transduction directly effects or
promotes any of the conditions such as infection which characterize human obesity (4).
There does appear to be a definite relationship between TNF-alpha, an important
inflammatory cytokine, and leptin responsiveness or production. Some have shown that
TNF-alpha is responsible for inducing leptin following exposure to LPS in mice and
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humans (5,6). Others have shown an inhibition ofleptin production by TNF-alpha in
certain other mice and human adipocytes (7). Still others have shown that IL-1 beta,
another important inflammatory cytokine, can mediate leptin induction during
inflammation (8). Indeed, under conditions which account for most human deaths from
obesity (infection, diabetes, etc.), there are abnormalities in TNF-alpha expression (6). In
yet other studies, exogenous leptin reversed or up-regulated abnormalities such as obesityrelated deficits in macrophage phagocytosis and in the expression of proinflammatory
cytokines (4). Taken together, these results indicate that leptin may up-regulate, or at
least
influenc{~
inflammatory immune responses, which may provide a common pathogenic
mechanism that leads to many of the complications resulting from obesity (5).
Thus, in addition to the interest in leptin as a weight management hormone,
interest has also been generated to investigate the role of leptin in the immune response,
particularly in starving or malnourished animals which have very low leptin levels.
Nutritional deprivation is known to suppress the function of the immune system in humans
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and animals. It was determined that levels ofleptin can be dramatically reduced by
inducing starvation or increased by inflammatory mediators. Both impaired cell-mediated
immunity and reduced levels ofleptin are both features oflow body weight in humans (9).
It is also known that malnutrition predisposes to death from infectious diseases.
It has been proposed that leptin has a specific effect on murine T-cell mediated
immune responses. It was discovered that leptin actually differentially regulated the
proliferation of naive and memory T cells. In addition, leptin increased Thl and reduced
Th2 cytokine production. When leptin was administered to mice that had been starved
(low levels ofleptin), it reversed the immunosuppressive effects of the acute starvation.
This indicated that the in YiYo. response to leptin was not immunoenhancing. It was also
suggested that a falling concentration of leptin may act as a signal to the body to conserve
energy in the face of limited reserves by shutting down some systems, such as the cognate
immune response which require considerable cellular proliferation (10).
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These findings led to the question we addressed in the following project. We
attempted to determine whether or not the presence of leptin would enhance lymphocyte
proliferation in Yitm in the presence of either a B or T cell mitogen. The B cell mitogen
used was lipopolysaccharide (LPS) and the T cell mitogen was Concanavalin A (Con A).
Interestingly, leptin appeared to significantly suppress rather than enhance proliferation
induced by both of these mitogens.
Materials and Methods
Experimental Animals
Three to five month old male and female BALB/C mice were used from the
breeding colony maintained at Ball State University. These mice were housed and fed
under conditions which meet the approval of the Ball State University Department of
Biology.
Solutions and Reagents
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Cells were cultured in RPMI media with penicillin, streptomycin, and antimycotic,
beta-mercaptoethanol 0.05 mM, and 10% FBS. Recombinant murine leptin produced in
E. coli was purchased from Linco Research, Inc. and dissolved in 100 ul sterile dd water
and 900 ul ofHBSS with 0.05% Triton-X to give 100,000 ng/ml stock. Con A produced
from Canavalia ensiformis type IV was purchased from Sigma Chemical and a 1 mg/ml
stock was made in sterile media. LPS from E. coli serotype 055:B5 was purchased from
Sigma chemical and a 1 mg/ml stock was made in sterile media.
Protocol for Proliferation Studies
Mice were killed by CO2 asphyxiation and the spleen removed. After washing 2X
in HBSS, the cells were adjusted to 2.5 X 106 or 4.5 X 106 cells/ml in RPMI media. The
cells were added to a microtiter plate and various concentrations of new (1997) and old
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(1993) ConA (1 ug/ml, 2 ug/ml, and 4 ug/ml) or LPS (Trial #1 : 10 ug/ml, 25 ug/ml, and
75ug/ml; Trial #2 : 5 ug/ml, 10 ug/ml, 25 ug/ml) were added to the cells. As controls,
media only was added to others. Leptin was added in various concentrations (40 ng/ml,
20 ng/ml. and 2 ng/ml) with either Con A or LPS to cell cultures to determine the
hormone's effect on proliferation. The cells were then incubated for two days in a CO 2
incubator. A11:er incubation, the cells were pulsed with MTT [5 mg/ml] or 3H-thymidine
and reincubat,ed for - 4 hrs or overnight, respectively. For the MTT based assays, the
plates were removed, spun in a floor centrifuge, flicked, and each well was resuspended
with 4% HCl acidified isopropal (100 ul) to dissolve the reduced dye by repeated
pipetting. For the 3H-thymidine based assays the cultures were harvested on glass fiber
filters with an automated microplate cell harvester. The optical densities of each well were
read at 570 run or 600 nm or by measuring CPM in a Beckman scintillation counter and
recorded.
Results
-.
Determination of optimum conditions/reagents for Con A mitogen stimulation of murine
splenocytes. We explored the effects of the mitogen Con A on the proliferation of murine
splenocytes. We incubated two concentrations of cells (2.5 x 106 or 4.5 x 106/ml). Both
concentrations of cells responded to Con A stimulation in a dose dependent manner when
measured using MTT uptake analysis (Fig 1).
The greatest amount of proliferation for the two different concentrations of cells
occurred at a Con A concentration of 2 ug/ml and was more than double that of when the
cells were grown in media alone. The Con A concentration of 4 ug/ml was also
stimulatory but not significantly higher than at 2 ug/ml. The minimum optimal stimulatory
concentration was confirmed to be 2 ug/ml in another experiment (Fig 2) in which only
2 x 106 cells/m! were used. In order to evaluate the value of another method of
assessment of cellular proliferation, 3H-thymidine uptake by stimulated cells was also used
as described in materials and methods. The data from this experiment is shown in figure 3.
For all further determinations we chose to use the MTT uptake analysis instead of the 3Hthymidine uptake because the MTT gave us a higher level of sensitivity. It was also much
easier to use because it takes less time and materials. By either assay method there
appeared to be no difference in older or newer Con A preparations.
Determination oj optimum LPS conditions/reagents. To examine the effects of the
mitogen LPS on the proliferation of murine B splenocytes we incubated the cells in either
media alone or with various concentrations ofLPS. The LPS was found to increase
murine splenocyte proliferation (Fig 4) with the optimum concentration at 25 uglml.
However, the LPS was not as effective in higher concentrations as Con A. This result
probably reflects the lower proliferative potential ofB cells as compared to T cells.
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Influence oj /eptin on Con A induced spleen cell mitogenesis. Since proliferation can be
stimulated by the addition of Con A to the murine splenocytes, we next examined what
effects the hormone leptin had on this proliferation. As we can see in figure 5, the leptin
had a dose-dependent immunosuppressive effect on the proliferation of the splenocytes.
The cells that were incubated in media and Con A only proliferated at a higher level than
those with leptin and Con A. The higher levels ofleptin had a much lower level of
proliferation than the lower levels. Thus, it appears that leptin suppressed the proliferation
of murine T splenocytes.
Influence oj /eptin on LPS induced spleen cell mitogenesis. We followed the same
procedure to examine the effects of leptin on LPS induced murine splenocyte proliferation
and found similar results. In figure 6 we can see that leptin also caused a dose-dependent
immunosuppression on the proliferation of the B splenocytes. Cells which were incubated
in LPS and m(~dia only showed higher proliferation levels than any of those with leptin.
Again, the higher the concentration of leptin was, the lower the level of proliferation.
Leptin suppressed the proliferation of murine B splenocytes.
Discussion
From this new found information we can now see that leptin has an
immunosuppressive effect on the proliferation of murine splenocytes in response to T and
B cell mitogens in Yitm. Several other studies have examined the role of leptin and its
function in the immune system and some have claimed that leptin actually enhances the
response. This is not what was observed in the data from this experiment.
1t would be interesting to perform this experiment in YiYo. to determine whether or
not there is some other factor in the mice that allows them to overcome leptin's in Yitm
immunosuppressive effects. This could be done by infecting or sensitizing animals
(starved or normal) to an antigen while giving the animal physiologically relevant doses of
exogenous leptin. Measurements of the animals' immune response to the
antigens/infection might reveal the role that leptin plays in immunity.
The existence and role of the hormone leptin is a fairly new and very exciting
discovery that has been implicated in many different body processes. It is very likely that
this hormone is under the control of many different factors and has numerous effects in
both humans and mice. Much remains to be learned about leptin and its role in the body.
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Figure 1. Determination of Optimum Conditions for Con A Mitogenesis Measured by MTT Uptake
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Figure 2. Con A Mitogenesis of Murine Splenocytes Measured by MTT Uptake Analysis
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Figure 4. LPS Mitogenesis of Murine Splenocytes Measured by MTT Uptake Analysis
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Figure 6. Influence of Leptin on LPS Mitogenesis Measured by MTT Uptake Analysis
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25
References
1.
Zhang, Y. et al. Positional Cloning of the Mouse Obese Gene and its Human
Homologue. Nature 372:425-432 (1994).
2.
Stephens, T.W. et al. The Role ofNeuropeptide Y in the Antibody Action of the
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3.
Pelleymounter, M.A. et at. Effects of the Obese Gene Product on Body Weight
Regulation in oblob Mice. Science 269:540 (1995).
4.
Loffreda, S. et al. Leptin Regulates Proinflammatory Immune Responses.
FASEB J 12:57-65 (1998).
5.
Finck, B. et al. In YiYu and In Yitm Evidence for the involvement of Tumor
Necrosis Factor-Alpha in the Induction ofLeptin by Lipopolysaccharide.
J Endo. 135:2278 (1998).
6.
Zumbach, M. et al. Tumor Necrosis Factor Increases Serum Leptin Levels in
Humans. J Clin Endocrin Metab. 82:4080-4082.
7.
Yamaguchi, M. et al. Autocrine Inhibition of Leptin Production by Tumor
Necrosis Factor-alpha (TNF-alpha) through TNF-alpha Type I Receptor In
Yitm. Biochem Biophys Res. Comm. 244:30-34 (1998).
8.
Faggioni, R. et al. IL-l beta Mediates Leptin Induction During Inflammation. Am
J Physiol. 274 (Regulatory Integrative Compo Physiol. 43) : R204-R208
(1998).
9.
Lord, G.M. et at. Leptin Modulates the T-Cell Immune Response and Reverses
Starvation-Induced Immunosuppression. Nature 394:897-901 (1998).
10.
Ahima, R.S. et al. Role ofLeptin in the Neuroendocrine Response to Fasting.
Nature 382:250-252 (1996).
Acknowledgments
I would like to thank Dr. Nancy Befhorouz for all of her help, not only on this
project, but throughout my college career. I would also like to thank my parents and
friends who have stayed behind me since day one. My final words of thanks go to my
girlfriend Traei, without her love and support I would have never made it this far.