By
From the Max-Delbrück-Center for Molecular Medicine, 13122 Berlin, Germany
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Abstract |
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The ability to reconstitute interleukin (IL)-4/
mice with bone marrow of IL-4+/+ mice was
investigated. The absence of the IL-4
/
gene in donor or recipient cells did not impair the reconstitution. All immunoglobulin (Ig) subsets occurred at normal serum levels except for IgE
and to some extent IgG1. IgE production did not recover in the reconstituted mice over prolonged time. However, these mice were competent for IgE production, because a single intrasplenic injection of IL-4 restored IgE levels, which then remained constant. Wild-type mice
reconstituted with wild-type bone marrow constantly had IgE serum levels comparable to untreated animals. In wild-type mice reconstituted with IL-4
/
bone marrow, IgE levels
dropped gradually and disappeared by week 12. We make three unrelated but nonetheless important conclusions: (a) (immunoregulation) the tightly regulated IL-4 gene should be expressed constantly in low amounts (and with apparent absence of antigen stimulation) to keep
the normal threshold of IgE; (b) (ontogeny of the immune system) an early unidentified source
of IL-4 must be postulated which is lost in adult mice; and (c) (bone marrow transfer/gene
therapy) under certain circumstances, the genotype of the recipient influences the reconstitution.
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Introduction |
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The production of immunoglobulin (Ig)E is under the
strict control of IL-4 (1). IL-4 is expressed by bone
marrow-derived cells (1). Its expression is tightly regulated
and considered to be essentially after antigen exposure. The
main source of IL-4 is CD4+ T cells of the Th2 subtype,
which paradoxically require IL-4 to be induced (1). It has
been suggested that NK1.1+ T cells initially provide IL-4
(4). However, the relative contribution of Th2 CD4+ T
cells and NK1.1+ T cells for IL-4-regulated immune responses and the time point of production of IL-4 by either
cell population in vivo are far from clear (4). The initial
production and contribution to immune-regulated processes of IL-4 have been studied in vitro and in vivo after
exposure of animals to antigens known to be strong inducers of IL-4. Yet IgE is constantly present in the serum in
considerable amounts (a few hundred nanograms per milliliter) even in the absence of apparent antigen. Its half-life in
serum is relatively short (5-12 h) (11). Therefore, new
IgE should be produced constitutively. Since the half-life
of IL-4 is even shorter (19 min after intravenous administration) (14), one should postulate that IL-4 is also constitutively produced. We prove here that this is indeed the case,
because in normal mice reconstituted with bone marrow
from IL-4/
mice, serum IgE levels declined during donor cell reconstitution.
Vice versa, if IL-4/
mice are reconstituted with IL-4+/+
bone marrow, the expectation would be that the mice do
establish normal IgE levels. This result would meet the
finding of others that reconstitution of gene-deficient mice
(e.g., genes for apolipoprotein E,
-glucuronidase, complement receptor CR2, TNF/lymphotoxin, or GM-CSF/
IL-3/IL-5 common receptor) with wild-type bone marrow restores the normal phenotype (15). Surprisingly, IL-4
/
mice reconstituted with wild-type bone marrow remained unable to produce the threshold IgE level, and thus
are defective for IL-4 production, even though they possess
the ability to secrete IL-4 and IgE. Our explanation is that
the cells initially producing IL-4 cannot be transferred with
adult bone marrow, dating initial IL-4 production back in
ontogeny.
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Materials and Methods |
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Mice and Bone Marrow Transfer.
C57BL/6 (IL-4+/+) mice were obtained from Bomholtgaard Breeding & Research Centre, Ry, Denmark. IL-4-deficient mice (IL-4ELISA for Serum Ig Detection.
At different time points (2, 4, 8, 12, and 16 wk, and in a second experiment, 48 wk after reconstitution), IgE levels were determined comparing serially diluted serum with commercially available Ig standards. The following reagents were used: R35-72 as capture antibody; purified mouse IgE, clone 27-74, as standard; and biotinylated R35-118 as secondary antibody (all from PharMingen Europe, Hamburg, Germany). For detection, avidin-peroxidase followed by 2,2' azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; both from Sigma Chemical Co., Deisenhofen, Germany) was used according to the manufacturer's recommendations, and the color reaction was read at 405 nm with a microplate ELISA reader (MR 5000; Dynatech Deutschland GmbH, Denkendorf, Germany). After long-term reconstitution (5 mo), IgG2a, IgG2b, IgG3, IgM, and IgA were detected in the sera of recipient mice using the Ig isotyping kit according to the manufacturer's recommendations (PharMingen Europe), and IgG1 levels with G1-6.5 as capture antibody, purified mouse IgG1, clone 107.3 as standard, and biotinylated R8-140 as secondary antibody (all reagents from PharMingen Europe). Except for IgG1, the amount of serum Igs in experimental animals was expressed as a percentage of the serum Ig of age-matched control animals.PCR Analysis for Presence of Host/Donor-type Blood Cells.
5 mo after reconstitution, 100 µl peripheral blood from four mice of each group was collected by retroorbital puncture. DNA was prepared using Sorb® Twin Prep according to the manufacturer's recommendations (InViTek GmbH, Berlin, Germany).Peripheral Blood FACS® Analysis of Reconstituted Mice.
5 mo after bone marrow transplantation, 5 × 105 peripheral blood cells of four mice from each group were stained with 0.5 µg mAbs against B220, CD4, CD8, and GR-1 for 30 min on ice. Isotype-matched rat Ig was used as a control (all antibodies from PharMingen Europe). Stained cells were fixed on the Q-Prep workstation with ImmunoPrep reagents, and analyzed using a flow cytometer (EPICS-XL; Coulter Electronics GmbH, Krefeld, Germany).Immunohistochemical Analysis of Cryosections and Bone Marrow Cytospins.
Embedded organs (thymus, Peyers patches) of animals 8 mo after transplant were cut on a Microtom-Kryostat HM500 OM (Microm Laborgeràte GmbH Life Sciences International GmbH, Walldorf, Germany). Cytospins (Shandon, Frankfurt, Germany) of bone marrow cells were air-dried overnight and stored atInfection with Nippostrongylus brasiliensis.
Mice (three C57BL/6 and three reconstituted IL-4+/+Injection of IL-4.
Three C57BL/6 and two reconstituted IL-4+/+ ![]() |
Results |
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Four
groups of bone marrow-reconstituted animals were generated: IL-4+/+ mice reconstituted with IL-4+/+ bone marrow (IL-4+/+ +/+); IL-4
/
mice reconstituted with IL-4+/+
bone marrow (IL-4+/+
/
); IL-4
/
mice reconstituted
with IL-4
/
bone marrow (IL-4
/
/
); and IL-4+/+
mice reconstituted with IL-4
/
bone marrow (IL-4
/
+/+).
Age-matched untransplanted C57BL/6 animals served as
controls. To test the reconstitution of mice by donor cells,
recipients were analyzed by PCR for wild-type and disrupted IL-4 gene in peripheral blood, hematopoietic cell
subset composition, and serum Ig levels 5 mo after bone
marrow transfer.
PCR analysis was done with primers close to the inserted neomycin sequence in the IL-4 gene, thus amplifying a 1,200-bp fragment for the defective and a 95-bp fragment for the wild-type allele. 5 mo after reconstitution, nearly
100% of peripheral blood cells in all four experimental groups
were of the donor type, comparing the IL-4+/+
/
and
IL-4
/
+/+ groups with the IL-4+/+
+/+ and IL-4
/
/
groups (Fig. 1). Only in some mice of the IL-4+/+
/
group were a few recipient cells left. Furthermore, hematological recovery of recipients was shown by similar numbers of B220+, CD4+, CD8+, and GR-1+ cells in the peripheral blood among the different groups (data not
shown). Additionally, mice were analyzed 7 mo after transplant for the presence of NK1.1+ and V
8.1, 8.2 TCR+
cells in the bone marrow and CD1d+ cells in the Peyers
patches and thymus. This was done because NK1.1+ T
cells preferentially using these TCRs occur at a comparably high frequency in bone marrow, are restricted to the CD1d
antigen, and could be important for IL-4 production in the
transplanted mice (4). Regardless of the genotype of the
donor or recipient, NK1.1+/V
8.1, 8.2 TCR+ and CD1d+
cells could easily be detected in the investigated specimen
(data not shown). Additional evidence for reconstitution
with functional donor cells was found by determination of
serum Ig isotypes IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM in
long-term (5 mo) reconstituted mice (Fig. 2). All isotypes
except for IgG1 were found in quite similar amounts in mice
of all experimental groups. In mice of the IL-4
/
/
group, IgG1 was strongly reduced, but in IL-4+/+
/
and
IL-4
/
+/+ animals, a statistically insignificant IgG1 reduction was observed. Whether this is related to IL-4 has
not been investigated. Taking the data together, there is no
reason to assume that the absence of the IL-4 gene in either
donor or recipient cells greatly impaired bone marrow reconstitution.
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We followed
IgE serum levels in the transplanted mice over time, considering that IgE production is a direct reflection of IL-4 expression (1). Normal untransplanted C57BL/6 mice
contained ~100 ng/ml IgE in serum at the age of 6-8 wk,
which increased to an average of 400 ng/ml over a period
of 3-4 mo (Fig. 3). IgE levels in mice of the IL-4+/+ +/+
group at all time points closely resembled that in age-matched C57BL/6 control animals. Mice from the IL-4
/
/
group
began and ended with no detectable IgE in the serum (detection limit 20 ng/ml). Mice in the IL-4
/
+/+ group had
normal serum IgE levels until 4 wk after transplantation, which then dropped and disappeared completely in all animals by week 12 (Fig. 3). Because the only difference between the IL-4+/+
+/+ and IL-4
/
+/+ groups is the
presence or absence of a functional IL-4 gene in donor
cells, we make three conclusions: (a) IL-4 is indeed only produced by bone marrow-derived cells and is mandatory
for IgE production; (b) recipient cells in the IL-4
/
+/+
group seem to produce IL-4 continuously early after irradiation until they are replaced by donor cells at week 12 at
the latest; and (c) the constant IgE levels in the IL-4+/+
+/+
group can best be explained by a constitutive IL-4 production in the mice. All animals were kept under specific
pathogen-free conditions and had no apparent contact
with bacteria, viruses, or parasites, though this does not exclude the possibility that the measured IgE was induced by
exogenous antigens undetectable by us.
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Considering the results with mice from the IL-4/
+/+
group, one would expect that IL-4+/+
/
mice start to
make IgE as a result of the functional IL-4 gene in bone
marrow-derived cells by week 12 at the latest. However, IgE levels in mice of this group remained undetectable in
almost all animals until week 16 and beyond after transplantation. Fig. 3 shows the serum IgE levels from one experiment using eight mice for each experimental group for
up to 16 wk. In a second experiment, 10 animals of the IL-4+/+
/
group were measured for serum IgE levels 48 wk
after transplantation. Again, IgE was undetectable in serum
of most animals (8/10). However, as we have seen above,
IL-4+/+ bone marrow-derived cells are very well able to
produce IgE, if transplanted into IL-4+/+ hosts where in
the early reconstitution phase, IL-4 is probably provided by
the host. We conclude that IL-4+/+ bone marrow-derived
cells do not have the ability to induce threshold IgE levels
in IL-4
/
hosts.
Despite the fact that IL-4+/+
/
mice seemed to be reconstituted in a normal
way (see above) and IL-4+/+ bone marrow-derived cells
were able to initiate IgE expression in an appropriate environment, we could not exclude a hematologic impairment
resulting from secondary effects, e.g., defective IgE expression in mice of the IL-4+/+
/
group resulting from partial graft failure in the IL-4
/
hosts undetectable by us.
Therefore, mice from the IL-4+/+
/
group received a
single intrasplenic injection of 500 U IL-4, and IgE serum
levels were followed over time (Fig. 4). Within 7 d, IgE
was detectable in serum, and at day 14 after IL-4 injection, IgE levels were comparable to normal C57BL/6 mice
treated in the same way. Remarkably, IgE levels remained
constant throughout the experiment (day 42), which can
best be explained by endogenous IL-4 induced through exogenous IL-4, which subsequently regulated a normal
threshold of IgE in serum. Additionally, 6 mo after reconstitution, IL-4+/+
/
animals were challenged with N. brasiliensis larvae, a strong natural IL-4 inducer. This led to
an ~10-fold increase in serum IgE in normal mice after 12 d
(Fig. 5). In IL-4+/+
/
mice, similar IgE levels were
reached. Thus, defective IgE production in mice carrying a
functional IL-4 gene in bone marrow-derived cells can be
overcome by a strong IL-4-inducing stimulus.
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Discussion |
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The question of which mechanism initiates IL-4 expression and subsequent IgE production has been studied
mainly after antigenic stimulation. The stimuli used were
strong inducers of IL-4 production, such as nematodes (1),
polyclonal stimuli like anti-CD3 and anti-IgD (21),
chronic antigen exposure (5), or adjuvant preferentially inducing Th2 responses such as alum (5, 22). Depending on
the experimental system, CD4+ T cells (5), NK1.1+ T cells
(21), or eosinophils (23) were responsible for initial IL-4
production. We addressed this question from a different
point of view, considering that serum IgE levels are present
in young animals without experimental antigenic stimulation. It has been shown that serum IgE is undetectable in
nonimmunized IL-4/
mice and that even nematode infection fails to induce IgE expression in the absence of a
functional IL-4 gene (2, 3). Therefore, we purposely measured serum IgE levels in the bone marrow-reconstituted animals, because it directly reflects IL-4 expression and because IL-4 is difficult to measure in naive animals. We used
low IgE responder C57BL/6 mice, which in a pathogen-poor environment contain 70 ng/ml IgE in serum at the
age of 6-8 wk, increasing to 400 ng/ml at the age of 5-6
mo on the average. Using bone marrow chimeras with IL-4
/
mice, we showed that these IgE levels depend completely on the ability of the mice to express IL-4, and that
their maintenance depends on the continuous presence of a
functional IL-4 gene in bone marrow-derived cells. This
was shown in mice of the IL-4
/
+/+ group, where IgE
serum levels declined rapidly, proportional to replacement
of IL-4-competent by IL-4-incompetent cells. Thus, taking into account sustained IgE serum levels in the IL-4+/+
+/+ mice over a long time period and the comparably short half-lives of both IgE (11) and IL-4 (14), it
becomes apparent that IL-4 should be constitutively expressed even though the animals were not exposed to any
overt antigenic stimulus. It is not known which IL-4-producing cells are responsible for threshold IgE serum levels. IgE expression in mice of the IL-4
/
+/+ group must not
necessarily reflect B cells induced by the recipient's IL-4 to
switch to IgE expression after irradiation. Recently, it has
been shown that the life-time of plasma cells which secrete ovalbumin-specific IgG1 antibodies in the bone marrow
can be as long as 90 d without DNA synthesis (24). The
observation that IgE serum levels in IL-4
/
+/+ mice disappeared within ~8 wk indicates that the half-life of IgE-secreting plasma cells in bone marrow-reconstituted mice
is shorter.
Our most important finding was that IL-4/
mice reconstituted with IL-4+/+ bone marrow failed to establish
normal IgE serum levels. Two control experiments showed
that this defect was caused by the absence in adult bone
marrow of cells from which the initially IL-4-providing
cells derive. In the first, if IL-4+/+ bone marrow was transplanted into IL-4+/+ mice, the animals continued to produce normal serum IgE levels. Therefore, IL-4+/+ bone
marrow-derived cells are able to produce IL-4 and IgE. In the IL-4+/+ recipient mice, IL-4 and concomitantly IgE
might be produced for a certain period after lethal irradiation and bone marrow transfer. This period can be estimated in the IL-4
/
+/+ group of mice, lasting between
6 and 8 wk after transplantation. During replacement of recipient by donor cells, donor bone marrow-derived cells
are educated by the recipient's IL-4 to produce IL-4 themselves and subsequently to maintain threshold serum IgE
levels. In the second control experiment, in mice of the IL-4+/+
/
group, defective IgE production could be rescued by a single injection of IL-4, ruling out partial graft
failure as the reason for the defect. Because these mice continued to produce IgE throughout the experiment (42 d),
the exogenous IL-4 should have induced IL-4 expression
in donor-derived cells. Once initiated, IL-4 expression acts
in an autostimulatory fashion. Therefore, our results demonstrate that bone marrow cells of adult animals are unable
to give rise to cells from which IL-4 first originates, and
thus are functionally not pluripotent. This raises the question of when and by which cells initial IL-4 is provided.
We offer two not mutually exclusive explanations. First,
IL-4 is expressed during early development. This is supported by the observation that IL-4 mRNA as well as IL-4
receptor mRNA could be detected in 10.5- and 12.5-d-old
embryos (25). In extracts of some tissues (e.g., liver) of
12.5-d-old fetuses, IL-4 protein could also be detected.
Furthermore, hematopoietic progenitor cells differentiated
in vitro from embryonic stem cells express IL-4 and IL-4
receptor mRNA (26). Second, IL-4 is provided by the
mother. This alternative is supported by the observation that pregnant mice (27) and women (30) produce increased amounts of IL-4, and IL-4 mRNA is detected in
uterine decidual plus placental tissue at days 8.5-12.5 of
gestation (25). We currently analyze these two possibilities.
However, regardless of whether induction of the IL-4-regulated network results from "maternal imprinting" or from
the expression of IL-4 by embryonic cells, the fetus is exposed to IL-4, and the initial source of IL-4 is not necessary any more in the adult because the system keeps itself alive.
IgE production in mice of the IL-4+/+
/
group can
also be rescued by challenge with larvae of N. brasiliensis,
further supporting that normal hematopoietic reconstitution had occurred in the animals. Even though it has been
shown that the IL-4 requirement for IgE production can be
bypassed by some antigens such as Plasmodium chabaudi or a
mouse retrovirus (31, 32), IgE production in the bone marrow-reconstituted mice after nematode exposure probably occurs in an IL-4-dependent way, because IL-4
/
mice
fail to produce detectable amounts of IgE after nematode challenge (2, 3). However, we think it is unlikely that strong IL-4 inducers such as nematodes are the driving
force for establishing the IL-4-regulated immune system in
early life.
For a variety of genetic diseases, bone marrow reconstitution with cells genetically modified to carry a functional copy of the defective gene is considered to be a therapeutic modality. Wild-type bone marrow transplantation into gene-deficient mice has been shown in several models to restore a normal phenotype (15). Our results differ in this regard and raise the caution that for certain genes, e.g., those involved in regulation of hematopoiesis, gene therapy approaches encounter problems not anticipated previously.
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Footnotes |
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Address correspondence to Thomas Blankenstein, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13122 Berlin, Germany. Phone: 49-30-9406-2816; Fax: 49-30-9406-2453; E-mail: tblanke{at}mdc-berlin.de
Received for publication 12 December 1997 and in revised form 20 February 1998.
We thank Dr. Harder from Bayer AG, Mohnheim, Germany for nematode larvae. IL-4/
mice were
kindly provided by W. Müller and K. Rajewsky, Institute for Genetics, University of Cologne, Cologne,
Germany. We also thank C. Westen and M. Rösch for excellent technical assistance.
This study was supported by the Deutsche Forschungsgemeinschaft, Projekt Bl 288/3-2.
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