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2
*
Department of Pediatrics, Divisions of Hematology/Oncology and the
Department of Pulmonary Medicine, Louisiana State University Health Sciences Center, New Orleans, LA 70112 and the
**
Department of Pediatrics, University of South Alabama College of Medicine, Mobile, AL 36617
2To whom correspondence should be addressed. E-mail: skuvib{at}lsuhsc.edu
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • iron deficiency • lymphocyte proliferation • concanavalin A • cell cycle analysis • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. The role of
iron on cell proliferation of activated lymphocytes has been
established in humans and laboratory animals (5
,6)
.
Although the major site of action of iron is the activity of
ribonucleotide reductase, it has been reported that iron may affect
lymphocyte proliferation independently and before the increase of
ribonucleotide reductase activity (7
8
9
10
11
12)
. We have
previously observed that iron deficiency reduced the hydrolysis of cell
membrane phosphatidyl inositol 4,5 bis-phosphate
(PIP2),3
total protein kinase C (PKC) activity, and PKC translocation from the
cytosol to the membrane-bound fractions (9
10
11)
. Both
these events take place within minutes of mitogen or antigen binding to
T cell receptor (CD3) (13)
. Lucas et al. (14)
also reported that iron depletion of normal human blood lymphocytes by
deferoxamine altered the progression through the cell cycle and
arrested cells before the cell cycle initiation phase (G1)/DNA
synthesis phase (S) border. They showed that iron chelation inhibited
the increase in certain gene transcriptions and mRNA translations,
which usually take place during the mid and late G1 phase. The products
of these genes (e.g., cdc2, product p34cdc2; cdk2
product p33cdk2) play crucial roles in the
progression of activated lymphocytes through the cell cycle
(15)
. However, deferoxamine can also bind other trace
elements, such as zinc, which also are required for DNA synthesis and,
subsequently, lymphocyte proliferation. When deferoxamine is added to
the medium, it is unknown whether its negative effects on progression
through the cell cycle is specifically due to iron chelation or to a
combination of effects of other trace elements. The effects of iron
deficiency induced in vivo on the progression of human and murine
splenic lymphocytes through the cell cycle have not been previously
reported. In the present study, we determined whether in vivo iron
deficiency is severe enough to alter the progression of activated
lymphocytes through the cell cycle when cells are grown in limiting
amounts of iron (serum). |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents were from the following sources: Sigma (St. Louis, MO): concanavalin A (Con A), ß-mercaptoethanol, hemoglobin standard, Drabkin’s reagents, iron assay kits; GIBCO (Grand Island, NY): Roswell Park Memorial Institute (RPMI) 1640 with 25 mmol/L HEPES, fetal calf serum (FCS), newborn calf serum (NBCS), nonessential amino acids, sodium pyruvate, penicillin/streptomycin; Bio-Whittaker (Walkersville, MD): L-glutamine; Harlan Teklad (Madison, WI): the iron-deficient (ID) test diet and the deficient diet supplemented with iron; NEN Dupont (Boston, MA): 3H-thymidine (6.7 Ci or 247.9 GBq/mmol); ICN (Costa Mesa, CA): Cytoscint and Pharmigen (Costa Mesa, CA): anti-CD3 antibody, anti-CD28 antibody.
Experimental design and mice feeding.
C57BL/6 female mice (n = 63, 21–23 d old) were
purchased from Charles River Breeding Laboratories (Wilmington, MA).
Upon receipt, they received for 7 d the control diet that
contained 50 mg (0.895 mmol) of iron per kg diet in the form of ferrous
sulfate and sterile deionized water. After the adjustment period, mice
were randomly assigned to the following dietary treatment groups:
control (C; n = 16), ID (n = 31) and pair-fed (PF; n = 16). Iron deficiency
was induced by feeding a diet that contained only 5 mg (0.0895 mmol) of
iron per kg diet. The C and PF groups received the same diet except
that it was supplemented with 50 mg of iron/kg (0.895 mmol/kg) diet.
Except for iron, the concentrations of protein, fat, carbohydrates,
vitamins and minerals of both diets were identical and have been
previously reported (10)
. C and ID mice had free access to
their diets 24 h/d and PF mice received the control diet in amounts
equal to the mean that the ID mice had consumed during the preceding
24 h. Mice were housed in sterile microisolator system cages
(product 109EI; Laboratory Products, Maywood NJ) and they received
sterile deionized water. The light to dark cycle was set for 12 h,
and the room temperature at 22°C. The study was approved by the
Institutional Committee for Animal Care and Use of Louisiana State
University School of Medicine. The feeding period lasted 68 d.
Evaluation of iron status at the end of the feeding period.
Twenty-four hours and 3 d before the experiments, 6
and 10 ID mice, respectively, with a hematocrit < 0.25 were
offered the control diet [iron repletion protocol = iron-deficient mice that were repleted for 1 (R1) and 3 (R3) d,
respectively (R1 and R3 groups)]. At the time of killing, mice were
anesthetized by ether inhalation for 30–60 s. After blood was drawn
from the retro-orbital plexus, they were killed by cervical
dislocation. Hemoglobin and hematocrit were measured by the
cyanmethemoglobin method and centrifugation, respectively
(16)
. The iron concentrations of the liver and the diets
were assayed as we previously reported (10)
.
Preparation of spleen cell suspension.
After spleens were removed under sterile conditions single-cell
suspensions were prepared by standard techniques in serum-free RPMI
1640 medium that was supplemented with 10 g/L bovine serum albumin, 50
mg/L streptomycin and 5 x 104 U/L penicillin
(10)
. It contained 0.113 umol/L of iron. Cells were washed
by centrifugation at 400 x g, 4°C for 10 min.
The supernatant was decanted and the pellets were resuspended in 1 mL
of ice-cold sterile deionized water to lyse red blood cells. Cells
were further washed twice under the same conditions. The pellets were
then resuspended in 2 mL of wash medium. Total and viable cells were
counted using a hemocytometer under a light microscope after cells were
diluted in a solution of trypan blue (4 g/L).
Cell cycle analysis.
The method described by Coligan et al. (17)
was used. In
the preliminary study, spleen cells from C, PF and ID mice were
incubated with or without Con A or anti-CD3 antibody for 72 h
before fixing cells with 70% ethanol. Cells were also stained with
trypan blue. Although the culture medium contained 10% FCS, analysis
of the cells either under light microscope (for cells stained with
trypan blue) or flow cytometry (for cells stained with propidium
iodide) revealed cell death of > 70% in all three study groups.
Consequently, the incubation period was reduced to 48 and 24 h.
Viable splenic lymphocytes (3 x 106) were combined with 1 mg of Con A/L, 50 ug/L anti-CD3 antibody or 50 µg/L anti-CD3 and 50 µg/L anti-CD28 in a 12 x 75 sterile culture tube. Culture medium was added to nonactivated cells to a total volume of 1 mL. The total volume was adjusted to 1 mL culture medium that was supplemented with either 10 or 50 mL/L NBCS. The composition of the culture medium was: 50 mg/L streptomycin, 5 x 104 U/L penicillin, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 0.1 mmol/L nonessential amino acids, 50 µmol/L ß-mercaptoethanol and RPMI 1640. The iron concentrations of the culture mediums that contained 10 mL and 50 mL/L serum were 0.76 µmol/L and 1.844 µmol/L, respectively. Cultures were incubated at 37°C, 5% CO2 in a humidified incubator for 24 or 48 h. At the end of incubation period, cells were washed twice by centrifuging at 400 x g, 4°C for 10 min. The pellets were fixed with ice-cold 70% ethanol overnight. After cells were centrifuged once at 581 x g, 4°C for 10 min, 1 mL propidium iodide (50 mg/L) was added to each tube. Two micrograms of RNase (DNase-free) were added to degrade double stranded RNA. Tubes were vortexed for 10 s, and they were incubated at room temperature for 60 min. The 60-min incubation was followed by an overnight incubation at 4°C. Between 10,000 and 20,000 cells (or events) were analyzed by flow cytometry (FACScalibur; Becton Dickinson, San Jose, CA) within 24 h after staining. Cell cycle analysis [resting phase of the cell cycle (G0) to G1 or resting phase, S or DNA synthesis, exit from the S phase (G2) to mitosis (M) phase] was performed using ModFitLT 2.0 software (Verity; Software House, Topsham, ME). Aggregates were discriminated by gate width versus area.
Estimation of the percentage of anti-CD3+ cells.
Viable spleen cells (2 x 106) were transferred to a 12- x 75-mm culture tube, followed by 50 µg/L anti-CD3 conjugated with fluorescein isothiocyanate. The total volume per tube was adjusted to 1 mL of wash medium. Tubes were incubated at 4°C in (crushed ice) for 30 min, protected from light. At the end of incubation period, cells were washed twice by centrifuging at 400 x g, 4°C for 10 min. Each pellet was resuspended in 300 µL of 4% formaldehyde. Tubes were incubated at room temperature (22°C) on a shaker for 30 min. Fixed cells were analyzed within 24 h on a FACScalibur flow cytometer (Becton Dickinson, Immunocytometer Systems, San Jose, CA) using CellQuest Software. Between 10,000 and 20,000 events were collected and an electronic gate, based on nonfluorescent parameters (forward and side scatter) was constructed around lymphocyte population.
Lymphocyte proliferation.
Cells (1 x 106) were mixed with 1 mg/L Con A, 50 µg/L anti-CD3 antibody or 50 µg/L anti-CD3 combined with 50 µg/L anti-CD28 antibodies. For background DNA synthesis, culture medium that was supplemented with 50 mL/L of serum (1.844 µmol/L iron) was added to cells instead of the mitogen. Mitogen-stimulated and unstimulated cells were incubated at 37°C, 5% CO2 in a humidified NAPCO incubator (Model 5100; Portland, OR) for 24 h before being pulsed with 37 kBq 3H-thymidine per 200,000 cells. After 24 h, the cultures were harvested (PHD Cell Harvester; Cambridge Technology, Watertown MA). Because progression through the cell cycle was studied at 48 h, the incubation period for lymphocyte proliferation was also limited to 48 h instead of the usual 72 h. Each filter disk was transferred to a scintillation vial that contained 2 mL of Cytoscint. The radioactivity incorporated into DNA was measured by counting each sample for 1 min in an LKB liquid scintillation counter (Model 1219; Turku, Finland).
Calculations and statistical analysis.
Descriptive statistics (mean ± SEM), analysis of
variance and Pearson’s correlation coefficient were calculated by the
use of Microstatistical program (Ecosoft; Indianapolis, IN) and as
described in the literature (18)
. When analysis of
variance detected significant differences among study groups,
Scheffé’s test was used to determine which pairs of means
differed. The level of significant difference was set at
P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At the time of killing, hemoglobin, hematocrit and liver iron stores of
ID mice were significantly lower than those of C mice (Table 1
; P < 0.05). Feeding ID mice the iron-supplemented
diet for 1 d (R1) and 3 d (R3) before killing slightly, but
significantly, increased hemoglobin and hematocrit (P
< 0.05) and liver iron stores in the R3 group (P
< 0.05). However, these indicators of iron status were not
corrected relative to C mice. There were no significant differences in
the hemoglobin, hematocrit or liver iron stores between the C and PF
groups.
|
). In contrast to the total
CD3+ cells, the CD3+ cell
numbers/mg spleen were significantly lower in ID and R3 groups than in
the C and PF groups (P < 0.05). No significant
differences were observed between the C and PF or the ID and R3 groups. Cell cycle analysis.
When cultures were incubated for 24 h in 10 mL/L of NBCS- supplemented medium, groups did not differ in the percentages of activated and nonactivated spleen cells in different phases of the cell cycle (data not shown). The percentage distribution of cells in various phases was as follows: 88.9%–90.3% of cells in G0–G1, 9.1%–9.9% in S and 1.1%–1.69% in G2–M. Groups also did not differ in the sum of cells in the S and G2–M phases or the mean proliferative indices {[S + (G2–M)]/(G0–G1)}.
When cultures were incubated for 48 h in 50 mL/L of
NBCS-supplemented medium, groups did not differ in the percentages
of nonactivated spleen cells in the resting phase (data not shown). In
Con A-treated cells, there were small, but nonsignificant
(P = 0.12–0.48), differences among groups in the
percentages of cells in the resting, S and M phases, the sum of S and
G2–M phases as well as the proliferative indices. The distributions of
Con A-treated cells in different phases of the cell cycle were:
81%–87% in G0–G1, 11.1%–16.9% in S phase and 1.5%–2.3% in
G2–M phase. In anti-CD3 (Fig. 1
) and anti-CD3/anti-CD28-treated cells (Fig. 2
), significant differences were observed among groups in the percentages
of cells in different phases of the cell cycle (P < 0.01). For both types of cultures, the percentage of cells in the
resting phase was 
80% in C and PF and 90% (P < 0.05) for ID and R3 mice. Conversely, significantly higher percentages
of spleen cells from C and PF than those from ID and R3 mice were in
the S and the G2–M phases (P < 0.05).
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90% to 80%
(P < 0.05; Fig. 3A
). Conversely, activation of spleen cells from C and PF mice
was associated with a significant increase in the percentages of cells
in the S (Fig. 3B
) and G2–M phases (Fig. 3C
;
P < 0.05). In ID and R3 groups, incubation of cells
with mitogens did not significantly affect the percentages of cells in
the various phases.
|
). In mitogen-treated cells, the proliferative indices of C and PF
groups varied between 0.220 and 0.259, whereas they were 
0.154 in
both the ID and R3 groups. When stimulated with anti-CD3 or anti-CD3
combined with anti-CD28 antibodies, indices were greater in C and PF
than in ID and R3 (P < 0.05).
|
). In nonactivated cells, there was no significant correlation between
indicators of iron status and the percentages of cells in different
phases of the cell cycle. In mitogen-treated cells, indicators of
iron status negatively correlated with the percentages of the cells in
the resting cells, whereas they positively correlated with those in the
S and G2–M phases (P < 0.05).
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In nonactivated cells and anti-CD3-treated cells, groups did not differ in 3H-thymidine incorporation into DNA. In Con A- and anti-CD3/anti-CD28-treated cells, there were small and nonsignificant differences (P = 0.117–0.39) among groups in the rate of DNA synthesis (data not shown). The lack of significant decreases in lymphocyte proliferative responses of spleen cells from ID mice in the present study compared with our previous work is very likely due to the shorter incubation period (48 h vs. 72 h) and the lower serum concentration (5% vs. 10%).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In theory, the cell cycle has discrete phases or steps: G0 or resting phase; G1, entrance into the cell cycle; S or DNA synthesis; G2, exit from the S phase and M, mitosis. However, progression through the cell cycle is a continuous rather than discrete process. In spite of this flaw, various changes in the rates of DNA, RNA and protein syntheses over time in activated versus nonactivated lymphocytes give a rough estimate of the percentages of cells that are in the S and M phases. The use of flow cytometry has the advantage over 3H-thymidine uptake by providing the percentages of cells in different phases of the cell cycle.
Previous work by our group (4
,9
10
11)
and many other
investigators (2
,19)
has provided strong evidence of
inhibition of lymphocyte proliferation by iron deficiency in most,
although not all, studies. The few studies that failed to show a
negative effect of iron deficiency were probably affected by the
severity of iron deficiency, iron concentration in the culture medium,
the type of stimulus used and the duration of incubation
(3
,20
,21)
.
In the present study, we determined whether iron deficiency alters the
progression of splenic lymphocytes through the cell cycle when
activated cells were incubated in 0.76 µmol/L and 1.844 µmol/L of
iron. The concentration of 1.844 µmol/L is between 40% and 50% of
the usual amounts when 10% FCS is added to culture medium
(10
,11)
. In spleen cells that were incubated with mitogens
for 24 h or those that were incubated without mitogens but for
48 h, iron deficiency did not affect the proportion of cells in
different phases of the cell cycle compared with the normal state of
iron status. The lack of significant differences among groups for
cultures that were incubated with mitogens for 24 h is not
surprising, because (at least in humans), activated T lymphocytes do
not synthesize DNA before 24 h of cell activation.
When cultures were incubated with mitogens, especially the combination
of anti-CD3 and anti-CD28 antibodies for 48 h, significant
differences were observed between cells from ID mice and those from C
and PF mice. These differences were anticipated because 48 h is
the time when DNA synthesis starts to increase and reaches a peak at
72 h. In spite of the presence of 1.844 µmol of iron/L, the
percentages of cells from ID mice that entered the S and G2–M phases
were lower than those from C and PF mice, suggesting that in vitro iron
repletion of deficient cells is not complete. Differences in the
proportion of T cells in spleen cell suspension are very unlikely to be
responsible for the altered progression of cells through the cell cycle
because the percentage of anti-CD3+ cells was
slightly higher in ID than in C, PF and R3 mice. The present results
are in agreement with those that we recently observed in thymocytes
(22)
in which iron deficiency altered the progression of
thymocytes in different phases of the cell cycle in vivo. Similar to
the data of the present study, the percentages of thymocytes in the
G0–G1 were higher in ID than in C and PF and the opposite was observed
for those of thymocytes in the S and the G2–M phases.
One discrepancy between the present study and previous studies is the
lack of a significant decrease in 3H-thymidine
incorporation of spleen cells from ID mice. However, the trend showed
that the proliferative responses to Con A and anti-CD3/anti-CD28
antibodies were lower in ID and R3 than in C and PF groups. The
discrepancy was likely due to differences in the duration of the
incubation and the low serum (iron) concentration. In all our previous
studies, the incubation period was 72 h instead of 48 h, and
100 mL of FCS or NBCS per liter were used. The use of 50 mL/L instead
of 100 mL/L of NBCS (or FCS) was an attempt to minimize the level of
iron in the culture medium and hence possible in vitro repletion of
spleen cells from ID mice. In fact, we have observed that the
proliferative responses of spleen cells from normal mice to Con A and
anti-CD3 antibody were higher when cultures were incubated in 100 mL/L
instead of 50 mL/L of FCS or NBCS (unpublished results). Our data on
reduced percentages of ID spleen cells in the S and G2–M phases are in
agreement with those of Lucas et al. (14)
who observed a
block of cells in the late G1 phase upon iron chelation by
deferoxamine.
In previous studies, we showed that iron deficiency reduced certain
early events in T cell activation pathways. The first is the hydrolysis
of cell membrane PIP2, a reaction that yields diacylglycerol (required
for PKC activation) and inositol 1,3,5-triphosphate (required for
release of sequestered calcium) (10)
. The second is PKC
activation (11)
, and the third [which was confirmed by
Galan et al. (3)
] is interleukin-2 secretion (IL-2)
(23)
. PKC is required for the phosphorylation of many
transcription factors, and certain receptors that are directly involved
in T lymphocyte proliferation, such as transferrin receptor (which
regulates iron delivery to activated and proliferating cells), and IL-2
receptor (which is required for IL-2 binding to lymphocytes). The
hydrolysis of cell membrane PIP2 and PKC activation are triggered
within minutes of mitogen or antigen binding to the T cell receptor
(CD3) (13)
, and IL-2 mRNA production starts within 1 h of lymphocyte activation. These three events play a pivotal role in T
cell activation and successful proliferation, and they take place
several hours before the S and G2–M phases. Based on our results and
those of other investigators, we propose the following mechanisms of
reduced lymphocyte proliferation associated with iron deficiency. The
defect in PIP2 hydrolysis leads to reduced production of second
messengers (diacylglycerol and inositol 1,3,5 triphosphate). As a
result, PKC activation is impaired, and this in turn prevents or
reduces the phosphorylation of many transcription factors. Based on the
work of Lucas et al. (14
,15)
, lack of iron reduces the
production and activity of cyclin A-dependent kinase 2
(p33cdk2), a kinase required for progression
through the G1 phase. With reduced IL-2 secretion in ID lymphocytes,
the second phase of increase of cyclin A/p33cdk2
activity (as well as other cyclin-dependent kinases), which
normally occurs during the S phase, is inhibited (24)
. As
a result, ID lymphocytes are blocked in the G1 phase; hence, a higher
percentage of ID cells are detected in the G0–G1 phase and lower
percentages in the S and G2–M phases compared with iron-sufficient
lymphocytes. The consequence of this blockade and the low concentration
of the autocrine IL-2 is a reduction in lymphocyte proliferative
responses to mitogens and antigens.
In summary, our results show that a lower percentage of activated splenic lymphocytes from ID mice enter and progress through the cell cycle. This alteration very likely contributes to reduced lymphocyte proliferative responses to mitogens and antigens and impaired cell-mediated immunity that are usually associated with iron deficiency in humans and laboratory animals. Experiments are planned to study the activity of some of the cyclins and the effect of exogenous IL-2 on the progression of ID lymphocytes through the cell cycle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: C, control; Con A, concanavalin A; FCS, fetal calf serum; G0, resting phase of the cell cycle; G1, cell cycle initiation phase; G2, exit from the S phase; ID, iron-deficient; IL-2, interleukin-2; M, mitosis phase; NBCS, newborn calf serum; PF, pair-fed; PIP2, phosphatidyl inositol 4,5 bis-phosphate; PKC, protein kinase C; R1 and R3, iron-deficient mice that were repleted for 1 (R1) and 3 (R3) d, respectively; RPMI, Roswell Park Memorial Institute; S, DNA synthesis phase.
Manuscript received August 8, 2000. Initial review completed November 30, 2000. Revision accepted April 9, 2001.
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