Vol. 274, Issue 4, R912-R920, April 1998
IGF-I alters lymphocyte survival and regeneration in thymus
and spleen after dexamethasone treatment
Pamela S.
Hinton,
Catherine A.
Peterson,
Elizabeth M.
Dahly, and
Denise M.
Ney
Department of Nutritional Sciences, University of Wisconsin-Madison,
Madison, Wisconsin 53706
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ABSTRACT |
Insulin-like growth factor I (IGF-I) is a
growth factor for the immune system, increasing lymphocyte number and
function via greater lymphocyte generation and/or survival. We
investigated the effects of IGF-I on lymphocyte survival and
regeneration in the thymus and spleen after dexamethasone (Dex)
treatment in rats maintained with parenteral nutrition and given
recombinant human IGF-I (800 µg/day) for 12 h, 48 h, and 5 days.
IGF-I did not prevent Dex-induced apoptosis of thymocytes but reduced
cell death in the spleen at 12 and 48 h. IGF-I exerted a modest
protective effect (10-15% reduction in cell loss) on all splenic
T and B cell subsets examined by flow cytometry. IGF-I enhanced
recovery of CD4+8+ immature T cells in the thymus and decreased the
proportion of CD8+ (cytotoxic/suppressor) T cells in the spleen. In
rats not treated with Dex, IGF-I significantly increased total
lymphocyte number and the number of CD4+8+ T cells in thymus and
spleen. Our results suggest that IGF-I may alter homeostasis in the
immune system by modulating lymphocyte generation and survival.
apoptosis; lymphopoiesis; insulin-like growth factor I; growth
factor; dexamethasone
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INTRODUCTION |
APOPTOSIS IS AN ESSENTIAL regulatory process that
occurs in the immune system, playing a fundamental role in recognition
of self/nonself. This "programmed" cell death allows for control of immune response and generation of immunological memory, as well as
central and peripheral tolerance. Apoptosis occurs in primary lymphoid
organs to eliminate T and B cells that have undergone nonfunctional
receptor rearrangements. In the thymus, deletion of self-reactive T
cells is essential to prevent the generation of autoreactive clones.
Autoimmunity is also regulated by apoptotic deletion of antiself T and
B cells in peripheral lymphoid organs such as lymph nodes and spleen.
In the thymus, immature T cells (CD4+8+) undergo negative selection
after activation via the T cell receptor-CD3 complex, whereas mature T
cells (CD4+ or CD8+) proliferate with activation. Endogenous
glucocorticoids may modulate the selection process, because
physiological levels of these hormones are sufficient to induce death
of immature CD4+CD8+ cortical thymocytes (7). In addition, both
activation- and glucocorticoid-induced cell death are operative in
peripheral lymphocytes; treatment with anti-CD3 antibody (12) or
dexamethasone (Dex) (20) results in apoptotic death of spleen cells.
The fate of a cell after activation is determined by cytokines (10,
21), cell-surface accessory molecules (26), and gene products that may
either induce or block apoptosis (5).
Insulin-like growth factor I (IGF-I) is being investigated as a
potential therapeutic agent for the treatment of growth
hormone-resistant and insulin-resistant disorders because of its acute
insulin-like metabolic effects and long-term anabolic actions (4).
Recently, IGF-I has been identified as a growth factor for the immune
system (5). In vivo treatment with IGF-I enhanced thymic reconstitution in diabetic (3), aged (6), steroid-treated (1), and cyclosporin-treated (2) animals. IGF-I may cause lymphocyte expansion by direct mitogenic
effects or by increasing cell survival. Although there is no direct in
vivo evidence that IGF-I has antiapoptotic effects on lymphocytes,
recent in vitro studies show that IGF-I prevents apoptosis of primary,
interleukin-3 (IL-3)-dependent hematopoietic cells and of pre-B cells
(23). Clark, in a recent review of IGF-I and immune function (5),
proposes a scheme whereby IGF-I modulates apoptosis in the immune
system. Information is also lacking on the role of IGF-I in the
positive or negative selection of thymocytes. However, the production
of IGF-I by thymic epithelial cells (28) and the increased IGF-I
receptor expression on T cells after in vitro activation with anti-CD3
antibody (14) suggests that IGF-I may play a role in the T cell
selection process.
Our objective was to address the following questions:
1) does in vivo IGF-I treatment
prevent Dex-induced cell loss in the thymus and spleen, in particular,
apoptosis of CD4+CD8+ double-positive thymocytes?
2) does in vivo IGF-I treatment
alter recovery of thymic and splenic lymphocytes after Dex-induced
apoptosis? and 3) does in vivo IGF-I
increase lymphopoiesis in rats during total parenteral nutrition (TPN)?
The TPN model used in these studies has the advantage of controlling
nutrient intake, because Dex treatment alters ad libitum consumption.
In addition, TPN is often required in clinical situations in which
glucocorticoids are elevated after severe injury or trauma, and IGF-I
treatment may have the utility to induce anabolism.
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METHODS |
Animal Protocol
The animal facilities and protocols were approved by the University of
Wisconsin-Madison Institutional Animal Care and Use Committee. Male
Sprague-Dawley rats (Harlan, Madison, WI) weighing 230-240 g were
housed in individual stainless steel cages with free access to water in
a room maintained at 27°C on a 12:12-h light-dark cycle. Animals
were adapted to the animal facility for 4 days before surgery and were
allowed to consume semipurified diet ad libitum. Immediately before
surgery (230-250 g body wt), animals were divided into treatment
groups matched for body weight. Animals were fasted 18 h before
surgery, anesthetized by intramuscular injection with 80 mg ketamine
and 8 mg xylazine/kg body wt and then underwent surgical placement of
catheters in the superior vena cava by way of the external jugular
vein, as previously described (17). Animals received 165 µg
dexamethasone 21-phosphate (Sigma, St. Louis, MO) in 100 µl PBS as an
intraperitoneal injection immediately after surgery. Recombinant human
IGF-I (rhIGF-I), provided by Genentech (Genentech, San Francisco, CA),
was coinfused continuously with the TPN solution at a dose of 800 µg/day beginning immediately after surgery. The infusion of the TPN
was introduced gradually, from 5-20 ml on
day 0 (day of surgery), to 30 ml on day
2, to 55 ml on
day
3. TPN solutions were
prepared aseptically using commercial preparations of amino acids
(Travasol 8.5% with electrolytes; Baxter Healthcare, Deerfield, IL),
dextrose, 20% medium-chain/long-chain triglyceride 3:1 lipid emulsion
(Clintec, Deerfield, IL), vitamins, trace elements, and electrolytes
(29). The TPN solution provided 48% of nonprotein energy from fat and
52% nonprotein energy from dextrose.
Experimental Design
Animals were killed at three time points: 12 h, 48 h, and 5 days after
Dex injection. For examination of the antiapoptotic effects of IGF-I,
animals were killed 12 and 48 h after the Dex injection. In a
preliminary study, DNA fragmentation indicative of apoptotic cell death
was detected in thymocytes and splenocytes 12 h after Dex treatment.
Two days after Dex injection, cell loss is complete, with a 90%
reduction in thymocyte number (22). Accordingly, at 12 and 48 h, three
groups of TPN animals were killed: animals treated with Dex, those
treated with Dex and IGF-I (Dex + IGF-I), and those receiving neither
treatment (TPN control).
In a preliminary study, we found that 6 days after Dex injection the
percentage of CD4+CD8+ thymocytes was partially recovered, i.e., 50%
CD4+8+ 6 days after Dex treatment compared with 90% CD4+8+ in normal
rats. For investigation of the possibility that IGF-I treatment would
accelerate the recovery of lymphocytes, animals were killed 5 days
after the Dex injection. Animals killed after 5 days included four
treatment groups: Dex, Dex + IGF-I, TPN-control, and IGF-I arranged in
a 2 × 2 factorial design (main effects, ± Dex, ± IGF-I).
Another group of rats that did not undergo any surgical treatment and
was allowed to consume the semipurified diet ad libitum served as a
normal comparison (normal rats).
Serum Hormone Concentrations
After 12 h, 48 h, or 5 days of TPN, rats were anesthetized and killed
by exsanguination via cardiac puncture. Serum was obtained, aliquoted,
and frozen at 
20°C for IGF-I analysis. Total serum IGF-I
concentrations were determined by radioimmunoassay (8) after IGF-I
binding proteins were removed by high-performance liquid chromatography
under acid conditions. Materials for the RIA included rhIGF-I as
standard, 125I-labeled IGF-I
(Amersham, Arlington Heights, IL), polyclonal antibody to human IGF-I
(National Hormone and Pituitary Program, Rockville, MD), goat
anti-rabbit immunoglobulin G, and normal rabbit serum (Antibodies,
Davis, CA). Serum extracts for total IGF-I were assayed in duplicate in
a single assay with an intra-assay coefficient of variation of <10%.
Lymphoid Tissue Response
The spleen and the thymus were removed and weighed, and single-cell
suspensions prepared by teasing the tissue apart and flushing the cells
through a 100-mesh screen (Micro Filtration Systems, Dublin, CA) using cold RPMI 1640 cell culture medium supplemented with
10 mM HEPES, penicillin (100 IU/ml), streptomycin (100 µg/ml), glutamine (2 mM), and 10% fetal calf serum (FCS, Hyclone, Logan, UT).
All cell culture reagents were purchased from Sigma. Cell counts were
done using a hemocytometer after trypan blue staining. In the animals
killed 12 h after Dex injection, portions of the thymus and spleen were
fixed in 10% buffered Formalin, embedded in paraffin, stained with
hematoxylin and eosin, and viewed under a light microscope and
representative areas were photographed.
Flow Cytometry
Flow cytometry was used for phenotypic and quantitative analysis of
thymocyte and splenocyte populations as previously described (11).
Aliquots of 1 × 106 cells
were suspended in 100 µl of antibody at the appropriate dilution and
were incubated on ice for 45 min. Antibodies were diluted in PBS with
1% FCS and 0.05% NaN3. Cells
were washed three times in PBS,
NaN3, and FCS and then fixed in
PBS with 2% paraformaldehyde and 0.02%
NaN3 for 15 min, washed twice, and
resuspended in 500 µl PBS, 1% FCS, and 0.05%
NaN3 for flow cytometric analysis.
Cells were stored at 4°C overnight until analyzed with a flow
cytometer (EPICS Profile Analyzer; Coulter, Hialeah, FL). Two-color
fluorescence parameters were collected with a log amplifier after
gating of the combination of forward and perpendicular light scatter
for lymphocytes and monocytes. Cells were stained with FITC-anti-CD4, PE-anti-CD8, FITC-anti-IgM, and PE-anti-CD44 (PharMingen, San Diego,
CA). Cells stained with an irrelevant antibody of the same isotype as
the test antibody served as negative controls.
Propidium iodide staining of DNA was used to determine the percentage
of apoptotic cells in the thymus and spleen. Briefly, 3 × 106 cells were pelleted by
centrifugation at 1,000 rpm and resuspended in 1 ml hypotonic
fluorochrome solution (50 µg/ml propidium iodide; Aldrich Chemical,
Milwaukee, WI and 0.1% sodium citrate and 0.1% Triton X-100; Sigma),
incubated at room temperature for 15 min in the dark, and stored at
4°C until analyzed by flow cytometry.
Statistical Analysis
Data were analyzed by one- and two-way ANOVA using the Statistical
Analysis System generalized linear models program (25). The data from
the "normal" group were not included in the statistical analysis;
they are meaningful as a point of reference for nonsurgical rats. Group
means were considered to be significantly different at
P < 0.05, as determined by the
protected least-significant difference technique unless otherwise
indicated. Statistical tests were performed on log-transformed data
when the variance was not equal among groups, i.e., on cell number
data. The results are expressed as means ± SE of untransformed
data.
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RESULTS |
Does IGF-I Prevent Dex-Induced Apoptosis in Thymus and Spleen?
Body and organ weights.
There were no differences between the Dex + IGF-I and Dex groups for
presurgery body weight or final body weight (data not shown). There was
a net loss of body weight for all three groups over the 48-h experiment
(Table 1). The relative weights of the thymus and spleen (g/kg body wt) are given in Fig.
1. Relative thymus weight of the Dex and
Dex + IGF-I groups was significantly reduced
(P < 0.05) ~30% compared with TPN
control and normal rats, as was absolute thymus weight (Dex, 0.21 ± 0.02 g; Dex + IGF-I, 0.19 ± 0.02 g; TPN, 0.31 ± 0.01 g; normal,
0.41 ± 0.01 g). Relative spleen weight was
significantly reduced by Dex treatment compared with TPN control and
normal rats. IGF-I treatment counteracted the effect of Dex; relative
spleen weight was significantly increased compared with the Dex group.
The same result was evident for absolute spleen weight (Dex, 0.53 ± 0.03 g; Dex + IGF-I, 0.70 ± 0.04 g; TPN, 0.80 ± 0.04 g; normal, 0.61 ± 0.02 g). The concentration of IGF-I in serum of
Dex-treated TPN rats was reduced ~30% compared with normal rats
(normal, 340 ± 16.2 µg/l) and was increased with IGF-I infusion
(Table 1).
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Table 1.
Serum IGF-I concentration and body weight change in rats maintained
with TPN and treated with IGF-I or Dex for 2 or 5 days
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Fig. 1.
Relative thymus and spleen weights [g/kg body wt (BW)].
Groups of rats (n = 6-10) were
injected with dexamethasone (Dex) or vehicle and treated with
insulin-like growth factor I (IGF-I), which was coinfused with total
parenteral nutrition (TPN) solution. Two or five days postinjection,
each thymus and spleen was removed and weighed. Normal rats were
provided ad libitum access to semipurified diet and did not undergo any
surgical treatment (normal rats: 1.70 ± 0.02 g thymus/kg BW, 2.5 ± 0.1 g spleen/kg BW). Data are means ± SE; means with
different letter superscripts are significantly different
(P < 0.05).
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Apoptosis.
Paraffin sections of thymus from animals treated with Dex showed severe
thymic atrophy (Fig. 2,
e and
f), disruption of normal thymic
architecture (i.e., cortex and medulla; Fig. 2,
a and
b), and condensed, darkly stained
nuclei in the cortical region indicative of apoptotic cell death
regardless of treatment with IGF-I (Fig. 2,
c-f).
Condensed nuclei characteristic of apoptotic cells were absent in TPN
control animals (Fig. 2, a and
b).
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Fig. 2.
Thymic histology. Groups of rats (n = 3) were injected with Dex or vehicle and treated with IGF-I, which was
coinfused with TPN solution. Twelve hours postinjection, each thymus
was removed, fixed, sectioned, stained with hematoxylin and eosin, and
examined for gross structural changes and condensed nuclei.
Top, structural changes:
a, TPN control;
c, Dex + IGF-I;
e, Dex.
Bottom, high magnification of cortical
thymocytes: b, TPN control;
d, Dex + IGF-I;
f, Dex.
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Sections of spleen from animals treated with Dex showed a decrease in
the distinction between white pulp (lymphoid area) and red pulp (red
blood cell storage and degradation), a decrease in white pulp area, and
condensed nuclei in the lymphoid area (Fig.
3, e and
f) compared with TPN controls (Fig.
3, a and
b). Propidium iodide staining of DNA
showed a subdiploid peak in the Dex group (7.5 ± 1.4% apoptotic
cells) but not in the Dex + IGF-I or TPN groups. IGF-I treatment
preserved splenic architecture and reduced the percentage of cells with
fragmented DNA (Fig. 3, c and
d). Flow cytometric analysis at 12 h
revealed that IGF-I exerted a protective effect on all splenic T and B
cell subsets examined, with a preferential effect to preserve CD4+8+ T
cells (data for flow cytometry at 12 h not shown because it was similar to the result at 48 h).
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Fig. 3.
Splenic histology. Groups of rats (n = 3) were injected with Dex or vehicle and treated with IGF-I, which was
coinfused with TPN solution. Twelve hours postinjection, each spleen
was removed, fixed, sectioned, stained with hematoxylin and eosin, and
examined for gross structural changes and condensed nuclei.
Top, structural changes:
a, TPN control;
c, Dex + IGF-I;
e, Dex.
Bottom, high magnification of cortical
thymocytes: b, TPN control;
d, Dex + IGF-I;
f, Dex.
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Lymphocyte populations.
The number of thymocytes was reduced ~80-85% in both the Dex + IGF-I and Dex groups compared with TPN control, as shown in Fig.
4. Immature thymocytes or CD4+CD8+ cells
were the population of thymic T cells targeted by Dex-induced apoptosis
(7). As presented in Fig. 5, the percentage
of CD4+CD8+ cells was reduced ~90% in both the Dex + IGF-I and Dex
groups (Dex, 10.4 ± 1.0% CD4+8+ cells; Dex + IGF-I, 9.2 ± 0.6% CD4+8+ cells) compared with TPN controls (88.7 ± 1.2% CD4+8+
cells) and normal rats (84.8 ± 1.2% CD4+8+ cells). The absolute
numbers of T cell subpopulations are given in Table
2. IGF-I treatment did not prevent
Dex-induced apoptosis when the percentage or absolute number of
CD4+CD8+ cells at 48 h after Dex treatment was compared.
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Fig. 4.
Thymus and spleen total cell numbers. Groups of rats
(n = 6-10) were injected with Dex
or vehicle and treated with IGF-I, which was coinfused with TPN
solution. Two or five days postinjection, each thymus and spleen was
removed and weighed and cells were counted. Normal rats were provided
ad libitum access to semipurified diet and did not undergo any surgical
treatment. (normal rats: 3.85 ± 0.55 × 108 thymocytes; 3.7 ± 1.4 × 108 splenocytes). Data are
means ± SE; means with different letter superscripts are
significantly different (P < 0.05).
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Fig. 5.
Thymic T cell subpopulations. Groups of rats
(n = 6-10) were
injected with Dex or vehicle and treated with IGF-I, which was
coinfused with TPN solution. Two (A)
or five (B) days postinjection, each
thymus was removed and thymocytes were counted and stained with
FITC-anti-CD4 and PE-anti-CD8. Staining was assayed by FACScan. Scatter
plots are from 1 representative animal per treatment group; group means ± SE are given in each quadrant. TPN control data are shown for
day 5 only, because values for day 2 were similar (normal rats: 1.7 ± 0.2% CD48, 5.7 ± 0.5% CD4+CD8, 8.2 ± 0.5% CD4CD8+, 84.4 ± 1.1% CD4+8+). Two days after Dex
injection, percentage of CD4+8+ immature T cells was reduced regardless
of cotreatment with IGF-I. IGF-I infusion significantly increased the
percentage of CD4+8+ cells compared with Dex-only group 5 days after
Dex treatment.
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Table 2.
Effects of IGF-I and/or Dex treatment on
thymocyte subpopulations in rats maintained with TPN for 2 or 5 days
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The number of cells in the spleen is shown in Fig. 4. IGF-I treatment
attenuated the Dex-induced reduction in splenocyte number by
10-15%; however, the number of splenocytes remained 40% of TPN
control. The percentages and numbers of splenic T cell subpopulations are shown in Fig. 6 and Table
3, respectively. The percentages and
numbers of mature CD4+ and CD8+ T cells were reduced in both the Dex
and Dex + IGF-I groups, whereas the percentage of CD4+8+ immature T
cells was significantly increased compared with TPN controls. IGF-I had
no effect on the percent distribution of CD4+ and CD8+ single- and
double-positive cells. However, IGF-I significantly increased the
number of CD4+CD8+ immature and CD8+ mature T cells present in the
spleen; a similar trend was seen for the numbers of mature CD4+ cells
(P < 0.1, Table 3).
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Fig. 6.
Splenic T cell subpopulations. Groups of rats
(n = 6-10) were injected with Dex
or vehicle and treated with IGF-I, which was coinfused with TPN
solution. Two or five days postinjection, each spleen was removed, red
blood cells were lysed, and splenocytes were counted and stained with
FITC-anti-CD4 and PE-anti-CD8. Staining was assayed by FACScan.
A: CD4+,
B: CD8+,
C: CD4+8+ (normal: 29.1 ± 1.6%
CD4+8, 15.7 ± 0.9% CD48+, 12.5 ± 1.6% CD4+8+).
Numbers are percentages (means ± SE). Means with different letter
superscripts are significantly different
(P < 0.05).
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Table 3.
Effects of IGF-I and/or Dex treatment on
splenocyte T cell subpopulations in rats maintained with TPN for 2 or 5 days
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The number of IgM-positive B cells in the spleen is shown in Table
4. IGF-I significantly attenuated the
Dex-induced decrease in the absolute number of IgM-positive B cells in
the spleen; however, the number of B cells remained 30% of TPN control
values. Neither Dex nor IGF-I treatment altered the distribution of
cells between IgMhi (immature) and
IgMlo (mature)
subpopulations of B cells compared with TPN control and
normal rats (data not shown). Consistent with the result at 12 h, the
protective effect of IGF-I in spleen was evident in all T and B cell
subsets examined.
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Table 4.
Effects of IGF-I and/or Dex treatment on
IgM+ splenocyte subpopulations in rats maintained with TPN for 2 or 5 days
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Does IGF-I Alter Lymphocyte Recovery After Dex?
Body and organ weights.
Body weight change over the 5 days is shown in Table 1. There were
significant main effects for IGF-I and Dex treatments by two-way ANOVA;
Dex treatment resulted in weight loss (+Dex, 5 ± 3 g;
Dex, 2 ± 3 g), whereas animals treated with IGF-I gained weight (+IGF-I, 6 ± 2 g; IGF-I, 8 ± 2 g). Thymus
weight was not recovered 5 days after Dex injection compared with TPN
control, whereas spleen weight was recovered independent of IGF-I
treatment (Fig. 1). The animals treated with IGF-I had significantly
greater concentrations of IGF-I in serum than other groups (Table 1).
Lymphocyte populations.
The number of thymocytes was still reduced ~80% in the Dex and Dex + IGF-I groups 5 days after Dex injection, as shown in Fig. 4. However,
the thymus was partially recovered at this time, because the
percentages of thymocytes differentiated by CD4 and CD8 were somewhat
restored to TPN control values (Fig. 5). IGF-I treatment significantly
accelerated the recovery of the percentage of immature CD4+CD8+
thymocytes after Dex-induced cell loss (Dex, 60.7 ± 5.9% CD4+8+
cells; Dex + IGF, 74.8 ± 6.1% CD4+8+ cells; TPN control, 88.7 ± 1.2% CD4+8+ cells; Fig. 5) and tended to increase the number of
CD4+8+ cells as well (Table 2). The percentages and numbers of single
positive mature CD4+ and CD8+ cells decreased from
day 2 to day
5 in the Dex-treated groups,
independent of IGF-I treatment.
Spleen weight and cell number were almost recovered to that of the TPN
control by day
5 in both the Dex and Dex + IGF-I
groups (Figs. 1 and 4). Spleen cellularity increased in the TPN
controls (~2-fold) between day
2 and
day
5, and the magnitude of the increase was similar to that of the Dex + IGF-I group (~3-fold) and less than
that of the Dex group (~6-fold). The numbers of all T cell subpopulations differentiated by CD4 and CD8 increased from
day 2 to day
5 in both Dex-treated groups to values
similar to the TPN control (Table 3). IGF-I treatment significantly
decreased the percentage (Fig. 6B) and number (Table 3) of
CD8+ splenocytes. As shown in Fig. 6C, the percentage of
double-positive CD4+CD8+ cells increased from
day 2 to day
5 in the Dex-treated groups; IGF-I did
not enhance recovery of the double-positive T cells. Five days after
Dex injection, the percentages and numbers of B cell subpopulations in
the spleen were also restored to values similar to TPN control (Table
4). IGF-I had no effect on the percent distribution of
IgMlo and
IgMhi B cells (data not shown).
Does IGF-I Increase Lymphopoiesis in Rats During TPN?
IGF-I treatment significantly increased relative thymus weight (Fig. 1)
and total thymocyte number (Fig. 4) in rats not treated with Dex for 5 days. IGF-I treatment did not alter the percent distribution of T cell
subsets distinguished by CD4 and CD8 (Fig. 5). However, IGF-I
significantly increased the number of CD4+CD8+ (Table 2) and T cells
expressing CD44 which is present on pre-T cells before the CD4+8+ stage
(27) (IGF, 1.3 ± 0.7 ×106 CD44+ cells; TPN,
0.63 ± 0.09 ×106 CD44+ cells).
IGF-I treatment significantly increased relative spleen weight (Fig. 1)
and total splenocyte number (Fig. 4). Both the percentages (Fig. 6) and
absolute numbers of single-positive CD8+ and double-positive CD4+8+,
but not CD4+, T cells were increased 50% by IGF-I (Table 3). IGF-I
significantly increased the number of total IgM+ B cells in the spleen
(Table 4) but did not alter the proportion of B cells in the spleen or
the percent distribution of IgM-positive cells between
IgMlo B cells (TPN, 51.2 ± 0.8% IgMlo B cells, IGF, 51.6 ± 3.8% IgMlo B cells) and
IgMhi B cells (TPN, 12.9 ± 0.6% IgMhi B cells; IGF, 13.8 ± 1.4% IgMhi B cells).
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DISCUSSION |
The capacity of the immune system to maintain homeostasis rests in the
ability to control cell proliferation and cell death. Evidence suggests
that IGF-I may exert immunotrophic effects by increasing lymphopoiesis
and cell survival. In vitro studies with cell lines of several
phenotypes, including pre-B and myeloid and erythroid bone marrow
progenitor, have shown that IGF-I prevents apoptosis that occurs in the
absence of other growth factors such as IL-3, granulocyte
colony-stimulating factor and granulocyte macrophage colony-stimulating
factor, and erythropoietin (19, 23). Likewise, IGF-I has been shown to
increase T and B cells in the thymus and spleen of aged (6), diabetic
(3), and parenterally fed (11) animals and to accelerate recovery of T
cells after cyclosporin treatment (2) and of T and B cells after bone
marrow transplantation (13). Our objectives were to more clearly
separate the effects of IGF-I on lymphocyte survival and regeneration
using a Dex model of apoptosis and to investigate the role of IGF-I in
T cell lymphopoiesis. The results of this study show that IGF-I is
unable to rescue thymocytes from Dex-induced apoptosis in vivo, but
that IGF-I increases cell survival by 10-15% in the spleen. In
addition, IGF-I alters the recovery of T cells in both the thymus and
spleen. IGF-I enhanced the recovery of CD4+CD8+ immature T cells in the
thymus, suggesting a role for IGF-I in T cell lymphopoiesis. The effect
of IGF-I on splenic recovery after Dex was to decrease the percentage
and number of CD8+ T cells.
Our results extend data from in vitro studies that demonstrate
anti-apoptotic effects for IGF-I (19, 23). Although our results did not
demonstrate any protective effect of IGF-I on thymocytes, the signals
initiating cell death and the cell types studied differed. We
investigated apoptosis using a glucocorticoid which acts through its
nuclear receptor to cause gene transcription, activation of an
endonuclease, and DNA degradation. In contrast, the in vitro studies
utilized cell lines that are dependent on growth factors for survival
and thus investigated a cell suicide pathway associated with cell cycle
arrest (24).
Although IGF-I did not prevent thymocyte loss due to Dex-induced
apoptosis, infusion of IGF-I attenuated the reduction in splenocytes
seen after Dex treatment (Figs. 1-4). This protective effect of
IGF-I was evident in all T and B cell subsets examined in the spleen
(Tables 3 and 4). The reason for the different effect in the thymus and
the spleen may be that the thymus, a primary lymphoid organ, normally
contains ~90% CD4+CD8+ immature T cells that are exquisitely
sensitive to glucocorticoids, whereas the spleen, a secondary lymphoid
organ, contains a mixed population of cell types, including,
predominantly, mature T and B lymphocytes. In addition, the ability of
IGF-I to rescue cells from apoptosis after Dex treatment may be
modulated by the activation state of the cell; IGF-I receptor
expression in T lymphocytes is increased after activation by mitogen
(15) and anti-CD3 antibody (14). The TPN model is unique in that the
systemic infusion provides an immune stimulus which results in
splenomegaly and possible activation of lymphocytes (11). This
activation may alter expression of the IGF-I receptor and the response
to exogenous IGF-I. Thus the ability of IGF-I to exert anti-apoptotic
effects may depend on the signal initiating cell suicide and the type
of cell, as well as the maturation and activation states of the cell.
We found that IGF-I accelerated the recovery of immature T cells in the
thymus. Five days of IGF-I treatment after Dex injection significantly
increased the percentage of CD4+CD8+ T cells (Fig. 5) with a
concomitant decrease in the percentage of CD4CD8 T cells
and no alteration in the distribution of mature T cells between CD4+
and CD8+ phenotypes. Furthermore, in rats not treated with Dex, IGF-I
significantly increased the number of total thymocytes, immature CD4+8+
T cells, and pre-T cells expressing CD44, a cell surface antigen
expressed early in T cell maturation before the CD4+8+ stage (27).
These findings suggest that IGF-I may act on the bone marrow to
increase pre-T cell seeding of the thymus. They are consistent with
results in aged mice which showed increased peanut
agglutinin binding in thymocytes after 7 days of IGF-I treatment (6) and in IGF-I-treated diabetic rats that had increased CD4+CD8+ thymocyte numbers (3).
Five days after Dex treatment, spleen weight and cellularity were
restored, independent of IGF-I treatment. Although the Dex + IGF-I
group had decreased splenocyte number compared with the TPN control
group at day
5, the increase in cellularity was
similar in these two groups between
days
2 and
5 (Fig. 4). The greater increase in
splenocyte cellularity between days
2 and
5 in animals treated with Dex without
IGF-I may reflect an enhanced regenerative response to the greater loss
of cells in these animals compared with treatment with Dex + IGF-I.
IGF-I significantly reduced the percentage and number of CD8+ T cells
in the spleen compared with the Dex group. Interestingly, in rats not
treated with Dex, IGF-I dramatically increased the percentage and
number of CD4+8+ and CD8+ but not CD4+ T cells in the spleen. This
differs from studies in aged mice (6) demonstrating an increase in CD4+
but not CD8+ T cells in the spleen after 7 and 14 days of IGF-I
treatment. Moreover, 7 wk of rhIGF-I treatment in aged female monkeys
increased the percentage of CD4+ T cells in the spleen, whereas in the
peripheral blood IGF-I had the opposite effect, increasing the
percentage of CD8+ T cells (18). These differences in the proportion of
mature CD4+ and CD8+ T cells may be due to the differences in treatment
duration and experimental models. Regardless, the increase in immature
CD4+8+ T cells reported here and the shifts in the mature CD4+ and CD8+
T cell populations suggest that IGF-I affects extrathymic T cell
production and development.
In conclusion, IGF-I did not prevent Dex-induced apoptosis of
thymocytes, although it produced a modest but statistically significant
reduction in splenocyte cell loss in this model. These data fit the
scheme proposed by Clark (5) whereby glucocorticoids released after
activation of the hypothalamic-pituitary-adrenal axis in response to
injury decrease systemic and local IGF-I expression and subsequently
increase apoptosis. In the current study, increasing systemic IGF-I via
parenteral infusion modestly reduced apoptotic cell death in the
spleen. Although this direct in vivo evidence suggests that IGF-I may
offer protection from apoptosis, the biological significance of this
finding remains unclear. Further investigations are required to
identify and quantify apoptotic lymphocyte populations, a difficult
undertaking in an organ such as the spleen which is designed to rapidly
degrade effete cells.
IGF-I is currently under investigation as a therapeutic agent to
promote anabolism during catabolic diseases (4) and to attenuate the
immunosuppression associated with the neurohormonal response to injury
(5). Additional studies are needed to assess whether exogenous IGF-I
increases the potential for the development of autoimmune disease or
lymphoid malignancy by allowing self-reactive or transformed
lymphocytes to escape programmed cell death.
| |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Susan M. Smith for providing advice
regarding methodology; Hui-Chen Lo, Mike Grahn, Karen Kritsch, Linda
Posinges, and Barry and Charmayne Hinton for technical assistance; and
Judith Kozminski for providing the computer graphics. We thank Genetech
(San Francisco, CA) for supplying the rhIGF-I and Clintec Technologies
(Deerfield, IL) for providing the parenteral solutions.
| |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grants R01-DK-42835 and T32-DK-07665 and by funds
from the College of Agricultural and Life Science, University of
Wisconsin-Madison, project 3096.
Current address of C. A. Peterson: College of Professional Studies,
242B CPS, University of Wisconsin-Stevens Point, Stevens Point, WI
54481.
Address for reprint requests: D. M. Ney, Dept. of Nutritional Sciences,
Univ. of Wisconsin-Madison, 1415 Linden Drive, Madison, WI 53706.
Received 12 June 1997; accepted in final form 10 December 1997.
| |
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