INFLUENCE OF GLUCOCORTICOSTEROID HORMONES ON
IMMUNE FUNCTIONS OF NORMAL AND CUSHING’S
SYNDROME HORSES
A Senior Scholars Thesis
by
KAITLIN ALYSSA GUTIERREZ
Submitted to the Office of Undergraduate Research
Texas A&M University
in partial fulfillment of the requirements for the designation as
UNDERGRADUATE RESEARCH SCHOLAR
April 2008
Major: Animal Science
INFLUENCE OF GLUCOCORTICOSTEROID HORMONES ON
IMMUNE FUNCTIONS OF NORMAL AND CUSHING’S
SYNDROME HORSES
A Senior Scholars Thesis
by
KAITLIN ALYSSA GUTIERREZ
Submitted to the Office of Undergraduate Research
Texas A&M University
in partial fulfillment of the requirements for the designation as
UNDERGRADUATE RESEARCH SCHOLAR
Approved by:
Research Advisor:
Associate Dean for Undergraduate Research:
April 2008
Major: Animal Science
Thomas H. Welsh, Jr.
Robert C. Webb
iii
ABSTRACT
Influence of Glucocorticosteroid Hormones on Immune Functions of Normal and
Cushing’s Syndrome Horses (April 2008)
Kaitlin Alyssa Gutierrez
Department of Animal Science
Texas A&M University
Research Advisor: Dr. Thomas H. Welsh, Jr.
Department of Animal Science
The effect of glucocorticosteroid hormones on the equine immune system was assessed.
Hypercortisolemia, associated with Cushing’s syndrome, is attributed with a decrease in
equine immunity. Immunity may be compromised due to the effect of both synthetic
(Dexamethasone) and naturally occurring (cortisol) glucocorticosteroid hormones. The
glucocorticosteroid receptor antagonist RU486 decreases the unfavorable effects of
hypercortisolemia.
This study was designed to see if RU486 could modulate the
negative effects of DEX. Whole blood samples were obtained via jugular venipuncture
from 15 horses (4 breeds; 12 stallions; 3 geldings; 5-to-15 years of age; 450-800 kg BW)
and were put into EDTA vacutainers. Separate cultures were established for each horse,
and lymphocytes were isolated. Lymphocytes were plated at 100,000 cells per well and
were incubated at 37oC for 96 hours. Lymphocytes were incubated in medium alone
(DMEM/F12 1:1), medium containing concanavalin A (0-to-5 µg/mL), 1 µM of DEX, 1
µM of RU486, or any combination thereof. Cellular proliferation was determined by
Promega CellTiter96 assay. Stimulation indices were determined by a comparison to
iv
conconavalin A at a concentration of 0 µg/mL. SAS was used to determine statistical
differences between treatments. Conconavalin A showed a dose dependent increase in
lymphoproliferation (P<.0001; initial and maximal increase at 0.3125 and 5 ug/ml
ConA, respectively). DEX inhibited ConA induced lymphoproliferation (P<.0001).
Specifically, DEX reduced basal proliferation by 20.2%. At 0.3125 and 5 µg/mL,
proliferation was reduced by 34.4% and 23.9% respectively. The addition of RU486
counteracted the adverse effects of DEX (P<.0001). RU486 increased basal proliferation
by 25.2% when compared to the inhibitory effect of DEX. Glucocorticosteroid
antagonists may be used to study how immune functions may be suppressed in horses
that are phenotypically hypercortisolemic due to the following factors: stress; DEX
therapy; Cushing’s syndrome, or metabolic syndrome.
v
ACKNOWLEDGMENTS
First, I would like to thank Dr. Welsh for all of his support and guidance. Over the past
year I have learned things that I will use over a lifetime. His kindness and knowledge
has been unremarkable, and I do not think that any of this could have been possible
without him.
I would also like to thank Jennie Lyons and Nicole Burdick. They are the reason I was
able to complete this project. They taught me how experiments were run and were
always there to lend a helping hand and be a voice of support. They are incredible
people, and have taught me so much.
Thank you to my family and friends for their support and love throughout the past year.
I have learned a lot, and they are the ones who helped me. I feel honored to have such
wonderful people in my life.
Finally, I would like to thank Texas A&M University and the Undergraduate Research
Office for providing me with such an incredible opportunity.
vi
NOMENCLATURE
ConA
ConcanavalinA
DEX
Dexamthasone
HPA
Hypothalamic-Pituitary-Adrenal Axis
CRH
Corticotrophin Releasing Hormone
ACTH
Adrenocorticotropin Releasing Hormone
MR
Mineralcorticoid Receptor
vii
TABLE OF CONTENTS
Page
ABSTRACT ....................................................................................................................... iii
ACKNOWLEDGMENTS ................................................................................................... v
NOMENCLATURE ........................................................................................................... vi
TABLE OF CONTENTS .................................................................................................. vii
LIST OF FIGURES ............................................................................................................ ix
CHAPTER
I
INTRODUCTION ....................................................................................... 1
Literature review ............................................................................. 2
II
METHODS.................................................................................................. 7
In vitro study of the effect of DEX and
RU486 on lymphocyte proliferation and
immunoglobulin production ............................................................ 7
In vitro comparison of lymphocyte proliferation
and immunoglobulin production in horses with
normal concentrations of cortisol and in horses
with consistently high concentrations of cortisol
(e.g., horses with Equine Metabolic Syndrome) ............................. 8
Proliferation- CellTiter96 ................................................................ 9
Statistical analysis ........................................................................... 9
III
RESULTS AND DISCUSSION ............................................................... 10
Incubation time .............................................................................. 10
Effect of treatments pertaining to equine
lymphocyte proliferation ............................................................... 12
viii
CHAPTER
Page
Effect of in vitro treatments in
Non-Cushing’s-like horses .............................................................. 18
Effect of in vitro treatments in
Cushing’s-like horses ...................................................................... 23
Comparison of lymphocyte proliferation in
Non-Cushing’s-like vs. Cushing’s-like horses ................................ 28
IV
CONCLUSIONS ....................................................................................... 36
LITERATURE CITED ......................................................................................... 37
APPENDIX A ....................................................................................................... 39
APPENDIX B ....................................................................................................... 40
APPENDIX C ....................................................................................................... 43
CONTACT INFORMATION ............................................................................... 45
ix
LIST OF FIGURES
FIGURE
Page
1
Lymphocyte proliferation over a
time course of 24-to-96 hours. .......................................................................... 11
2
Stimulatory effect of increasing concentrations
of ConA on in vitro proliferation of equine
lymphocyte proliferation (P<.0001). ................................................................. 13
3
The suppressive effect of DEX on
in vitro equine lymphoproliferation (P<.0001). ................................................ 14
4
The effect of RU486 on equine lymphocyte proliferation ................................ 16
5
The effects of ConA, DEX, and RU486
on equine lymphocyte proliferation .................................................................. 17
6
Effect of ConA on lymphocyte
proliferation in Non-Cushing’s-like horses ....................................................... 20
7
The effect of DEX on cellular
proliferation in Non-Cushing’s-like horses ....................................................... 21
8
The antagonistic effect of RU486
on DEX in Non-Cushing’s-like horses .............................................................. 22
9
The effect of ConA on lymphocyte
proliferation in Cushings-like horses ................................................................ 25
10 The effect of DEX on proliferation in
Cushing’s-like horses ........................................................................................ 26
11 Antagonistic effect of RU486 on DEX,
and agonistic effect on ConA in Cushing’s-like horses .................................... 27
x
FIGURE
Page
12 A comparison of lymphocyte proliferation
in the presence of ConA in
Cushing’s-like vs. Non-Cushing’s-like horses .................................................. 29
13 A comparison of equine lymphocyte proliferation,
with the addition of DEX, among Cushing’s-like
and Non-Cushing’s-like horses ......................................................................... 30
14 The effect of equivalent concentrations of DEX
and RU486 on lymphocyte proliferation of
Cushing’s-like or non-Cushing’s-like ............................................................... 32
15 The effect of RU486 on equine lymphocyte proliferation ................................ 35
1
CHAPTER I
INTRODUCTION
Equine Metabolic Syndrome is a hormonal disorder caused by the prolonged exposure of
the body’s tissues to the glucocorticosteroid hormone cortisol. This situation may be the
consequence of adrenal gland over production of cortisol due to an imbalance of the
hypothalamic-pituitary-adrenal (HPA) axis. Horses that have Equine Metabolic
Syndrome or exhibit Equine Metabolic Syndrome- like symptoms generally have higher
concentrations of cortisol in their peripheral circulation.
Cortisol is a glucocorticosteroid hormone, the synthesis and secretions of which is
triggered by stress. The HPA axis is critical in maintaining physiological homeostasis.
Cortisol stimulates gluconeogenesis and it has profound regulatory effects on numerous
physiological systems. Chronic activation of the HPA axis due to extended exposure to
stress causes an increase in glucocorticosteroid production from the adrenal cortex and
catecholamine production from the adrenal medulla. Glucocorticosteroids prevent or
suppress functions of the mammalian immune system (Bauer, 2005; Chrousos, 1995).
Increased concentrations of glucocorticosteroid hormones allow pathogens to more
readily establish an infection (Chrousos, 1995).
_______________
This thesis follows the style of The Journal of Animal Science.
2
As horses with Equine Metabolic Syndrome have higher secretions of cortisol, it is now
important to determine the effect of prolonged exposure of the equine immune system to
cortisol. Data collected from this experiment will help to fill this void of knowledge.
Specifically, this project will explore the effect of DEX, a synthetic glucocorticosteroid,
on mitogen- induced proliferation in normal horses and horses with Equine Metabolic
Syndrome.
Literature Review
Equine Metabolic Syndrome
Equine Metabolic Syndrome is a hormonal disorder caused by the prolonged exposure of
the body’s tissues to cortisol, also referred to as hypercortisolism and Cushing’s
syndrome. The tissue’s prolonged exposure to cortisol is due to an imbalance of the
hypothalamic-pituitary adrenal (HPA) axis, causing an increase in secretion of
adrenocorticotropin (ACTH). The ACTH produced is semiautonomous and resets the
HPA feedback; therefore, ACTH is still secreted in the presence of abnormally high
levels of cortisol. Some horse and pony breeds are genetically predisposed to metabolic
syndrome (Johnson, 2002).
Horses affected with this Metabolic Syndrome are usually between eight and twenty
years of age and characterized as obese. Distinct excess body fat of these horses is
distributed around the neck and haunches. Brood mares exhibit irregular estrous cycles,
and as a result, are extremely difficult to breed. Geldings affected with Equine
3
Metabolic Syndrome tend to develop swollen sheaths as a result of enhanced
subcutaneous adiposity. In comparison to horses, ponies readily deposit fat around the
neck and haunches, consequently leaving them more susceptible to laminitis. It is shown
that ponies are more glucose intolerant than horses (Field and Jeffcott, 1989). Glucose
intolerance is defined as delayed hypoglycemia after an intravenous, oral glucose load,
or following a grain meal, because peripheral tissues or the liver are insensitive to insulin
action. Horses that have Equine Cushing’s Syndrome or exhibit Cushing-like symptoms
have higher concentrations of cortisol circulating throughout their system. With the
naturally high levels of cortisol, stressors do not need to be applied in order to achieve
the disruption of the HPA axis, making them a unique model to measure the effect of
prolonged exposure to glucocorticosteroids on the immune system.
Stress and Cortisol
Cortisol is a glucocorticosteroid that is synthesized by the zona fasciculata of the adrenal
cortex. Glucocorticosteroids enhance the action of hormones on body tissues and are
essential in metabolic regulation during periods of injury and stress (Gerrard et al.,
1985). Cortisol is regulated by the hypothalamic-pituitary-adrenal (HPA) axis. The
hypothalamus secretes corticotrophin releasing hormone (CRH), which acts on the
pituitary, causing the pituitary to release adrenocorticotropin (ACTH). ACTH stimulates
the adrenocortical cells to secrete cortisol, which diffuses into the blood stream and is
carried throughout the body. A dysregulation in the HPA axis causes an increase in
concentration of ACTH, consequently increasing cortisol concentrations.
4
Stress is a disruption of physiological homeostasis caused by environmental events and
conditions. There are two glucocorticosteroid actions: modulating actions and
preparative actions. Modulating actions alter an organism’s response to a stressor, while
preparative actions alter an organism’s response to a subsequent stressor, or aid in
adapting to chronic stressors (Sapolsky et al., 2000). The response to the application of
a stressor is mediated by the endocrine system. When responding to a stressor, the first
wave is quick, lasting only seconds. Secretion of epinephrine and norepinephrine is
increased from the Sympathetic Nervous System. The hypothalamus also acts acutely
by releasing CRH into portal circulation causing the pituitary to enhance ACTH
(Sapolsky et al., 2000). The second wave of the endocrine response to a stressor is long,
lasting several minutes. It involves steroid hormones and a decrease in gonadal
secretion as a result of stimulated glucocorticosteroid secretion (Sapolsky et al., 2000).
Immunity
Immunity is an animal’s ability to ward off foreign matter such as bacteria, fungi and
infectious microbes. Both the innate and adaptive immune systems form two different
systems of immunity. Innate immunity is the body’s first response to foreign matter
(Chaplin, 2006). Its germ line includes the skin, phagocytes, neutrophils, natural killers
and cytokines (Gaylean et al., 1999). Adaptive immunity is more complex than innate
immunity and requires that the antigen first be recognized and processed. Adaptive
immunity is introduced by the body’s recognition of antigens and responds through
5
vaccinations, lymphocytes, and antibodies. Adaptive, or acquired, immunity is further
divided into humoral and cell mediated immunity, where cell mediated immunity
requires that T-lymphocytes provide defense against intracellular pathogens (Gaylean et
al., 1999). Humoral immunity is driven by B-lymphocytes that respond to antigens and
become antibody producing cells and memory cells (Gaylean et al., 1999).
Stress and the Immune System
Stress causes an increase in glucocorticosteroid concentrations, compromising the
immune system, making the animal more susceptible to disease. These increased
concentrations allow pathogens to easily establish an infection. The HPA axis is critical
in maintaining physiological homeostasis and its dysregulation is associated with several
immune-mediated diseases (Bauer, 2005). Chronic activation of the HPA axis due to
extended exposure to stress causes an increase in glucocorticosteroid and catecholamine
production from the adrenal medulla. Glucocorticosteroids prevent or suppress
functions of the immune system (Chrousos, 1995). An increase in ACTH causes a boost
in production of cortisol. Glucocorticosteroid receptors bind cortisol, interfering with
the regulation of cytokine producing immune cells (NF-KB). Cortisol receptors are
located in lymphocytes and can change cellular trafficking, proliferation, cytokine
secretion, and antibody production (Padgett and Glaser, 2003). Concentrations of
cortisol achieved during periods of stress are known to inhibit lymphocyte proliferation
and suppress the secretion of certain cytokines (Chrousos, 1995). Glucocorticosteroid
hormones are lipophilic and readily pass through the plasma membrane of cells in the
6
body. GR and MR (mineralcorticoid receptor) are glucocorticosteroid receptors. Low
circulating levels of GC bind MR due to corticosterone’s preference of MR over GR.
Therefore, only at times of stress (high concentrations of glucocorticosteroids) are the
GR receptors bound. Immune cells, such as macrophages and T lymphocytes, have GR
as the primary receptors for glucocorticosteroid hormones, indicating that immune
function is mediated by the GR (Padgett and Glaser, 2003). The Sympathetic Nervous
System (SNS) is also involved in the body’s reaction to stress. CRH stimulates not only
the pituitary, but the SNS as well, prompting the secretion of norepinephrine from the
SNS and epinephrine from the adrenal medulla (Madden, 2003). The SNS and adrenal
medulla alter the autonomic nervous system resulting in catecholamine production. In
order to adapt to stress, cortisol causes a shift in homeostasis, disarming the immune
system, leaving it more susceptible to infection and disease.
7
CHAPTER II
METHODS
In Vitro Study of the Effect of DEX and RU486 on Lymphocyte Proliferation and
Immunoglobulin Production
Peripheral blood sample was obtained from seven normal horses to isolate lymphocytes.
To do this, approximately 20 mL of blood was collected from each horse via jugular
venipuncture into EDTA-coated vacutainer tubes. Samples were placed on ice and
transferred back to the lab. The red blood cells were lysed, with the intention of getting
rid of contaminants, and the lymphocytes were isolated. Seven mL of blood were
layered over 5 mL of Ficoll-Plaque and centrifuged at 4○ C and 1000G for 25 minutes.
The lymphocyte layer were removed with a sterile 5- mL pipette and transferred to a
clean 15- mL tube. HBSS was added to the 14 mL mark, and the sample centrifuged
again at 200G for 10 minutes. The supernatant was aspirated, and 2 mL of 0.2% NaCl
quickly added for trituration, then an additional 2 mL of 1.6% NaCl was added. Again,
HBSS was added to the 14 mL marker of the tube, and then centrifuged at 200G. The
supernatant was aspirated and the cell pellet re-suspended in 1 mL of culture medium
and diluted with 20 µL for determination of cell number. Treatments consisted of serial
dilutions (ranging from 0.16-to-10 ug/ml) of the blastogenic mitogen Concanavlin
(ConA, lot# 033K8936, Sigma-Aldrich, St. Louis, MO). Treatments and cell suspension
were added to the wells to yield a total volume of 100 μL and a final concentration of
5
1x10 cells/well. Treatment groups consisted of medium alone (control), increasing
8
doses of ConA, DEX (ranging from 0.001-to-1 uM), RU486 ((ranging from 0.001-to-1
uM) and appropriate combinations of ConA, DEX and RU486. Cells were incubated at
o
37 C, 5% CO2, and 50% relative humidity for 92 hrs. Media was harvested to
determine IgM concentrations, and cell proliferation rate was determined as noted
below.
In Vitro Comparison of Lymphocyte Proliferation and Immunoglobulin
Production in Horses with Normal Concentrations of Cortisol and in Horses with
consistently High Concentrations of Cortisol (e.g., Horses with Equine Metabolic
Syndrome).
Peripheral blood sample was obtained from seven normal horses and from six horses
with metabolic syndrome to determine if abnormally high concentrations of endogenous
cortisol adversely affect lymphocytes. Peripheral blood samples were taken once from
each horse to determine their endogenous concentration of cortisol. Lymphocytes were
isolated from horses with either normal and abnormally high concentrations of cortisol.
The ability of these cells to proliferate and to produce IgM in response to the blastogenic
mitogen Concanavlin (ConA) were contrasted in vitro to assess to what degree, if any,
that high endogenous concentration of cortisol affects immune functions. Specifically,
cells were treated with media alone or increasing doses of ConA (ranging from 0.16-to10 ug/ml) to determine proliferation rate. Medium concentration of IgM were also
determined.
9
Proliferation- CellTiter96
Once the cells were isolated, they were transferred to a 96-well microtiter incubation
plate. The plate was put into a 5% CO2 humidified incubator for 92 hours. After the 92hour period, 15 µL dye solution was added to each well, and the plate incubated for
another four hours at 37○ C. A STOP solution was added to each well and incubated at
room temperature for one hour. Absorbance was recorded at 570 nm with a reference
wavelength of 630-750 nm.
Statistical Analysis
RIA and ELISA data was analyzed by Assay Zap (Biosoft, Cambridge, UK). Treatment
differences were determined by ANOVA procedures of SAS (SAS Inst., Inc., Cary, NC).
10
CHAPTER III
RESULTS AND DISCUSSION
Incubation Time
After preparation of the plates was completed, they were incubated for 96 hours at 37oC,
5% CO2. A time course study was performed to see how incubation time influenced
proliferation, and at what time would it be optimal to incubate the plates for in order to
see adequate stimulation indices (Figure 1). Proliferation was observed at 24, 48, 72 and
96 hours. Higher levels of proliferation were observed at 72 hours, with ample levels of
proliferation seen at 96 hours. Due to only slight proliferation at 72 hours, and
incubation time of 96 hours was used throughout the study.
11
2.5
Stimulation Index
2.0
ConA 24hr
ConA 48hr
ConA 72hr
ConA 96hr
1.5
1.0
0.5
[ConA] ug/mL
Figure 1. Lymphocyte proliferation over a time course of 24-to-96 hours.
5.
00
00
2.
50
00
1.
25
00
0.
62
50
0.
31
25
0.
00
00
0.0
12
Effect of Treatments Pertaining to Equine Lymphocyte Proliferation
Lymphocytes were incubated in medium alone (DMEM/F12 1:1), medium containing
Concanavalin A (0-to-5 µg/mL), 1 µM of DEX, 1 µM of RU486, and specific
combinations of these factors as depicted by the appropriate figure legend.
Conconavalin A (ConA) caused a dose dependent increase in lymphoproliferation
(P<.001; Figure 2). Lymphocyte proliferation increased as the concentration of ConA
in the medium ranged from 0-to-5 µg/mL. At a concentration of 0 µg/mL of ConA, the
mean basal stimulation index was 1.207 and increased by 2.56-fold to have a stimulation
index of 3.092 when the medium concentration of ConA was 5 µg/mL.
Dexamethasone (DEX) is a synthetic glucocorticosteroid that is antagonistic to cellular
proliferation. DEX is a synthetic form of the endogenous glucocorticosteroid hormone
cortisol. In vitro, it behaves as cortisol would in vivo, suppressing immune function by
decreasing the number of lymphocytes produced. A constant concentration of DEX, 1
µM, was added to varying concentrations of ConA. The addition of DEX to cell culture
medium containing ConA decreased lymphocyte proliferation by 21.7% (P<.0001;
Figure 3). Specifically, it decreased basal proliferation by 20.2%, and at ConA
concentrations of 0.3125 µg/mL and 5 µg/mL by 31.7% and 23.9% respectively.
RU486 is synthetic antiglucocorticosteroid that negates the negative effects of DEX. In
the presence of ConA, RU486 allows for the further expression of cell proliferation
(Nordeen et. al, 1993). RU486 coupled with ConA outperformed cellular proliferat
13
5
Stimulation Index
4
3
2
1
5.
00
00
2.
50
00
1.
25
00
0.
62
50
0.
31
25
0.
00
00
0
[ConA] ug/mL
Figure 2. Stimulatory effect of increasing concentrations of ConA on in vitro
proliferation of equine lymphocyte proliferation (P<.0001).
14
4
ConA
Dex
Stimulation Index
3
2
1
5.
00
00
2.
50
00
1.
25
00
0.
62
50
0.
31
25
0.
00
00
0
[ConA] ug/mL
Figure 3. The suppressive effect of DEX on in vitro equine lymphoproliferation
(P<.0001).
15
of ConA alone, except at a basal concentration of ConA of 0 µg/mL, where ConA had a
higher stimulation index than RU486. At ConA concentrations of 0.3125 and 5 µg/mL,
RU486 had higher proliferation percentages than ConA alone of 17.0 and 13.1
respectively.
As displayed in Figure 3, DEX suppresses lymphocyte proliferation, and as shown in
Figure 4, RU486 increases lymphocyte proliferation at concentrations of ConA above o
µg/mL. The next study was performed to see if RU486 could counteract the suppressive
action of DEX on proliferation. Addition of equivalent concentrations of RU486 and
DEX (1 µg/mL) were added to varying concentrations of ConA (0-to-5 µg/mL), in
medium containing equine lymphocytes. The addition of RU486 was able to overcome
the inhibitory action of DEX (P<.0001; Figure 5).
Specifically, RU486 overcame the suppressive effects of DEX and increased basal
proliferation by 6.4%. At 0.3125 µg/mL, ConA coupled with RU486 and DEX
increased proliferation by 4.5%, whereas DEX reduced proliferation at the same
concentration by 34.4%. When DEX alone is compared to DEX and RU486 combined
together in one treatment at a concentration of 0.3125 µg/mL, proliferation was
increased 1.6 fold. Also, at a concentration of 5 µg/mL, RU486 coupled with DEX
overcame the suppressive action of DEX by 21.3%; however, it was not able to stimulate
as much proliferation as the cells in medium and ConA alone (absent of RU486 and
16
4
ConA
RU486
Stimulation Index
3
2
1
[ConA] ug/mL
Figure 4. The effect of RU486 on equine lymphocyte proliferation.
5.
00
00
2.
50
00
1.
25
00
0.
62
50
0.
31
25
0.
00
00
0
17
4
ConA
Dex
Dex+RU486
Stimulation Index
3
2
1
5.
00
00
2.
50
00
1.
25
00
0.
62
50
0.
31
25
0.
00
00
0
[ConA] ug/mL
Figure 5. The effects of ConA, DEX, and RU486 on equine lymphocyte proliferation.
18
DEX). This indicates that RU486 is capable of counteracting the suppressive action of
DEX on cellular production, allowing the body to produce lymphocytes.
Effect of In Vitro Treatments in Non-Cushing’s-like Horses
Figures 2-to-5 illustrate the effects the in vitro treatment of lymphocyte proliferation on
both Non-Cushing’s-like and Cushing’s-like horses. The effect of DEX and RU486 on
Non-Cushing’s- like horses was also assessed. The trends are very similar to those
previously stated.
Initially, a ConA dose curve was established using medium and ConA alone with
lymphocytes. As the concentration of ConA administered to the cells increased,
lymphocyte proliferation increased (P<.0001; Figure 6). The concurrent addition of
DEX decreased cellular proliferation at all concentrations of ConA in Non-Cushing’slike horses (P<.05; Figure 7).
19
Endogenous levels of cortisol in Non-Cushing’s-like horses is assumed to be at
physiologic, non-detrimental concentration, allowing for the observation that when a
synthetic form of cortisol (DEX) is added to the in vitro blood sample attained from
horse, cellular proliferation is decreased. Lymphocyte proliferation was decreased by
19.2% at basal concentrations. At concentrations of 0.3125 and 5 µg/mL, DEX reduced
proliferation by 39.9% and 15.3% respectively. The suppression of proliferation by
DEX in vitro is indicative of the suppression of proliferation by endogenous cortisol,
which can have a negative impact on immune function by decreasing the body’s
capability of producing lymphocytes to ward off infection.
RU486 was added to the DEX in Non-Cushing’s-like horses to see if it could counteract
the negative impact of DEX on cellular proliferation. RU486 when added to DEX was
able increase proliferation (P<.01; Figure 8). RU486 coupled with DEX had higher
stimulation indices than ConA. This indicated that RU486 was able to overcome the
suppressive action of DEX in Non-Cushing’s-like horses, and increase proliferation by
14.9%.
20
4
Stimulation Index
3
2
1
5.
00
00
2.
50
00
1.
25
00
0.
62
50
0.
31
25
0.
00
00
0
[ConA] ug/mL
Figure 6. Effect of ConA on lymphocyte proliferation in Non-Cushing’s-like horses.
21
4
ConA
Dex
Stimulation Index
3
2
1
5.
00
00
2.
50
00
1.
25
00
0.
62
50
0.
31
25
0.
00
00
0
[ConA] ug/mL
Figure 7. The effect of DEX on cellular proliferation in Non-Cushing’s-like horses.
22
4
ConA
Dex
Dex+RU486
Stimulation Index
3
2
1
5.
00
00
2.
50
00
1.
25
00
0.
62
50
0.
31
25
0.
00
00
0
[ConA] ug/mL
Figure 8. The antagonistic effect of RU486 on DEX in Non-Cushing’s-like horses.
23
Effect of In Vitro Treatments in Cushing’s-like Horses
Equine Metabolic Syndrome is a hormonal disorder caused by the prolonged exposure of
the body’s tissues to the glucocorticosteroid hormone cortisol. This situation may be the
consequence of adrenal gland over production of cortisol due to an imbalance of the
hypothalamic-pituitary-adrenal (HPA) axis. Horses that have Equine Metabolic
Syndrome or exhibit Equine Metabolic Syndrome- like symptoms generally have higher
concentrations of cortisol in their peripheral circulation. Treatments were applied to
lymphocytes obtained from these horses, and the effects were observed.
Cushing’s-like horses also displayed a similar ConA induced, dose-dependent increase
in proliferation (P<.0001; Figure 9).
The effect of exogenous DEX on proliferation of lymphocytes in horses with already
higher concentrations of circulating cortisol was assessed. DEX treatment applied in
vitro to Cushing’s-like cells decreased proliferation (P<.0001; Figure 10). At basal
concentrations, proliferation was decreased by 20.2%. At concentrations of 0.3125 and
5 µg/mL, proliferation was decreased by 21.7 and 38.0% respectively. As the
concentration of ConA increased, so did the suppressive action of DEX.
24
DEX and RU486 were added to cells cultured from Cushing’s-like horses. As
previously stated, Cushing’s-like horses already have higher concentrations of
endogenous cortisol. When additional synthetic cortisol (DEX) was added in vitro with
RU486, the RU486 was able to induce proliferation, despite the higher levels of
endogenous cortisol and exogenous DEX (P<.01; Figure 11). However, at basal
proliferation of 0 µg/mL of ConA, RU486 was not able to repress the suppressive action
of DEX. The medium consisting of DEX alone proliferated 6.5% more than that of
medium containing DEX and RU486. At higher concentrations of ConA, RU486 was
able to overcome DEX and increase stimulation. Specifically, at 2.5 and 5 µg/mL,
RU486 was able to increase proliferation by 24.4 and 19.2% respectively. In the
presence of ConA, RU486 is able to inhibit the action of DEX and elicit a proliferative
response, despite the already present higher concentrations of cortisol from the
Cushing’s-like horses.
25
4
Stimulation Index
3
2
1
5.
00
00
2.
50
00
1.
25
00
0.
62
50
0.
31
25
0.
00
00
0
[ConA] ug/mL
Figure 9. The effect of ConA on lymphocyte proliferation in Cushings-like horses.
26
4
ConA
Dex
Stimulation Index
3
2
1
[ConA] ug/mL
Figure 10. The effect of DEX on proliferation in Cushing’s-like horses.
5.
00
00
2.
50
00
1.
25
00
0.
62
50
0.
31
25
0.
00
00
0
27
4
ConA
Dex
Dex+RU486
Stimulation Index
3
2
1
5.
00
00
2.
50
00
1.
25
00
0.
62
50
0.
31
25
0.
00
00
0
[ConA] ug/mL
Figure 11. Antagonistic effect of RU486 on DEX, and agonistic effect on ConA in
Cushing’s-like horses.
28
Comparison of Lymphocyte Proliferation in Non-Cushing’s-like vs. Cushing’s-like
Horses
In the presence of ConA, lymphocyte proliferation from Cushing’s-like and NonCushing’s-like horses was compared. It was hypothesized that normal horses would
have higher levels of proliferation than Cushing’s like horses; however, Cushing’s-like
horses had higher stimulation indices than Non-Cushing’s-like horses (Figure 12). At a
concentration of 0 µg/mL, Cushing’s-like horses proliferated 8.3% more than NonCushing’s-like horses. At concentrations of 0.625 and 5 µg/mL, Cushing’s-like horses
had higher levels of proliferation (18.1 and 15.1% respectively) than non-Cushing’s-like
horses. While Cushing’s-like horses have higher levels of lymphocyte proliferation than
Non-Cushing’s-like horses, they may not be producing adequate amounts of antibodies
to be able to sufficiently ward of potential pathogens.
At lower concentration levels (0-to-0.625 µg/mL), Cushing’s-like horses had higher
proliferation levels in the presence of DEX, while at higher ConA concentration levels
(1.25-5 µg/mL), non-Cushing’s-like horses had higher proliferation levels than
Cushing’s-like horses (Figure 13). A noted feature of this study is the decrease in
lymhoproliferation at a ConA concentration of 0.3125 µg/mL. The additional amounts
of glucocorticosteroids added to the already present high levels of cortisol in the
Cushing’s-like horses accounts for the lower levels of proliferation at higher
concentrations of ConA. With higher concentrations of cortisol already present in the
29
4
Normal
Stimulation Index
3
Cushings
2
1
5
2.
5
1.
25
.3 0
12
5
.6
25
0
[ConA] ug/mL
Figure 12. A comparison of lymphocyte proliferation in the presence of ConA in
Cushing’s-like vs. Non-Cushing’s-like horses.
30
3
Normal
Stimulation Index
Cushings
2
1
5
2.
5
1.
25
.3 0
12
5
.6
25
0
[ConA] ug/mL
Figure 13. A comparison of equine lymphocyte proliferation, with the addition of DEX,
among Cushing’s-like and Non-Cushing’s-like horses.
31
peripheral blood system, the HPA axis is potentially deregulated. An abnormality in the
regulation of the HPA axis, like chronic activation of the axis due to stress, can lead to
an increase in glucocorticosteroid (cortisol) production. Cortisol is bound by
glucocorticoreceptors resulting in an interference with the regulation of cytokine
producing immune cells (NF-KB). Concentrations of cortisol achieved during periods of
stress are known to inhibit lymphocyte proliferation and suppress the secretion of certain
cytokines (Chrousos, 1995). The addition of the synthetic form of cortisol (DEX), to the
already high concentrations of endogenous cortisol, increased the available
glucocorticosteroid hormones to be bound to receptors on lymphocytes. This increase in
binding further reduces lymphocyte proliferation, resulting in the supplementary
reduction of cytokine secretion. The lymphocytes, although in the presence of increased
concentrations of a proliferative stimulant, were not able to increase proliferation due to
the competitive binding of glucocorticosteroids. Therefore the concentration of
glucocorticosteroids must have been greater than that of ConA, to prevent proliferation,
whereas the non-Cushing’s-like horses did not have excessive amounts of
glucocorticosteroids present, which accounts for their ability to proliferate more than the
Cushing’s-like horses.
Equivalent amounts of DEX and RU486 (1 µg/mL) were added to medium containing
equine lymphocytes and varying amounts of ConA. Lymphocyte proliferation of nonCushing’s-like horses was greater than that of Cushing’s-like horses (Figure 14). At a
basal concentration of Con A (0 µg/mL), lymphoproliferation was 36.6% greater in non
32
4
Normal
Stimulation Index
3
Cushings
2
1
5
2.
5
1.
25
.3 0
12
5
.6
25
0
[ConA] ug/mL
Figure 14. The effect of equivalent concentrations of DEX and RU486 on lymphocyte
proliferation of Cushing’s-like or non-Cushing’s-like.
33
Cushing’s-like horses. At concentrations of 0.3125 and 5 µg/mL, lymphoproliferation
was greater in non-Cushing-like horses by 24.5 and 18.9 % respectively. The absence of
a proliferative stimulant allows for only endogenous hormones to induce proliferation.
Higher concentrations of cortisol are present in Cushing’s-like horses, resulting in
suppression of lymphoproliferation. RU486 competes with DEX for binding sites on the
intracellular glucorticoid receptor (Garzo et al., 1988). RU486 was only able to
counteract the negative effects of DEX and cortisol by competitively binding with
already high concentrations of endogenous cortisol.
However, RU486 was able to
counteract the negative effects of glucocorticodteroids and increase stimulation above
that of DEX with ConA and lymphocytes.
The effect of RU486 on lymphoproliferation was assessed in Non-Cushing’s-like and
Cushing’s-like horses. At all concentrations of ConA, Cushing’s-like horses had higher
stimulation indices than non-Cushing’s-like horses (Figure 15). Basal proliferation was
very similar for both, with Cushing’s-like horses having higher proliferation levels of
7.5%. At concentrations of 0.625 and and 5 µg/mL, Cushing’s-like horses had higher
34
proliferation counts by 23.6 and 7.5% respectively. Interestingly, RU486 may have an
agonistic role in the presence of ConA. Not only does this data support this idea, the
results are not unprecedented. While RU486 is antagonistic to glucocortisoids and
progesterone, it had been shown that it is agonistic in the presence of proliferative
stimulants (Beck et al., 1993). To overcome the detrimental effects of DEX, RU486
binds with high affinity to glucocorticoid receptors, preventing DEX from binding,
stabilizing the complex (Nordeen et al., 1993). Much like this study, other studies have
also shown the dual nature of RU486; its antagonistic nature in the presence of
glucocorticoids and progesterone, binding their receptors, preventing the suppression of
lymphocytes, as well as its ability to enhance the action of agonists (Nordeen et al.,
1993).
35
5
Normal
Stimulation Index
4
Cushings
3
2
1
[ConA] ug/mL
Figure 15. The effect of RU486 on equine lymphocyte proliferation.
5
2.
5
1.
25
.3 0
12
5
.6
25
0
36
CHAPTER IV
CONCLUSIONS
Cortisol is a glucocorticosteroid that is triggered by stress and is produced from the
adrenal cortex, resulting in increased blood pressure and suppressed immune system
function. The depression of the immune system disables the body from properly
warding off infection, greatly compromising the horse’s health. Understanding how the
immune system is compromised is imperative in order to make advances to overcome
this problem. It has been shown that endogenous cortisol and its synthetic counterpart,
DEX, suppress the immune system, decreasing the production of lymphocytes, therefore
decreasing the amount of antibody produced, resulting in depression of immune
function. This study has illustrated the ability of RU486 to overcome the detrimental
effects of DEX. By preventing DEX from binding glucocorticoid receptors, RU486
stabilizes the complex, preventing the suppression of lymphocytes, and enhancing the
action of agonists. In the presence of RU486, the capability of lymphocytes exposed to
higher levels of endogenous cortisol to proliferate more than lymphocytes out of horses
that were not exposed to high concentrations of endogenous cortisol presents an
interesting area for further research. A possible explanation for this may be that while
horses with high concentrations of endogenous cortisol have higher lymphoproliferation
levels, the lymphocytes may not be producing enough antibodies in order to properly
defend the immune system. The enhanced simulative effects of RU486 give rise to
interesting areas that allow for further research.
37
LITERATURE CITED
Bauer, M. E. 2005. Stress, glucocorticoids and ageing of the immune system. Stress
8:69-83.
Beck C., N. L. Weigel, M. Moyer, S. Nordeen, and D. Edwards. 1993. The progesterone
antagonist ru486 acquires agonist activity upon stimulation of camp signaling
pathways. Proc. Natl. Acad. Sci. USA 90: 4441-4445.
Chaplin, D. D. 2006. Overview of the human immune response. J. Allergy Clin.
Immunol. 117:S430-S435.
Chrousos, G. P. 1995. The hypothalamic-pituitary-adrenal axis and immune-mediated
inflammation. N. Engl. J. Med. 332:1351–1362.
Field, J. R., and L. B. Jeffcott. 1989. Equine laminitis--another hypothesis for
pathogenesis. Med. Hypothesis 30:203-210.
Garzo, V. G., J. Liu, A. Ulmann, E. Baulieu, and S. S. Yen. 1988. Effects of an
antiprogesterone (ru486) on the hypothalamic-hypophyseal- ovarian-endometrial
axis during the luteal phase of the menstrual cycle. J. Clin. Endocrinol. Metab.
66: 508-517.
Gaylean, M. L., L. J. Perino, and G. C. Duff. 1999. Interaction of cattle health/immunity
and nutrition. J. Anim. Sci. 77:1120-1134.
Gerrard, T. L., T. R. Cupps, R. J. M. Falkoff, G. Whalen, and A. S. Fauci. 1985. Effects
of in vitro corticosteroids on B- cell activation, proliferation and differentiation.
J. Clin. Invest. 75:754-761.
Johnson, P. J. 2002. The equine metabolic syndrome peripheral cushing's syndrome.
Vet. Clin. Equine 18:271-293.
Madden, K. S. 2003. Catecholamines, sympathetic innervation, and immunity. Brain,
Behav., and Immunity. 17:S5-S10.
Nordeen S. K., B. J. Bona, and M. L. Moyer. 1993. Latent agonist activity of the steroid
antagonist, ru486, is unmasked in cells treated with activators of protein kinase a.
Mol. Endo. 7: 731-742.
Padgett, D. A., and R. Glaser. 2003. How stress influences immune response. Trends in
Immunology 24:444-448.
38
Sapolsky, R. M., L. C. Krey, and A. U. Munck. 2000. How do glucocorticoids influence
stress responses? Integrating permissive, supressive, stimulatory, and
preparative actions. Endocrine Reviews 21: 55-89.
39
APPENDIX A
PROLIFERATION MEDIA PROTOCOL
1. Media contained DME/F12 + 10% Heat Inactivated Fetal Bovine Serum + 1X PennStrep + 1 X L-Glutamine + 10 µM BME.
a. For a 50 mL mix
i. 44 mL of DME/F12
ii. 5 mL of FBS
iii. 0.5 mL P/S
iv. 0.5 mL L-Glut
v. 3.52 µL BME
40
APPENDIX B
DOSING STANDARDS PROTOCOL
2. Label 4 sets of 8- 15 mL conical tubes (orange tops).
a. ConA H  A/ [high] [low]
b. ConA + Dex H  A/ [high] [low]
c. ConA + RU486 H  A/ [high] [low]
d. ConA + Dex + RU486 H  A/ [high] [low]
3. Thaw FBS, P/S, and L-Glutamine in a water bath at 37oC. Vortex.
4. Make proliferation media.
5. Dose standards (ConA). Do this for all 4 sets of treatments.
41
Dose (Tube
Volume of Media
Volume of ConA
Lable)
Concentration in
Final Concentration
media
in well
H
1.96 mL
40 µL
20 µg/mL
10 µg/mL
G
1 mL
1 mL from
10 µg/mL
5 µg/mL
5 µg/mL
2.5 µg/mL
2.5 µg/mL
1.25 µg/mL
1.25 µg/mL
0.625 µg/mL
0.625 µg/mL
0.3125 µg/mL
0.3125 µg/mL
0.15625 µg/mL
0 µg/mL
0 µg/mL
tube H
F
1 mL
1 mL from
tube G
E
1 mL
1 mL from
tube F
D
1 mL
1 mL from
tube E
C
1 mL
1 mL from
tube D
B
1 mL
1 mL from
tube C
A
1 mL
0 mL
6. Make Dex and RU486 doses.
a. Romve 4 µL of ConA/media solution from all 8 “Dex” labeled tubes. Add 4
µL of Dex to each tube.
b. Romve 4 µL of ConA/media solution from all 8 “RU486” labeled tubes. Add
4 µL of RU486 to each tube.
42
c.
Romve 8 µL of ConA/media solution from all 8 “Dex + RU486” labeled
tubes. Add 4 µL of Dex to each tube and 4 µL of RU486
7. Plate media 50 µL per well in 3 replicates ([0 µg]A  [20 µg]H).
a. Label 2 plates.
i. EDTA Proliferation
ii. EDTA IgM
Plate Layout
1
2
3
4
5
6
7
8
9
10
11
12
A[0µg/mL]
ConA +
Dex
B[0.15625µg/mL]
C[0.3125µg/mL]
ConA
D[0.625µg/mL]
ConA +
RU486
E[1.25µg/mL]
F[2.5µg/mL]
G[5µg/mL]
H[10µg/mL]
8. Store plates in incubator until ready to use.
ConA +
Dex +
RU486
43
APPENDIX C
ISOLATION OF PBMC’S AND PROLIFERATION PROTOCOL
9. Collect equine blood in six 10 ml plasma EDTA vaccutainers (purple top). Place on
ice.
10. Add 5 ml of Ficoll Paque to seven sterile 15 ml conical tubes. Layer about 7 ml of
blood very slowly on top of the Ficoll Paque. Do not allow the blood and the Ficoll
to mix.
11. Centrifuge at 1000xg for thirty minutes.
12. Using a sterile 10 ml pipette, very carefully, remove the buffy coat layer (try not to
aspirate off any of the red blood cell layer) and transfer and transfer to a clean 50 ml
conical tube. Add 1X HBSS to the 40 ml mark on the 50 ml conical. Centrifuge at
200 xg for 10 minutes.
13. Aspirate supernatant (leaving the white blood cell pellet). Add 2 ml of 0.2% NaCl,
triturate to brak up the cell pellet. Lyse cells for no more than 1 minute. Add 2 ml
of 1.6% NaCl. Add 1X HBSS to the 40 ml mark on the 50 ml conical. Centrifuge at
200 xg for 10 minutes.
14. If you still have red blood cell contamination, repeat step 5. If not:
15. Aspirate supernatant and re-suspend cell pellet in 1 ml of media.
16. Remove 10 µL of cells and count in 90 µL of trypan blue using a hemacytometer.
17. Dilute cells to desired concentration in 50 µL and transfer to a 96-well plate using a
multichannel pipette. Final volume in all wells will be 100 µL (including doses).
44
18. Incubate in a 37o C humidified incubator with 5% CO2 for 92 hours.
19. Add 15 µL of Dye solution (Promega Cell Titer 96 Non-Radioactive Cell
Proliferation Assay) to all wells of the plate designated for proliferation. Return to
the incubator for 4 hours.
20. Add 100 µL of Solubilization/Stop solution to all wells. Incubate at room
temperature overnight. Read absorbance at 570 nm with a reference wavelength of
630-750 nm.
21. Remove IgM plate from incubator at 96 hours, seal and store at -80oC.
45
CONTACT INFORMATION
Name:
Kaitlin Alyssa Gutierrez
Professional Address:
c/o Dr. Thomas H. Welsh, Jr.
Department of Animal Science
MS 2471
Texas A&M University
College Station, TX 77843-2471
Email Address:
katieg@tamu.edu
Education:
B.A. Animal Science, Texas A&M University, May 2009
Undergraduate Research Scholar