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3
*
Graduate Program in Department of Human Nutrition, Foods and Exercise and
Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic University, Blacksburg, VA 24061
3To whom correspondence should be addressed at VMRCVM, Department LACS, Phase II, Duckpond Dr., Blacksburg, VA 24061.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • CD4 • CD8 • starvation • lymphocytes • calcium • cats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The incidence of sepsis,
prolonged ventilation and increased mortality rates has been associated
with malnutrition (Reinhardt et al. 1980
). The primary
goal of nutritional status assessment is to identify nutrient
deficiencies. In doing so, steps can be taken to improve the overall
nutritional status, which would enhance the capability of the body to
fight infection or illness. Nutritional assessment can be defined as a
systematic method of gathering data, classifying the degree of
malnutrition and instituting the appropriate treatment and intervention
techniques (Gilbride et al. 1984
).
Current methods of nutritional status assessment include
anthropometric, biochemical, dietary and clinical evaluations. No
definitive test of nutritional status exists due to the complexity of
the human diet and the multiple effects that nutrients have on various
tissues, organs and physiological functions (Manning and Shenkin 1995
). In addition, non-nutritional aspects of the response
to illness, such as fever, infections and drugs, may affect assessment
tests (Blackburn and Thornton 1979
). Tests of
immunological functions can be indicative of nutritional status,
because many are sensitive to overall nutritional status as opposed to
deficiencies of individual nutrients (Puri and Chandra 1985
). This is appropriate because it is rare for a patient to
be deficient in only one nutrient. The purpose of the present study was
to identify immune system indicators associated with acute nutrient
deprivation in a feline model. Specifically, the identification and
quantification of cluster of differentiation
CD44
and CD8 markers in
T-lymphocytes, lymphocyte proliferation and intracellular calcium
concentration were examined in cats before, during and after a 7-d
period of acute nutrient deprivation. A feline model for acute
starvation was chosen because cats are less tolerant of energy and
protein deficiencies due to their general inability to regulate enzymes
and to use alternative metabolic pathways and substrates. They also
better represent the human anatomy than other available animal models
due to the uniformity of their adult body size (Walker 1982
). This type of animal model allows for more control with
regard to dietary intervention as well as environmental and physical
variables that may affect the immune system.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Twenty-three (4–7 y old) healthy, neutered adult domestic shorthaired
cats weighing 3–6 kg were used for this study. These cats were part of
an established research colony at the Virginia-Maryland Regional
College of Veterinary Medicine (VMRCVM). The study protocol was
approved by the Virginia Tech Animal Care Committee. Cats were randomly
chosen in groups of eight, eight and seven to undergo the food
deprivation and refeeding study, because smaller groups of cats were
easier to monitor during the 14-d period. Each cat acted as its own
control, and each group was separately housed in a climate- and
environmentally controlled room equipped with individual steel cages
(61 cm x 61 cm) during the study period. During the 14-d period,
food was withheld on days 0–7 and then subsequently fed (Table 1
) to meet daily energy requirements on days 7–14. Fresh water was
available at all times throughout the study period, and daily water
intake was monitored.
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30 mL of lactated Ringer’s solution after each sampling
period. General attitude, health and body condition were assessed each
morning and evening by monitoring temperature, respiration, pulse,
urination, defecation, capillary refill time and alertness. Hydration
status was determined daily by checking the dryness of the mucous
membranes and skin elasticity. Body weight was measured and recorded at
the same time of day on days 0, 4, 7, 11 and 14. In addition, total
protein, albumin, blood urea nitrogen, alkaline phosphatase, hemoglobin
and hematocrit were determined (Olympus AV400 Analyzer, Dallas, TX) on
days 0, 4, 7, 11 and 14 to further assess the health status of the
cats. Peripheral blood differential counts were performed in the
clinical pathology laboratory at VMRCVM on the same days (0, 4, 7, 11
and 14). Cell isolation.
To maintain sterile conditions for lymphocyte (cell culture)
proliferation, cell separation was performed under a laminar flow hood.
Peripheral blood mononuclear cells (PBMC), consisting of lymphocytes
and monocytes, and polymorphonuclear cells (PMNC) were isolated
according to a double density Ficoll separation method (Toth et al. 1992
) with centrifugation. Briefly, 3 mL of Histopaque
1.119 (Sigma Chemical, St. Louis, MO) was added to a 15-mL conical tube
with a 20-gauge needle. With a separate 20-gauge needle, 3 mL of
Histopaque 1.077 (Sigma Chemical) was layered over the 1.119 layer.
Next, 6 mL of whole blood was layered over the 1.077 layer with an
automatic pipette. The conical tube was centrifuged at 700 x g for 20 min with no brake at room temperature. After
centrifugation, separation of the cells led to two opaque interfaces
consisting of PBMC and PMNC. The top layer (PBMC) of each sample was
aspirated with a Pasteur pipette and transferred to a 50-mL conical
centrifuge tube for washing.
Isolated cells were washed twice with Hanks’ Balanced Salt Solution (HBSS; Gibco Laboratories, Burlington, Ontario, Canada) and centrifuged at 200 x g for 10 min. Cells were resuspended in 10 mL of complete medium [RPMI 1640, fetal bovine serum (FBS), L-glutamine, sodium pyruvate and penicillin-streptomycin; Sigma Chemical] and centrifuged at 400 x g for 20 min with the brake on to remove platelets. Cells were again suspended in 2–4 mL of HBSS for cell counting with a hemocytometer and adjusted to 1 x 109 cells/L with HBSS for time 0 [before stimulation with concanavalin-A (Con-A)] CD4/CD8 quantification, to 3 x 109 cells/L for proliferation assay and to 2 x 109 cells/L for time 72 h (after stimulation with Con-A) CD4/CD8 quantification.
Immunophenotyping.
T-lymphocytes were identified through the detection of their surface
markers with flow cytometric analysis according to the method described
by Ackley et al. (1990
). Briefly, 25 µL of a 1:25
dilution of primary mouse anti-cat unlabeled purified CD4 or CD8
monoclonal antibody (Southern Biotechnology, Birmingham, AL) was added
to 500 µL of adjusted cells (1 x 109 cells/L). Each
tube was incubated for 30 min at 4°C and centrifuged for 10 min at
room temperature at 200 x g. The supernatants were
discarded, and the pellets were resuspended in 500 µL of HBSS. Two
microliters of fluorescein isothiocyanate (FITC)-conjugated rabbit
anti-mouse IgG (H + L) secondary antibody (Southern Biotechnology,
Inc., Birmingham, AL) was added to each tube and incubated for 30 min
at 4°C. Samples were centrifuged at room temperature at 200 x g for 10 min. Cell pellets were resuspended in 500 µL
of paraformaldehyde (2.0%) and analyzed on the flow cytometer.
CD4+ and CD8+ expressions of PBMC were
calculated by subtracting the value of the negative control (FITC)
sample from the value obtained from the sample that had been incubated
with both primary (CD4 or CD8) and secondary (FITC) antibodies.
Lymphocyte proliferation assays.
One hundred microliters of mononuclear cells (3.0 x 109 cells/L) in complete medium was cultured in triplicate in two sterile 96-well round-bottom plates (Corning Glassworks, Corning, NY). One hundred microliters of either complete medium alone or Con-A (a stimulus for lymphocyte proliferation) (5 mg/L media; Sigma Chemical) was added. The plates were incubated in a humidified incubator at 37°C at 5% CO2 for 72 h.
In one plate, lymphocyte proliferation was determined according to the
Alamar Blue staining methods of Gogal et al. (1997)
.
After 48 h, 20 µL of Alamar Blue (Accumed International,
Westlake, OH) was added to all wells in the plate and returned to the
incubator. The proliferation of cultures was determined 24 h later
by measuring absorbance at 570 and 600 nm in a microplate reader
(Molecular Devices, Menlo Park, CA). When Alamar Blue is added to cell
cultures, it is in an oxidized (blue color) form, and as the cells
proliferate, the dye is reduced (red color). The absorbance at 570 and
600 nm determines the optical density (OD) of reduced and oxidized
forms of Alamar Blue. The subtraction of OD at 600 nm from OD at 570 nm
accurately determines the true absorbance (specific absorbance), which
reflects proliferation. The specific absorbance of unstimulated cells
(medium alone) was subtracted from the specific absorbance of the cells
incubated with Con-A to yield a 
-specific absorbance. The mean
-specific absorbance of triplicate determinations was calculated.
To determine CD4+/CD8+ quantification of
lymphocytes at 72 h, cells from the second plate were aspirated
and resuspended in 500 µL of HBSS. T-lymphocyte subsets were
quantified according to the flow cytometric methods of Ackley et al. (1990)
. Data for CD4/8 subset immunophenotyping are
reported as prestimulation (0 h) and poststimulation (72 h) with Con-A.
Calcium flux.
Two fluorescent probes, Fluo-3 and SNARF-1 (Molecular Probes, Eugene,
OR), were dissolved in dimethylsulfoxide (Sigma Chemical) at
concentrations of 1 and 2 mmol/L for stock solutions. A 1:10 dilution
of Fluo-3 and SNARF-1 stocks in RPMI 1640 and 1% FBS (Sigma Chemical)
was made and used as working solutions to load the cells for flow
cytometric analysis. PBMC were resuspended to a concentration of 1.0
x 109 cells/L in RPMI 1640 and 1% FBS. Fluo-3 and
SNARF-1 were loaded into the cells according to the procedure
previously described by Rijkers et al. (1990)
with modifications. PBMC were incubated with 10 µL of each Fluo-3 and
SNARF-1 working solution and incubated for 30 min in a 37°C water
bath. Cells were washed twice in RPMI plus 1% FBS, centrifuged at 200
x g for 10 min and resuspended in calcium
(Ca2+)-free HBSS at a final concentration of 1 x 109 cells/L. Cells were analyzed on a flow cytometer by
measuring the Fluo-3/SNARF-1 ratios over time. Data were collected for
a total of 120 s. Intracellular Ca2+ flux was
expressed as a mean ratio (Fluo-3 ratio of activated versus resting
population).
Statistical analysis of data was carried out with a multiple dependent
t test to determine significant differences between
baseline values and each subsequent measure, and analysis between d 7
and each subsequent measure was carried out with SAS Statistical Software (1996)
. Differences with values of
P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cats tolerated the starvation period without incident. Measures of
health, including total protein, alkaline phosphatase and blood urea
nitrogen, remained within normal limits in all cats as reported by
workers at the Clinical Pathology Laboratory. All cats lost weight
(P < 0.0001) throughout the food deprivation period
and gained weight (P < 0.0001) during the refeeding
period (Table 2
). Compared with day 0 values, albumin levels (P < 0.05) increased on day 4 of the food deprivation treatment and
decreased (P < 0.05) on days 11 and 14 compared with
day 7.
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Blood leukocyte numbers decreased (P < 0.05) on day 4
compared with day 0 (Table 2)
. There were no significant changes in
blood leukocyte numbers during the refeeding period. The total
lymphocyte number decreased (P < 0.05) during the
starvation period and increased (P < 0.05) at days 11
and 14 of the refeeding period compared with day 7. The percentage of
lymphocytes decreased (P < 0.01) throughout the latter
part (day 7) of the food deprivation period and tended to increase
(P = 0.09) throughout the refeeding period.
Changes in lymphocyte cell membrane surface markers.
The percentage of CD4+ lymphocytes decreased
(P < 0.05) on day 4 of the food deprivation period
compared with day 0 (Fig. 1
). An increase (P < 0.01) in the proportion of
CD4+ cells was observed on day 11 compared with
day 7. There was no significant change in percentage of
CD4+ cells on day 14. An increase in the
percentage of CD8+ cells was observed on day 7
(P < 0.05) compared with day 0 and on day 11 compared
with day 7.
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).
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The proliferative capacity of lymphocytes in culture tended to decrease
(P = 0.07) during the 7-d food deprivation period and
subsequently increased (P = 0.01) during refeeding. The
-absorbance values as measured for days 0, 4, 7, 11 and 14 were
0.328 ± 0.06, 0.291 ± 0.06, 0.235 ± 0.04, 0.307
± 0.04 and 0.300 ± 0.05, respectively.
A nonsignificant decrease (P = 0.10) in the
percentage of cells expressing CD4+ marker after
stimulation with Con-A was observed throughout the starvation
period. The percentage of CD4+ cells was greater
(P < 0.01) on day 14 than on day 7 (Fig. 3
). The percentage of cells expressing CD8+ cell
markers after proliferation was greater on day 4 (P < 0.01) than on day 0. The percentage of CD8+ cell
expression tended to decrease (P = 0.10) for the
remainder of the food deprivation period and into the early refeeding
period. By the end of the refeeding period (day 14),
CD8+ cell expression was greater than on day 0
(P < 0.01).
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Intracellular Ca2+ changes.
The overall concentration of intracellular Ca2+ ([Ca2+]i) increased during the food deprivation period (mean differences 1.76 ± 2.13 and 9.27 ± 3.02 for days 4 and 7, respectively), with a significant increase (P < 0.01) observed on day 7 compared with day 0. The activated/resting ratio was 14.6 versus 22.3 for days 0 and 7, respectively. A further increase in [Ca2+]i compared with day 7 was observed on day 11 (P = 0.06); then [Ca2+]i tended to decrease (P = 0.14) by day 14.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, a decrease in percent lymphocytes, as determined by
differential counts, occurred throughout the food deprivation treatment
and tended to increase during refeeding. Our findings are consistent
with those of other studies (Dhabher et al. 1995
,
Komaki et al. 1997
) that also examined starvation and
refeeding treatments in animals. Similar findings were also reported in
humans who consumed a very low energy diet for 6 weeks followed by a 1-
to 2-wk refeeding period (Field et al. 1991
). The
effects that a decrease in total lymphocytes has on immunodeficiency
are still unknown at this time.
Depressed CD4/CD8 ratios are associated with decreased immunity. In
humans, a CD4/CD8 ratio of <1.5 has been associated with
immunosuppression (Chailleux et al. 1985
). In our study,
the CD4/CD8 ratio (1.0) during acute starvation was below normal
feline ratios (1.2–2.6) (Dean et al. 1991
,
Komaki et al. 1997
). The change in the CD4/CD8 ratio in
our study was attributed to an increase in the percentage of
CD8+ cells, as well as a decrease in the
percentage of CD4+ cells. The increase in the
CD8+ cell population in our study was consistent
with other findings (Nuevonin and Salo 1984
,
Ogawa et al.1993
). The route of circulation may in part
explain an increase in CD8+ cells.
CD8+ cells migrate from the thymus to the spleen,
where nonspecific CD8+ cells proliferate
(Garre et al. 1986
). Recirculating cells are limited in
number and are short lived. This along with the facts that acute
starvation acts initially on the proliferation of
CD8+ cells in the thymus (Malave et al. 1980
) and that CD8+ cells further
proliferate and differentiate in the spleen may explain why the
proportion of CD8+ cells may remain normal
(Chandra 1983
) or become elevated in circulating blood.
A decrease in percent CD4+ cells was seen by day
4, followed by an increase on day 11. The CD4+
cell population in the present study decreased significantly
(P < 0.05) during the starvation period, which
parallels other investigations (Barlough et al. 1991
,
Komaki et al. 1997
). The development and route of
circulation of CD4+ cells may influence their
susceptibility to change. When CD4+ cells migrate
from the thymus to the lymph nodes, they are more mature than
CD8+ cells and therefore proliferate less than
CD8+ cells. In contrast to
CD8+ cells, CD4+ cells
recirculate and are long lived. The secondary effects of malnutrition
affect thymus-derived cells (Garre et al. 1986
).
During the adaptive phase of undernutrition (24–48 h), differentiation
of cells is impaired. This may explain why the proportion of
CD4+ cells initially decreased in the circulating
blood in our study. As the nutrient deprivation period continued, the
proportion of CD4+ cells was unchanged, whereas
the proportion of CD8+ cells increased (Fig. 1)
.
In humans, many cell types produce interleukin-1 (IL-1) in response to
damage, infection or antigens. IL-1 positively influences
CD4+ cell proliferation, IL-2 receptor expression
and cytokine production (Roitt et al. 1996
). In our
study, food deprivation for >4 d may have stimulated IL-1 production
in the cats, which in turn stimulated CD4+
proliferation and helped to prevent a further decline in the percentage
of CD4+ cells, as observed at day 7. Stimulated
CD4+ cells secrete cytokines, which are involved
in cell-mediated immunity responses. IL-10 does not appear with the
initial stimulation of CD4+ cells but does after
repeated in vitro stimulation, and this may allow for the
down-regulation of CD4+ production
(Palmer and van Seventer 1997
). The secretion of IL-10
by CD4+ cells may have prevented a continued
increase in percent CD4+ cells from day 11 to day
14 of the refeeding period (Fig. 1)
. An evaluation of IL concentrations
such as IL-1 and IL-10, as well as IL-2 receptor expression, may aid in
the explanation of the alterations in CD4+
expression. Unfortunately, at the time of this study, the technology to
examine these cytokines in cats was unavailable.
In retrospect, the measurement of corticosterone would have contributed
greatly to this study, because it is reported that malnutrition is
associated with increased secretion of corticosterone, a major
corticosteroid produced in mammals (Stinet 1983
,
Winick and Nobel 1966
). Starvation-induced stress
has been shown to increase corticosteroid production in humans, and an
inverse correlation between serum cortisol (a corticosteroid) and the
proportion of CD4+ cell subsets during fasting
has been reported in humans (Komaki et al. 1997
).
Lymphocytes express receptors for corticosteroid, and therefore
cortisol affects circulating lymphocytes in humans (Fauci et al. 1976
). Serum cortisol can therefore directly influence
lymphocyte and lymphocyte proliferation. An increase in serum cortisol
during food deprivation could be responsible for substantial
alterations in immunosuppression (Komaki et al. 1997
).
Initial decreases in CD4+ cells in our study may
have resulted from increased secretion of cortisol by cats.
Corticosteroids act as a major feedback mechanism on immune responses.
An examination of corticosteroid concentration may provide additional
information for the determination of changes in lymphocyte subset
populations.
Lymphocytes can respond to corticotropin-releasing factor to
generate their own adrenocorticotropic hormone. Lymphocyte-secreted
adrenocorticotropic hormone in turn induces corticosteroid release
(Roitt et al. 1996
) and may cause inhibition of
lymphocyte proliferation. Increased corticosteroid secretion had a
lympholytic effect in response to 72-h starvation in mice (Wing et al. 1987
). We also observed a decrease in total lymphocytes
in response to starvation. Although we did not measure corticosterone
secretion, this is a possible mechanism in the cat for alterations in
immune responses. Corticosteroids may explain the reductions in
lymphocyte proliferation that occurred during the 7-d starvation
period.
Con-A is a plant lectin used to study lymphocytes as both
CD4+ and CD8+ T-cell
populations respond to this mitogen (Reinherz and Schlossman 1980
). The in vitro mitogen-response assay detects defects
in some aspects of mononuclear and lymphocyte cell function
(Field et al. 1991
). In our study, lymphocyte
proliferation decreased throughout the food deprivation period and
increased during refeeding. Lymphocyte proliferative capacity appeared
consistent with the observed in vitro changes in the numbers of
CD4+ cells during food deprivation and refeeding.
Dowd et al. (1986)
implied that T-cell subsets
stimulated by Con-A are differentially affected in a variety of
clinical conditions, such as altered nutritional states. Our study
supports these findings because we observed an increase in the
percentage of CD8+ cells compared with day 0,
whereas the percentage of CD4+ cells decreased.
Con-A–stimulated T-cell subsets may be differentially affected
during acute nutritional deprivation.
Altered nutritional states, such as food deprivation, decrease the
availability of nutrients needed for cell proliferation. A decrease in
mitogen-induced lymphocyte proliferation was observed in rats fed a
protein-deficient diet (Mengheri et al. 1992
) and in
hospitalized humans with low serum zinc and transferrin levels
(Dowd et al. 1986
). A decrease in mitogenic response
suggests a decrease in the rate of DNA synthesis. Mechanisms for this
decrease in mitogen response are unclear (Field et al. 1991
); however; low serum zinc levels have been shown to
depress lymphocyte proliferation, suggesting that it is
required for optimal lymphocyte transformation (Dowd et al. 1986
, Messer et al. 1982
).
A decrease in dietary zinc associated with the food deprivation period
may have been in part responsible for the decrease in lymphocyte
proliferation observed in our study cats. Zinc has also been shown to
regulate transcription through its interaction with metallothionein as
well as to stimulate the production of interferon-
and IL-2, which
are required for lymphocyte proliferation and differentiation
(Hannigan 1994
). In rats fed a zinc-deficient diet,
T-lymphocyte proliferative capacity was decreased (James et al. 1987
). Similar results occurred in our food deprivation
study. Decreased cellular zinc concentrations resulting in altered
metallothionein, interferon- and IL-2 concentrations may explain the
results in the present study.
Altered lymphocyte proliferation observed in our study may also have
been associated with increases in the CD8+
regulator cell population. Neuvonen and Salo (1984)
reported that during acute starvation, increased subsets of regulator
cells contributed to a decrease in lymphocyte proliferative response.
The depression in lymphocyte proliferation observed during the food
deprivation period in our study may also have been due to a reduction
in total circulating T cells, which has been reported previously
(Chandra 1977
, Salimonu et al. 1982
).
Although the mechanism is not clear, the decrease in lymphocyte
proliferation exhibited by our study cats may be linked to T-cell
number, altered expression of
CD4+/CD8+ subsets, altered
cellular zinc concentrations or a combination.
Intracellular Ca2+ is determined by stimulating
the lymphocytes with ionomycin to release intracellular
Ca2+ stores. The amount of intracellular
Ca2+ is indicative of the activation state of the
cell. One of the early responses of T-cell receptor stimulation is
a rise in [Ca2+]i
(Verhuegen et al. 1997
). In the present study,
[Ca2+]i increased through
day 11 and decreased on day 14. We interpret the changes in
[Ca2+]i stores as
reflecting alterations in expression of the CD8+
subset population during starvation and refeeding. The literature
indicates a relationship among
[Ca2+]i, protein kinase C
and IL-2 with regard to cell activation and proliferation
(Nishizuka 1984
, Roitt et al. 1996
,
Verheugen et al. 1997
); suggesting that changes in
[Ca2+]i should correlate
closely with cell function and nutrient status. However, data derived
from cats in our study did not suggest such a relationship in that
[Ca2+]i changes did not
mirror lymphocyte proliferative activity. Additional studies on this
topic may help to better determine the value of
[Ca2+]i changes as an
indicator of nutrient status.
In conclusion, our data show that immunosuppression can result in cats
that have undergone 7 d of acute starvation. Immunosuppression
began to be alleviated with refeeding. The decreases in lymphocyte
number, proliferation and CD4/CD8 ratio are all indicative of
immunosuppression; the use of these variables as measures of
nutritional status is hopeful for both human and veterinary patients.
The incidence of sepsis (Chandra 1983
), prolonged
ventilation and increased mortality rates has been associated with
malnutrition in the critically ill (Reinhardt et al. 1980
). Patients who are immunosuppressed as a result of
malnutrition must be identified so nutritional intervention can be
implemented to enhance the capability of the body to fight infection
and disease. The many complex interactions between starvation and
immunity offer a great challenge to researchers. A greater
understanding of this area may help decrease patient mortality rates.
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FOOTNOTES |
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2 Food and Nutrition Board, Institute of Medicine, 2101 Constitution Avenue, Washington, D.C. 20418.
4 Abbreviations used: CD, cluster of differentiation; [Ca2+]i, intracellular calcium concentration; Con-A, concanavalin-A; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HBSS, Hanks’ balanced salt solution; IL, interleukin; OD, optical density; PBMC, peripheral blood mononuclear cells; PMNC, polymorphonuclear cells; VMRCVM, Virginia-Maryland Regional College of Veterinary Medicine.
Manuscript received January 18, 2000. Initial review completed March 3, 2000. Revision accepted June 26, 2000.
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