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*
Trudeau Institute, Saranac Lake, NY 12983;
University of California at San Diego, La Jolla, CA 92039; and
Middlebury College, Middlebury, VT 05753
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thymidine and its analogues are taken up into the cell by a process of facilitated diffusion under the influence of a permease enzyme and are phosphorylated to deoxynucleotide monophosphate by the salvage pathway enzyme, thymidine kinase (1). The BrdU monophosphate derived from the salvage pathway competes with thymidine (5-methyldeoxyuridine) monophosphate of the de novo pathway derived from the deamination and methylation of cytidine deoxynucleoside monophosphate. The levels of some enzymes in the de novo pathway may vary and are under regulatory control. Levels of salvage enzymes can also vary. Thus, different cell lineages and developmental stages may have substantial differences in the relative amounts of the enzymes involved in the de novo and salvage pathways and may vary quite substantially in the efficiency of BrdU labeling (2, 3). This is generally ignored by immunologists, however, and it is assumed that the amount of proliferation is proportional to the amount of BrdU incorporated.
Another caveat is that continuous exposure to BrdU may select for a subset of cells with lower levels of the salvage enzymes or higher levels of de novo pathway enzymes (4). Thus, function in the presence of BrdU may appear to be retained or recover, but could be the property of BrdU-resistant cells that can no longer label with BrdU.
A third caveat is that BrdU may alter the metabolism of the cell into which it is incorporated. This is of little consequence if the assay of BrdU incorporation is conducted immediately after incorporation and is merely used to determine the number of cells in the S phase of the cell cycle. In some studies, however, the BrdU-labeled cells are followed for considerable lengths of time. If the experiment calls for a pulse protocol, the BrdU may be diluted by subsequent division, and the deleterious effects of BrdU may be ameliorated. In other protocols, BrdU is made available continuously and may begin to seriously affect the behavior of the BrdU-labeled cells (5, 6, 7, 8, 9, 10, 11, 12).
There is a very large literature on the nature of these effects, which range from death of the mouse, death of the BrdU-labeled cells, induction or inhibition of development in BrdU-labeled cells in vitro, and in changes in the expression of cell surface molecules (for a brief sampling, see Refs. 13, 14, 15, 16, 17, 18, 19, 20). Other studies, however, have appeared to show that the incorporation of BrdU has no effect on lymphocyte homeostasis or function, and the worrying studies are largely ignored by immunologists.
In the experiments described in this work, we have measured a number of parameters of the performance of cells variously labeled with BrdU in different experimental models. It is clear from our results that the incorporation of BrdU can, under some circumstances, seriously impair the performance of the labeled cells and raise a note of caution in the interpretation of studies that make use of long-term exposure to BrdU.
Young mice continuously exposed to BrdU fail to gain weight, and the lymphocytes recovered after a prolonged period of exposure are fewer in number than in control mice. The recovery of normal levels of T and B lymphocytes after irradiation is severely impaired. Ag-specific cells responding to Ag in an adoptive transfer model fail to expand as much in the presence of BrdU as in the absence, and the Ag-specific effectors produced in the presence of BrdU are less able to secrete cytokines upon restimulation in vitro. Polarized populations of Tc1 and Tc2 effectors generated in vitro proliferate less in the presence of BrdU, and the resulting effectors make less cytokines per cell upon restimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male or female C56BL/6 mice were used at 6 wk of age, unless
otherwise specified. In some experiments, OT-1 mice (C57BL/6 mice
bearing the 
- and ß-chains of the TCR specific for the OVA peptide
SIINFEKL presented on Kb) were used
(21).
Antigens
OVA was obtained from Sigma (St. Louis, MO).
Flow cytometry
The following mAbs were used for immunofluorescent staining: Cy-chrome anti-CD8 (PharMingen, San Diego, CA), FITC anti-CD44 (PharMingen; clone IM7), FITC anti-CD4 (GK1.5), PE anti-mouse CD19 (PharMingen, 1D3), and FITC anti-BrdU (Becton Dickinson, San Jose, CA), used as described in the package insert. After staining with the appropriate mAbs, FACS analysis was conducted on a FACScan (Becton Dickinson) by using the CellQuest software. All plots shown were gated on the propidium iodide-negative, live population.
Irradiation
Mice received whole body irradiation in an AECL Gammacell 40 irradiator, at rate of 62 Gy/h, for a total dose of 2 or 5 Gy.
Preparation of CD8 T cells
CD8 T cells from the spleens and lymph nodes of C57BL/6 or OT-1
TCR-transgenic mice were enriched by passing through nylon wool and
treating with anti-CD4 (RL172.4), and anti-heat-stable Ag
(J11D), anti-class II MHC (D3.137, M5114, CA4) mAbs, and
complement. Small resting CD8 T cells were harvested from the bottom
interface of a four-layer Percoll gradient (Sigma). The freshly
isolated T cell populations were 90–95%
CD8+V2+ T cells. CD8 T
cells were cultured in RPMI 1640 (Irvine Scientific, Santa Ana, CA)
supplemented with penicillin, streptomycin, glutamine (PSG), 2-ME,
HEPES, and 10% FCS (HyClone Laboratories, Logan, UT).
Preparation of Tc1 and Tc2 effector cells
For effector generation, CD8 T cells from the OT-1 transgenic
mice (2 x 105 cells/ml) were stimulated
with plate-bound anti-CD3 plus anti-CD28 in the presence of
IL-2 (20 U/ml, supernatant from the X63Ag.IL-2 murine cell line), IL-12
(9.2 U/ml, kindly provided by Dr. Stanley Wolf, Genetics Institute,
Cambridge, MA), and anti-IL-4 mAb (10 µg/ml, 11B11) for Tc1
cultures, and in the presence of IL-2 (20 U/ml), IL-4 (200 U/ml,
X63.Ag.IL-4 supernatant), and anti-IFN-
mAb (XMG1.2, 20 µg/ml)
for Tc2 cultures. On day 4 of culture, effectors were 99%
CD8+V2+.
Adoptive transfer
A total of 107 naive CD8 T cells from OT-1 mice was injected i.v. into C57BL/6 recipients 1 day after irradiation.
Analysis of cytokine production
Enriched CD8 T cells (2 x 105
cells/ml) were stimulated with plate-bound anti-CD3 (at 10
µg/ml). IFN-, IL-4, and IL-5 were measured by specific ELISAs, as
described previously (22).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It had been previously reported (3) that C57BL/6 mice
die eventually if BrdU is administered daily. Ten 6-wk-old female
C57BL/6 mice were divided into two equal groups. One group was placed
on drinking water containing 1 mg/ml BrdU prepared fresh daily, and the
other group served as the non-BrdU control. The mice were weighed
before the start of the experiment and at weekly intervals, for 9 wk.
It can be seen (Fig. 1
) that the control
group gained weight progressively, as expected, while the BrdU group
languished and declined slightly in weight throughout the 9-wk period.
We made no attempt to determine how BrdU administration limited weight
gain. The mice were sacrificed at wk 9, and spleen cell suspensions
were prepared. The number of spleen cells recovered from mice in the
BrdU-exposed group was 6.65 ± 1.1 x
107, approximately half that from the control
group, which was 12.5 ± 2.3 x 107. In
a second experiment, the effect was even more marked with 3.1 x
107 ± 0.75 x 107
cells from the treated mice and 13.25 x 107
± 2 x 107 in the controls.
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Samples of the two spleen cell suspensions were adjusted to standard numbers and stimulated with plate-bound anti-CD3 and anti-CD28 for 64 h, and the thymidine uptake was determined in the last 16 h. There was no significant difference in the thymidine uptake between cells from control mice (4439 ± 1221 cpm) and BrdU-exposed mice (4426 ± 846 cpm).
The effect of continuous exposure to BrdU on the recovery of T and B lymphocyte populations from whole body irradiation
Next, we focused on cells of the immune system involved in homeostasis-driven proliferation. Mice were irradiated with 2 or 5 Gy from a cobalt source, and the number of surviving lymphocytes in the spleens of these and unirradiated mice was counted at day 1. Previous studies have shown that the number of lymphocytes falls rapidly in the period immediately after irradiation and reaches a minimum at 24 h. The fall in the log of the numbers of CD4 T cells, CD8 T cells, and B cells recovered from the spleen after exposure to 2 or 5 Gy was proportional to the irradiation dose, as previously shown, and the relative fall in the numbers of CD4 T cells, CD8 T cells, and B cells was approximately equal, with 1–5% survivors after 5 Gy, as seen by previous investigators (23). Recovery in the number of cells took place progressively in the absence of BrdU after 2 or 5 Gy (data not shown), and at an accelerating rate. The recovery of CD4 cells was the most marked, and there were 2.75x as many CD4 cells at day 28 as before the irradiation. The recovery of CD8 T cells was less dramatic, reaching 2.1x the preirradiation value, and B cells reached only 1.75x during the period of observation.
The recovery of the mice that received 5 Gy was delayed when mice were
placed on drinking water with 1 mg/ml BrdU throughout the entire period
of the recovery (Fig. 2). The total cell
numbers were only 75% of the preirradiation number by day 28 in mice
administered BRDU (Fig. 2A). CD4 cells recovered more
strongly at 140% at day 28, but still only half of that seen in the
controls that were not exposed to BrdU (Fig. 2B). Comparable
results were seen with CD8 T cells (Fig. 2C), and B cells
recovered less well (Fig. 2D). It should be noted that in
the case of the CD4 and CD8 T cells, recovery appeared to be delayed
and the cells did increase in number at the same accelerating rates at
the later time points that were seen earlier in the controls.
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Mice were irradiated with 5 Gy and allowed to recover in the
presence or absence of BrdU, as before. Mice were sacrificed at days 3,
7, 12, and 20, and spleen cell suspensions were prepared and stained
with Abs to CD4, CD8, CD19, CD44, and BrdU. Cells generated during the
recovery in the presence of BrdU showed a progressive increase in the
level of BrdU incorporation (data not shown), and they were not widely
different in each of the three lymphocyte populations by day 20 (Fig. 3). There were no clear-cut
differences between CD44low and
CD44high populations (data not shown). Again, a
significant number of CD4-, CD8-, and CD19-positive cells appeared to
be unlabeled, even after 20 days of BrdU.
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We next examined the effect of BrdU on the Ag-driven proliferative
response of naive cells transferred into irradiated recipients and
immediately challenged with Ag. A total of 107
naive CD8 cells from V2-positive OT-I mice was injected in
each of 10 normal syngeneic C57BL/6 mice that received 5 Gy 1 day
previously. BrdU was administered to five of the mice, and five served
as controls. The mice were challenged by i.p. injection of 50 µg of
OVA in CFA, and the mice were sacrificed at day 7. The numbers of donor
cells were determined by multiplying the total spleen cells count by
the percentage of V2-positive, CD8-positive T cells. It can be seen
that mice on BrdU generated less than half the number of V2 cells
seen in the control by day 7 (Fig. 4).
About 80% of the V2-positive cells were labeled at day 7, and 90%
in a separate cohort run until day 14 (data not shown).
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Naive CD8 T cells from OT-1 mice were transferred into irradiated
recipients in the same experiment as above. CD8 cells were prepared
from the same 7-day spleens, adjusted to standard cell numbers, and
challenged in vitro with 3-day B cell blasts pulsed with the OVA
peptide. Supernatants were harvested at 24 and 48 h and were
assayed by ELISA for IFN-, IL-4, and IL-5 to see whether the
effectors from BrdU-treated mice make the same amount of cytokines per
cell as those from the control mice. It can be seen (Fig. 5) that the cells from BrdU-treated mice
were less effective per comparable number of cells in the secretion of
IFN- upon restimulation and make less that half the amount of
cytokine made by the same number of cells from the control mice. No
IL-4 or IL-5 was secreted by either set of cells (data not shown).
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Previous studies (13) conducted many years ago had
shown that low concentrations of BrdU inhibited the development of Ab
formation in cultures of rabbit spleen cell suspensions from rabbits
that were stimulated with Ag in vivo 2 days before culture. It was
concluded, at the time, that the BrdU inhibited the expansion of the
population of Ab-forming cells. The design of this experiment was
archaic, and a somewhat different protocol was adopted in this study.
CD8-enriched populations from the spleens of OT-1 TCR transgenic mice
were stimulated in vitro with plate-bound anti-CD3 and
anti-CD28 for 4 days in the presence of varying concentrations of
BrdU. The cells were cultured under one of two conditions to
polarize the response with respect to cytokine secretion
(24). One group was stimulated in the presence of IL-12
and anti-IL-4 to produce Tc1 effectors, while the other group was
stimulated with IL-4 and anti-IFN- to generate Tc2 effectors.
The total number of cells generated in the presence of BrdU was reduced
(Fig. 6 ).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the first model, we examined the gross effect of continuous exposure
to BrdU in young female mice 6 wk old at the start of the experiment.
Those fed BrdU failed to gain weight and were also sickly in
appearance. We made no attempt to determine which cells were involved
in the arrest of normal weight gain, but presume multiple mechanisms
may be involved. We did, however, examine whether there were any
detectable departures from the normal in the relative numbers of T and
B lymphocyte populations, and none were discerned. The mice were
sacrificed after 9 wk of exposure, and cells were also stained with Abs
to BrdU, and moderate levels of BrdU incorporation were revealed. We
made no measurement of incorporation at intermediate times in this
experiment, but saw somewhat higher levels of label in the recovery
from irradiation model (68–86% seen in Fig. 3
) and the adoptive
transfer model (80–90%) in which earlier measurements were made. It
should be noted, however, that the three models differ also with
respect to the stimulus to division. In the long-term exposure
experiment, there was no additional stimulus to divide other than the
normal turnover and overall growth of the animals, while in the
recovery from irradiation model, cells were responding to homeostasis
signals, driving the restoration of normal lymphocyte numbers after
irradiation, and in the adoptive transfer model the proliferation of
all the V2 cells was Ag driven. When the cells from the long-term
BrdU-fed mice were stimulated in vitro with anti-CD3 and
anti-CD28, they showed the same rate of thymidine incorporation as
did cells from the control mice.
It is possible that the cells surviving in mice exposed to BrdU for a very long time were selected for those that had lost the salvage pathway and were no longer able to incorporate deoxynucleotides. Our experiments did not allow us to determine whether such selection had occurred or whether cells already labeled with BrdU were able to incorporate thymidine.
In the second model, we looked at the recovery of lymphocyte
populations after whole body irradiation. It is believed that a
substantial part of the recovery comes from the division of lymphocytes
that survived irradiation, while other cells are regenerated from
surviving stem cells. The lymphocyte populations in the normal mice
expanded 
100-fold during the recovery period and must have undergone
a minimum of seven rounds of division, and much more if there was
significant cell death or other heterogeneity in the process. In either
event, the recovery of lymphocyte numbers after irradiation occurs more
slowly in mice exposed to BrdU than do those from control mice, and the
recovery is delayed, as shown in Fig. 2. This is true for CD4-, CD8-,
and CD19-positive lymphocytes, and there are no obvious differences
between the effect on T cells and B cells. It would appear that after
the initial lag in recovery, the expansion of the CD4- and CD8-dividing
cells proceeds at the same rate as in the control, but this was not
seen for B cells. It is possible that this later growth is due to cells
that have reduced levels of BrdU incorporation due to changes in the
relative use of the de novo and salvage pathways for the synthesis of 5
methyl deoxyuridine monophosphate. The fact that a substantial fraction
of the expanded lymphocyte population is still unlabeled with BrdU even
after 20 days exposure to BrdU in the drinking water (shown in Fig. 3)
is compatible with this possibility.
In the third model, we looked at the effect of BrdU on the response of
naive CD8 T cells to Ag. In this study, we used naive cells from OT-1
TCR transgenic mice adoptively transferred into irradiated mice, which
were then challenged with OVA. In this model, the cells that we
followed were not themselves irradiated in contrast to the cells in the
previous model. Again, the expansion was reduced by the incorporation
of BrdU (Fig. 4), showing that the effect was not due to a combination
of irradiation and BrdU, but to BrdU alone. In addition, the labeled
cells were shown to be less able to secrete cytokines when challenged
in vitro, as shown in Fig. 5. Whether this was due to a higher
incidence of cell death on in vitro challenge or to some other effect
was not determined.
Finally, we reexamined the effect of BrdU in vitro. In this study, it was clear that BrdU severely inhibited cell growth and again led to a compromised ability of the Tc1 and Tc2 effectors to make cytokines.
It is clear that cells that have proliferated in the presence of BrdU, under the conditions commonly used to enumerate dividing cells, are less able to undergo further division and are less able to perform effector functions. This is not likely to affect the validity of the interpretations drawn in previous studies, if BrdU is used only to determine the number of cells in cycle over a brief labeling period.
The deleterious effects observed in inhibition of weight gain or homeostatic regeneration after exposure to irradiation were the result of several weeks of exposure to BrdU. In the studies in which we examined the effect of Ag-driven expansion in vivo, however, the time was only 7 days, while the in vitro studies lasted only 4 days. The effect of BrdU in vitro appears to be much more severe than in vivo and may not be relevant to in vivo models.
In studies in which cells are labeled for 7 days or more in vivo, it is likely that the subsequent performance of such cells is compromised, both in their potential for further division and for carrying out their effector function. Alternatively, it may be that the cells that do function in the mouse after prolonged exposure to BrdU represent a subset that either was or became resistant to BrdU as a result of changes in the relative use of the de novo and salvage pathways for nucleotide synthesis. This may or may not affect the validity of the interpretation of such experiments, according to the nature of the conclusions drawn.
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Acknowledgments |
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Footnotes |
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2 Summer intern from Saranac Lake High School, Saranac Lake, NY 12983.
3 Address correspondence and reprint requests to Dr. Richard W. Dutton, Trudeau Institute, Box 59, Saranac Lake, NY 12983.
4 Abbreviation used in this paper: BrdU, 5-bromodeoxyuridine.
Received for publication March 28, 2000. Accepted for publication July 20, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/
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