Download Induced disruption of the transforming growth factor

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IMMUNOBIOLOGY
Induced disruption of the transforming growth factor beta type II receptor gene
in mice causes a lethal inflammatory disorder that is transplantable
Per Levéen, Jonas Larsson, Mats Ehinger, Corrado M. Cilio, Martin Sundler, Lottie Jansson Sjöstrand,
Rikard Holmdahl, and Stefan Karlsson
Recent studies in mouse models deficient in transforming growth factor beta
(TGF-␤) signaling have documented
TGF-␤ as one of the major regulators of
immune function. TGF-␤1–null animals
demonstrated massive autoimmune inflammation affecting multiple organs, but
attempts to transfer the phenotype to
normal animals by bone marrow transplantation only resulted in minor inflammatory lesions. We wanted to ask whether
a lethal inflammatory phenotype would
develop following transplantation of bone
marrow deficient for the TGF-␤ type II
receptor (T␤RII) gene to normal recipient
animals. The T␤RII-null mutation would
generate a cell autonomous phenotype
that cannot be reverted by the influence
of endocrine or paracrine TGF-␤ derived
from the recipient animal. We have generated conditional knockout mice in which
the T␤RII gene is disrupted upon induction with interferon-␣␤ or polyI:polyC. We
show that induction of T␤RII gene disruption in these mice by polyI:polyC results
in a lethal inflammatory disease. Importantly, bone marrow from conditional
knockout mice transferred to normal recipent mice caused a similar lethal inflammation, regardless of whether induction
of TGF-␤ receptor deficiency occurred in
donor animals before, or in recipient animals after transplantation. These results
show that TGF-␤ signaling deficiency within
cells of hematopoietic origin is sufficient to
cause a lethal inflammatory disorder in mice.
This animal model provides an important
tool to further clarify the pathogenic mechanisms in animals deficient for TGF-␤ signaling and the importance of TGF-␤ to regulate
immune functions. (Blood. 2002;100:
560-568)
© 2002 by The American Society of Hematology
Introduction
Transforming growth factor beta (TGF-␤) is recognized as a highly
pleiotropic family of growth factors involved in the regulation of
numerous physiologic processes including development, hematopoiesis, wound healing, and immune response. The 3 isoforms of
this growth factor that have been identified in mammals (TGF-␤1,
-␤2, and -␤3) are encoded by distinct genetic loci and share a high
level of homology. They act on virtually all cell types and mediate
similar cellular responses in vitro, like regulation of proliferation,
differentiation, apoptosis, and extracellular matrix synthesis.1-3 In
vivo, however, they demonstrate partly unique sets of physiologic
functions due to different tissue distribution and temporal expression during development.4-6 The TGF-␤ isoforms exert all their
cellular functions through formation of a tetrameric complex with
the 2 cell surface receptors T␤RI and T␤RII. Complex formation
leads to phosphorylation of T␤RI on serine/threonine residues and
propagation of the intracellular signal to the nucleus through a
chain of phosphorylations of Smads, which regulate gene expression in cooperation with other transcription factors.7
A growing body of evidence suggests TGF-␤ to be one of the
major regulators of immune function, acting both by suppressive
and stimulatory mechanisms on leukocytes to achieve a balanced
immune response.8-10 The suppressive mode of action has been
highlighted by studies demonstrating inhibition of interleukin 1
(IL-1)–, IL-2–, and IL-7–dependent thymocyte proliferation by
TGF-␤11-16 through autocrine and paracrine mechanisms,13,17,18
whereas immunostimulatory functions were suggested by the
capacity of TGF-␤ to induce cytokine expression in T cells and to
promote effector expansion by inhibition of apoptosis.19-21 Moreover, the influence of TGF-␤ on the development and function of
other cells of the immune system, such as B cells, macrophages,
and dendritic cells, has been reported.10 Striking evidence for the
importance of TGF-␤ in immune regulation was reported from
studies on TGF-␤–null animals that demonstrated postnatal lethality and massive multifocal inflammation affecting multiple organs.9,22,23 The uncontrolled inflammatory reaction has been ascribed to autoimmune mechanisms including autoantibodies and
autoreactive T cells.24-27 However, attempts to develop the phenotype by transplanting TGF-␤1–null bone marrow to healthy
recipient mice unexpectedly resulted in minute inflammatory signs
that did not cause clinical symptoms.25 This raised the possibility
that the presence of immune cells deficient for TGF-␤1 is not
sufficient to cause the inflammatory phenotype. Alternatively,
TGF-␤1–deficient donor cells may be responsive to endocrine or
paracrine sources of TGF-␤1 produced by recipient tissues.
Further evidence strongly suggests a role of TGF-␤ in the
regulation of inflammation using dominant-negative transgenic
From the Departments of Molecular Medicine and Gene Therapy, Pathology,
and Medical Inflammation Research, Lund University, Sweden; and the
Department of Endocrinology and Paediatrics, Malmö University Hospital,
Lund University, Malmö, Sweden.
Foundation, New York, NY.
Submitted June 22, 2001; accepted March 5, 2002.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by Cancerfonden, Stockholm, Sweden; Astra Draco, Lund, Sweden;
The Foundation for Strategic Research, Stockholm, Sweden; and the Crafoord
Foundation, Lund, Sweden. C.M.C. is supported by the Juvenile Diabetes
560
Reprints: Stefan Karlsson, Molecular Medicine and Gene Therapy, Lund
University, Sölvegatan 17, S-22184, BMC A12, Lund, Sweden; e-mail:
[email protected].
© 2002 by The American Society of Hematology
BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
mouse models for T-cell–specific TGF-␤ type II receptor (T␤RII)
deficiency: abrogation of TGF-␤ signaling in CD4- and CD8expressing cells generated a phenotype reminiscent of the inflammation of TGF-␤1–null animals27 whereas CD2-specific T␤RII
deficiency resulted in a lymphoproliferative disorder involving
peripheral expansion of CD8⫹ populations, but without an inflammatory component.28 However, neither of these models produced a
phenotype as dramatic as detected in the TGF-␤1–null mice,
indicating a more general requirement of TGF-␤ in other cells of
the immune system, or alternatively, that the transgenic approaches
do not generate complete lack of TGF-␤ signaling.
Here, we asked whether TGF-␤ deficiency within bone marrow
cells is sufficient to generate a fully developed and lethal inflammatory phenotype. For this purpose a conditional knockout model was
developed, using the Cre/lox system,29-31 designed to disrupt the
T␤RII in adult animals upon induction with interferon-␣/␤ or
polyI:polyC, which releases interferon. This approach has several
unique and important features, rendering it suitable to further
evaluate the role of TGF-␤ in inflammation. These include the
absence of embryonic and early postnatal lethality as was observed
in the TGF-␤1– and T␤RII-null animals22,23,32 and a total loss of
TGF-␤ signaling. In addition, the mutation causes a cellautonomous TGF-␤ signaling deficiency (ie, signaling cannot be
restored in hematopoietic cells by endocrine or paracrine mechanisms when tissues are transplanted to normal recipient mice). A
transplantation approach makes it possible to restrict the primary
phenotype to hematopoietic cells as well as to analyze the role of
TGF-␤ in immune cells before their homeostasis is perturbed by
the onset of inflammatory disease. Specifically, the approach could
be used to identify the cell lineages and subpopulations of the
immune system that are dependent on TGF-␤ to exert a proper
inflammatory response. Furthermore, mechanistic studies on these
cells should contribute to a deeper understanding of inflammation
at the cellular and molecular levels. We show in this study that
induction of conditional T␤RII knockout mice by polyI:polyC
causes a lethal inflammatory disease affecting multiple organs. In
addition, our results demonstrate that TGF-␤ signaling deficiency
within cells of hematopoietic origin is sufficient to cause a lethal
inflammatory disorder.
Materials and methods
Targeting of the T␤RII genomic locus
A cDNA probe encompassing 580 base pair (bp) of the sequence encoding
the extracellular domain of mouse T␤RII was used to screen a 129/Sv
Lambda FIX genomic phage library. This cDNA probe was amplified from
a mouse kidney cDNA library (Clontech, Palo Alto, CA) using the
oligonucleotides 5⬘-GGTCTATGACGAGCGACGGG-3⬘ and 5⬘-TGACCAACAACAGG TCGGGA-3⬘. One 18.9-kbp genomic T␤RII clone containing exon 4 and 5 was obtained from the Lambda FIX library and used to
build the targeting construct. Briefly, a 1.3-kbp SalI-XbaI fragment
containing the neo gene, controlled by the HSV-tk promoter and flanked by
loxP sequences (gift from H. Gu, National Institutes of Health, Bethesda,
MD), was inserted into the BamHI-site immediately upstream of exon 4 of a
5.8-kbp HindIII-KpnI 5⬘ subclone. An EcoRV-SacI single loxP fragment
(H. Gu) was blunt end ligated into the KpnI site at the 3⬘ end of the same
subclone. For negative selection, a 3.0-kbp BamHI-SalI fragment containing the herpes simplex virus thymidine kinase gene (HSV-tk) (R. Jaenisch,
Massachusetts Institute of Technology, Boston) was inserted into the 3⬘ end
of a 3⬘ subclone that included exon 5. The construct was then assembled by
cleaving out the 3⬘ subclone with BamHI and ligating it into the BamHI site
at the 3⬘ end of the 5⬘ subclone. The construct was linearized using NotI and
INDUCED T␤RII GENE DISRUPTION
561
electroporated into the embryonic stem (ES) cell line RI which was
subsequently grown under selection (300 ␮g/mL neomycin G418 and 4 ␮M
gancyclovir) using standard culture conditions for ES cells.33 Surviving
colonies were screened for homologous recombination by polymerase
chain reaction (PCR) using the 5⬘ external homology primer P1: 5⬘TTCCTTCCGGCCTGAGTTGTTATTG-3⬘ and the neo primer P2: 5⬘TTGGCTGCAGGTC GCTTCGGTGGT-3⬘. Retention of the single loxP
site in homologous recombinants was confirmed by PCR using the loxP
primer P7: 5⬘-ATTAAGGGTTATTGAATATGATCGG-3⬘ and the downstream exon 4 primer P6: 5⬘-CGACTTGACCTGTTGCCTGT-3⬘.
In order to excise the neo gene from the T␤RII locus, the Cre
recombinase expression plasmid pIC-Cre was transiently transfected into
correctly targeted clones. G418-sensitive clones, identified by replica
plating onto 96-well dishes, were screened for the combined absence of neo
and presence of exon 4 (“floxed” clones) using the PCR primer pairs P1/P2
and P6/P7, respectively. The same strategy identified clones lacking both
exon 4 and neo (knockout clones). Cells from each clone were separately
injected into C57BL/6 blastocysts to generate chimeric male mice that were
mated with C57BL/6 females to obtain germline transmission of the
mutated alleles. Germline offsprings of the “floxed” genotype were
routinely screened using 2 primers flanking the 5⬘ loxP site (P3: 5⬘TATGGACTGGCTGCTTTTGTATTC-3⬘ and P4: 5⬘-TGGGGATAGAGGTAGAAAGACATA-3⬘) whereas animals containing the null allele were
identified using the primers P3 and P5 (P5: 5⬘-TATTGGGTGTGGTTGT
GGACTTTA-3⬘). “Floxed” mice were mated with transgenic mice carrying
the Cre-recombinase gene under control of the interferon inducible
promoter Mx1 to generate animals of the T␤RII flox/flox ⫻ Mx1-Cre
genotype. The presence of Mx1-Cre was verified using the following Cre
primers: Cre forward: 5⬘-ACGAGTGATGAGGTTCGCAA-3⬘ and Cre
reverse: 5⬘-AGCGTTTTCGTTCTGCCAAT-3⬘.
Bone marrow transfer
Donor mice were killed in CO2 and bone marrow was flushed from femur
and tibia using a 27-gauge needle and phosphate buffered saline (PBS) plus
2% fetal calf serum. The cell suspension was filtered trough a 70 ␮M cell
strainer (Falcon, BD, Franklin Lakes, NJ) and 2 ⫻ 106 cells were injected in
a volume of 500 ␮L into the tail vein of lethally irradiated (950 cGy)
recipient mice.
Colony assays
For granulocyte macrophage–colony-forming units (CFU-GMs), cells were
plated in methylcellulose cultures containing IL-3 (10 ng/mL), IL-6 (10
ng/mL), and stem cell factor (SCF) (50 ng/mL) (Myelocult 3534; Stem Cell
Technologies, Vancouver, BC, Canada). For erythroid–blast-forming units
(BFU-Es) and megakaryocyte–colony-forming units (CFU-Megs), cells
were grown under serum-free conditions (Myleocult 3236; Stem Cell
Technologies) in the presence of SCF (50 ng/mL), erythropoitin (4 U/mL),
and thrombopoietin (50 ng/mL). TGF-␤ (10 ng/mL) was added to some of
the cultures to confirm an effective block in TGF-␤ signaling. After 7 days,
colonies were scored under the microscope. Bone marrow cells for clonal
PCR were grown under the same conditions as CFU-GMs. Individual
colonies were isolated after 10 days in culture and analyzed using a PCR
strategy including the primers P3, P4, and P5.
Histologic analysis
Organs were processed for routine histology by fixation in PBS buffered 4%
paraformaldehyde followed by paraffin embedding and sectioning. Sections
were stained with Erlich eosin for microscopic examination.
Flow cytometry
Fluorescein isothiocyanate (FITC)– and phycoerythrin (PE)–conjugated
monoclonal antibodies (mAbs) against CD4, CD8, CD25, CD62, CD69,
T-cell receptor (TCR)-␣␤, and B220 (Pharmingen, BD, San Diego, CA),
diluted in PBS/1% fetal calf serum, were used to stain cells from the spleen
and lymph nodes. Cell suspensions were prepared by grinding and flushing
organs through a 70-␮m nylon cell strainer (Falcon). For selection of donor
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562
LEVÉEN et al
BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
populations, each staining contained biotin/streptavidin allophycocyanin
(APC)–conjugated mAbs against Ly5.2. For stainings, 106 cells were placed
in a 96-well plate and incubated with blocking Abs (mouse IgG 1␬, 10
␮g/mL; Sigma) for 10 minutes at room temperature. After washing with
PBS/1% fetal calf serum, antibodies (concentrations determined by titration) were incubated with the cells on ice and protected from light for 20
minutes. Washing was followed by incubation on ice for 20 minutes with a
secondary reagent (streptavidin-APC, 2 ␮g/mL; Pharmingen) to conjugate
biotin-Ly5.2 Abs with APCs. Stained cells were washed and analyzed by a
4-color FACS calibur (Becton Dickinson, San Jose, CA).
Enzyme-linked immunosorbant assay
For determination of anti–histone/double-stranded DNA (dsDNA) autoantibodies, plates (Costar, Corning, NY) were coated with 10 ␮g/mL histone
(unfractionated whole histone type II-A from calf thymus; Sigma, St Louis,
MO) and thereafter with 50 ␮g/mL dsDNA prepared from calf thymus
(Sigma). The plates were blocked overnight with 2% fetal calf serum in
PBS, washed, and then incubated 2 hours with 10⫻ serial dilutions of
mouse sera starting with a dilution of 1/50. After washing, the plates were
incubated for 1 hour with the secondary antibody, peroxidase-conjugated
goat anti–mouse IgG antibody (Jackson Immuno Research Laboratories,
West Grove, PA), washed, incubated with ABTS (2,2⬘-azino-di-[3ethylbenzthiazoline sulfonate (6)], diammonium salt; Roche, Basel, Switzerland), and read in a Titertek multiscan spectrophotometer at 405 nm. All
tests were carried out in duplicate and the standard deviations did not
exceed 10%. The titer values were converted to units per milliliter, using a
seriediluted positive control of pooled serum from 6-month-old MRL-lpr
mice. One unit corresponds to the titer of the positive sera divided by 4.
Immunohistochemistry
Paraffin-embedded sections for analysis of lymphocytes and macrophages were
processed according to standard protocols34 before staining with primary
antibodies and peroxidase/3-3⬘diaminobenzidine. Primary antibodies: rat anti–
mouse CD3 and CD45R (both from Cederlane, Hornby, Canada). As secondary
antibodies, biotinylated goat anti-rat was used. For determination of IgG-immune
complexes in the kidneys, deparaffinated sections were blocked with 1.5%
bovine serum albumin (BSA), washed, and blocked for 20 minutes for
endogenous peroxidase using 2% hydroperoxidase in methanol. After washing,
the sections were incubated for 30 minutes with biotinylated rabbit anti–mouse
IgG antibody (Dako, Glostrup, Denmark) diluted 1/100, washed, and incubated
for 30 minutes with peroxidase conjugated complex (ExtrAvidin; Sigma) diluted
1/2000. The stainings were developed for 7 minutes using the DAB-kit (Vector
Laboratories, Burlingame, CA).
Results
Generation of ES cells and mouse strains carrying “floxed”
or null T␤RII alleles
T␤RII exon 4 was selected as the target for mutagenesis because
this exon encodes the majority of the kinase and the entire
transmembrane domain of the receptor, both of which are essential
for receptor function.35 We used the Cre/loxP gene targeting
approach to achieve a “flox” mutation of T␤RII (ie, exon 4 is
flanked by loxP) in ES cells that would not interfere with gene
function until recombination with Cre-recombinase. To generate
mice with “floxed” T␤RII alleles, ES cells were subjected to
homologous recombination with a gene construct containing T␤RII
exon 4 flanked by loxP sequences. The neo cassette was subsequently excised by transient transfection with a Cre-expression
plasmid. In this way “floxed” ES clones were generated and, in
addition, the procedure resulted in clones containing one T␤RIInull allele (T␤RII⫹/⫺) (ie, clones lacking both neo and exon 4), to
be used for functional verification of gene targeting. “Floxed” and
Figure 1. Conditional targeting of the T␤RII gene. (A) The wild-type T␤RII locus
was targeted by homologous recombination with a gene construct containing
insertions of the neo gene flanked by loxP sites (arrowheads) upstream of exon 4, a
single loxP site downstream of exon 4, and the HSV-tk gene at the 3⬘ flank of the
construct. Homologous recombinants were identified using the PCR primers P1
(external) and P2 whereas the primers P6 and P7 verified retention of the single loxP
site. Transient Cre-expression in targeted ES cells generated clones with a “floxed” or
a null allele, respectively. PCR for screening of “floxed” and null mutants following
Cre/lox-recombination in ES cells was done by using the P1 and P2 primers to verify
excision of neo and the P6 and P7 primers to determine the presence or absence of
exon 4. The primer pairs P3/P4 and P3/P5 were used to screen for germline
transmission of the “floxed” and the null alleles, respectively. (B) PCR screening of
neo-resistant clones for homologous recombination using P1 and P2 to amplify a
3.6-kbp recombinant sequence. M indicates 1 kbp molecular weight marker. (C)
Germline transmission of the “floxed” allele in samples 1 and 2 as shown by the
presence of a 575-bp PCR product amplified from tail DNA by the P3 and P4 primers.
R1 indicates T␤RII⫹/flox ES-cell DNA; R2, T␤RII⫹/⫺ ES-cell DNA; R3, wild-type DNA.
T␤RII-null mouse strains, one of each, were created from successfully mutated ES cells by injecting them into C57BL/6 blastocysts.
Homologous recombination in ES cells and germline transmission
of the mutated alleles in mice were analyzed by PCR (Figure 1) and
verified by Southern blot analysis (data not shown).
In order to functionally confirm successful targeting of exon 4,
T␤RII⫹/⫺ mice were mated. Genotyping of the litters showed that
T␤RII⫹/⫹ and T␤RII⫹/⫺ pups were born in the expected 1:2 ratio,
whereas no pups with the T␤RII⫺/⫺ (T␤RII null) genotype were
found, indicative of an embryonic lethal T␤RII⫺/⫺ phenotype.
Furthermore, microscopic analysis of embryos at day 9.5 to day
10.5 postcoitus demonstrated a phenotype identical to the one
reported previously32,36 (ie, absence of yolk sac vasculogenesis and
erythropoiesis as well as reduced embryonic size and enlargement
of the pericardium; data not shown). Mice homozygous for the
“floxed” allele showed, in contrast, no signs of disease, were born
at the expected 1:3 ratio from heterozygous matings, and reproduced normally. These “floxed” mice were crossed with a transgenic mouse strain carrying the Cre-recombinase gene under the
control of the inducible Mx1 promoter, resulting in animals with
the T␤RII flox/flox ⫻ Mx1-Cre genotype. These animals express
the Cre transgene only upon induction either with interferon-␣␤ or
the interferon inducer polyI:polyC.37
Bone marrow cells from T␤RIIⴚ/ⴚ mice are unresponsive
to TGF-␤
The efficiency whereby Cre/lox recombination occurs in various
tissues upon induction was tested by semiquantitative PCR analysis
of organs from T␤RII ⫹/flox ⫻ Mx1-Cre mice treated 3 times at
2-day intervals with polyI:polyC (250 ␮g). Bone marrow from
these mice was, in addition, tested separately by seeding into
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
semisolid methylcellulose culture medium, followed by PCR
analysis of individual colonies 10 days later. The results were in
general agreement with a previous study37 and showed efficient
Cre/lox recombination (ie, percentage floxed alleles transformed to
null alleles) in the liver (95% efficiency) and freshly isolated,
unfractionated bone marrow (100%). The latter result was strongly
supported by the fact that 60 out of 60 bone marrow–derived
hematopoietic colonies were positive for the T␤RII-null allele,
while negative for the floxed allele (data not shown). These results
suggest that bone marrow cells are ideal targets for inducible
gene disruption.
Inhibitory functions for TGF-␤ in hematopoiesis have been
implicated by numerous in vitro studies showing a suppressive
mode of action on proliferation, mainly at the stem cell and
progenitor levels.38 However, hematopoietic homeostasis was not
affected in symptomatic animals of our transplantation model (see
below), as measured by bone marrow cellularity or red and white
blood cell counts in circulating blood. We further tested the
importance of TGF-␤ signaling in hematopoiesis by colony assays
on bone marrow cells from induced T␤RII flox/flox ⫻ Mx1-Cre
animals. Cells were plated on semisolid culture medium with or
without TGF-␤. However, no significant differences to controls
(bone marrow from induced T␤RII flox/⫹ ⫻ Mx1-Cre animals) in
terms of numbers and size of total colonies, proportions of
erythroid (BFU-E), megakaryocytic (CFU-Meg), and myeloid
(CFU-GM) lineages were observed in cultures without TGF-␤
(data not shown). Addition of TGF-␤ resulted, as expected, in
significantly reduced CFU-GM colony numbers from control bone
marrow (mean colony numbers from untreated bone marrow,
199 ⫾ 56; from TGF-␤–treated, 115 ⫾ 34; P ⬍ .05; n ⫽ 3 for each
group), whereas colonies from induced T␤RII flox/flox ⫻ Mx1Cre bone marrow remained unresponsive (mean colony numbers
from untreated bone marrow, 162 ⫾ 30; from TGF-␤–treated,
173 ⫾ 12; P ⫽ .31; n ⫽ 3). These results further strengthen the
evidence that TGF-␤ signaling is functionally abolished in this
animal model.
Induced T␤RII disruption causes multifocal inflammation
in multiple organs
We tested the pathologic consequences of induced T␤RII gene
disruption by treating 4 T␤RII flox/flox ⫻ Mx1-Cre and 3
T␤RII⫹/flox ⫻ Mx1-Cre mice with polyI:polyC at an age of 8
weeks. The mice were injected 3 times intraperitoneally with
polyI:polyC (250 ␮g) at 2-day intervals and all 4 T␤RII flox/flox ⫻
Mx1-Cre mice developed a wasting syndrome that was fatal by 8 to
10 weeks after induction, whereas the T␤RII⫹/flox ⫻ Mx1-Cre
control mice did not show any signs of disease. The symptomatic
mice were killed for histopathologic examination at the terminal
stage of disease. The clinical picture included dramatic weight loss,
immobility, unsteady movements, and signs of inflammation of the
eyes. Histopathologic examination of organs (liver, kidney, spleen,
stomach, small intestine, colon, esophagus, heart, lung, and thymus) demonstrated massive focal infiltration of inflammatory cells,
predominantly consisting of lymphocytes and granulocytes, in the
stomach (4/4), pancreas (3/3), and liver (2/4), accompanied by
tissue destruction of variable severity. One animal showed myocarditis, characterized by foci of lymphocytes and plasma cells in the
myocardium. This animal was also affected by extensive inflammation of renal glomeruli and interstitium and demonstrated esophagitis with lymphocytes and granulocytes invading lamina propria.
The thymus of all induced T␤RII flox/flox ⫻ Mx1-Cre mice was
reduced in size (see results from transplanted mice below) in
INDUCED T␤RII GENE DISRUPTION
563
agreement with previous observations of TGF-␤-signaling–
deficient mice.27,39 In conclusion, T␤RII flox/flox ⫻ Mx1-Cre mice
develop a severe inflammatory disorder affecting multiple organs
following induction with polyI:polyC.
The phenotype is transferred by bone marrow transplantation
Due to the multifunctional nature of TGF-␤ in a wide variety of
organs and tissues, we wanted to restrict the mutagenesis to cells of
hematopoietic origin. This was achieved by induction of T␤RII
flox/flox ⫻ Mx1-Cre mice followed by transfer of their bone marrow
(T␤RII⫺/⫺) to normal and lethally irradiated (950 cGy) C57BL/6
recipient mice. In the first series of transplantations, 2 T␤RII flox/flox ⫻
Mx1-Cre mice were induced at 7 weeks of age to serve as donors of
T␤RII⫺/⫺ bone marrow for a total of 10 C57BL/6 recipients aged 12
weeks (5 recipients for each donor). Another 5 C57BL/6 animals
received T␤RII⫹/⫺ control bone marrow from 2 induced T␤RII⫹/
flox ⫻ Mx1-Cre mice (3 and 2 recipients, respectively, for each donor).
All 10 recipients of T␤RII⫺/⫺ marrow demonstrated weight loss,
starting approximately at 3 to 4 weeks after transplantation, that
progressed to a lethal condition 3 to 6 weeks later. The 5 control mice
appeared healthy until they were killed at 7 to 15 weeks after
transplantation (Figure 2). Another set of transplantations, where
induction was done in recipient mice after transplantation with
TGF-␤-receptor–deficient bone marrow, resulted in the same lethal
phenotype (data not shown) showing that the transplanted bone
marrow cells aquired their pathogenic properties as the consequence of intrinsic genetic failure and not through interaction with
other T␤RII-deficient tissues.
The symptomatic profile and histopathology of T␤RII⫺/⫺ bone
marrow recipients was reminiscent of the phenotype of induced
T␤RII flox/flox ⫻ Mx1-Cre mice, although the distribution of
inflammatory loci among organs differed somewhat. The most
frequent inflammatory lesions in transplanted mice were found in
the stomach, pancreas, and lung, whereas the liver, small intestine,
and colon were affected at lower frequencies and the heart and
esophagus were normal (Table 1). In general, the histopathology of
the stomach involved destruction of both the squamous and the
glandular epithelium with inflammation primarily affecting the
mucosa and to a lesser extent, the submucosa (Figure 3). Morphologic and immunohistochemical analyses revealed that the inflammatory cells included a mixture of B and T cells (Figure 4),
Figure 2. Clinical progression of the inflammatory disease. Animal weight
change following transplantation with T␤RII⫺/⫺ (filled line) and T␤RII⫹/⫺ control bone
marrow (dotted line) is shown. The initial numbers of transplanted animals were 10
(T␤RII⫺/⫺ donor bone marrow) and 5 (T␤RII⫹/⫺ donor bone marrow). The mean
weights of recipients of T␤RII⫺/⫺ bone marrow and T␤RII⫹/⫺ bone marrow at
transplantation were 19.8 ⫾ 2.6 g and 19.8 ⫾ 1.0 g, respectively. Each point
represents the average weight change compared to the weight at transplantation.
Animal deaths occurred among T␤RII⫺/⫺ bone marrow recipients from 6 to 9 weeks
after transplantation. Control animals remained healthy until they were killed for
histologic examination by 7 to 15 weeks after transplantation.
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
LEVÉEN et al
T␤RIIⴚ/ⴚ bone marrow recipients show an activated
T-cell phenotype
Table 1. Histopathologic lesions in transplanted mice
3.5 weeks AT
Organ
T␤RII
⫺/⫺
6-9 weeks AT
S
T␤RII
⫺/⫺
S
T␤RII
⫹/⫺
Stomach
1 (6)
⫹
8 (8)
⫹⫹⫹⫹
0 (4)
Pancreas
1 (6)
⫹
8 (8)
⫹⫹⫹⫹
0 (5)
Spleen
0 (7)
Lung
1 (8)
⫹
8 (8)
⫹⫹⫹
0 (4)
7 (7)
⫹⫹⫹
0 (4)
ND
3 (4)
⫹⫹⫹⫹
0 (4)
Liver
0 (8)
6 (9)
⫹⫹
0 (5)
Colon
0 (8)
6 (9)
⫹⫹
0 (5)
Small intestines
0 (8)
2 (9)
⫹⫹
0 (5)
Kidney
0 (8)
1 (9)
⫹⫹
Heart
0 (8)
0 (9)
0 (4)
Esophagus
0 (7)
0 (6)
0 (3)
Thymus
0 (5)
The number of mice with lesions in the specified organs are shown and the total
number of animals examined are indicated within parentheses. The genotype refers
to the donor bone marrow following polyI:polyC induction. S denotes the most
frequent subjective estimate of severity, where ⫹ indicates mild, ⫹⫹ indicates
moderate, ⫹⫹⫹ indicates pronounced, and ⫹⫹⫹⫹ indicates very severe. AT
indicates after transplantation.
macrophages, and polymorphonucleated granulocytes. In 4 of 9
animals ulcers of the stomach were found, and in 6 of 9 animals
colitis was observed as indicated by mucosal invasion of inflammatory cells (mainly B and T cells), with degeneration of the
epithelium and focal ulcers. The small intestine showed only minor
lymphocytic infiltration in the mucosa of 2 of 9 animals. A
consistent finding was a severe inflammation in the pancreas
involving large focal infiltrates of inflammatory cells, mainly B and
T cells, with destruction of the glandular parenchyma. Occasionally, extension of lymphocytes to the islands of Langerhans
(insulitis) was observed. Equally consistent were inflammatory
reactions in the lung characterized by peri- and intrabronchial
accumulation of B and T cells, macrophages, and foci of plasma
cells. All lungs from T␤RII⫺/⫺ bone marrow recipients demonstrated perivascular inflammation consisting of B and T cells,
plasma cells, and some granulocytes. Focally, invasion of the
vascular wall was observed. In 2 animals, increased densities of
lymphocytes were found in the alveoli. Sections of the liver from 6
of 9 animals showed moderate to severe portal and perivenular
infiltrates of B and T cells with focal spread to the liver parenchyma. Furthermore, polymorphonucleated granulocytes were
sometimes found in the epithelium of bile ducts, suggestive of
cholangitis. The thymus was reduced in size (thymus weight/body
weight was 2.38 ⫾ 0.81 ⫻ 10⫺3 and 3.02 ⫾ 0.73 ⫻ 10⫺3 for ⫺/⫺
and ⫹/⫺ donor bone marrow, respectively; P ⬍ .04) and exhibited an
atrophic cortex with depletion of T cells. Thymus athrophy was even
more pronounced in terms of cellularity which was reduced by 80%
(0.52⫾0.66 ⫻ 106 and 2.6⫾1.28 ⫻ 106 thymocytes/organ for ⫺/⫺ and
⫹/⫺ donor bone marrow, respectively; P ⬍ .00001) compared with
control mice. The spleen could not, however, be evaluated since all
animals, including the controls, showed expansion of the red pulp and
extramedullary hematopoiesis, most likely caused by the irradiation.
In contrast to the symptomatic animals described above, a
series of 8 recipients of T␤RII⫺/⫺ marrow that was analyzed by
3.5 weeks after transplantation, when the animals still appeared
healthy but their body weight was beginning to decrease,
showed only occasional (3/8 animals) and mild signs of
inflammation in the pancreas, the stomach, or the lung (Table 1).
These results show that T␤RII-deficient bone marrow is sufficient to cause a progressive inflammatory disease and that loss
of TGF-␤ signaling in other tissues, like the endothelium, is not
required for the pathogenesis.
Further investigation of peripheral lymphoid tissues from B6SJL
recipients (Ly5.1⫹) of induced bone marrow were done in order to
determine the influence of T␤RII deletion on lymphocyte homeostasis and activation. Spleen and lymph nodes from symptomatic
animals were analyzed by flow cytometry to determine the relative
proportions of B and T cells within these organs. Antibodies against
Ly5.2 were used to select for donor populations. In the spleen,
donor populations (Ly5.2⫹) contributed on average to 79 ⫾ 10%
and 82 ⫾ 17% of total cells for T␤RII⫺/⫺ and T␤RII⫹/⫺, respectively. In the lymph nodes, T␤RII⫺/⫺ and T␤RII⫹/⫺ donor cells
contributed to 63 ⫾ 6% and 76 ⫾ 16%, respectively, of the total
cell populations. Using B220 and TCR␣␤ as markers for B cells
and T cells, respectively, the results showed a significant increase in
the fraction of B cells in the lymph nodes (47 ⫾ 4% and 26 ⫾ 4% for
⫺/⫺ and ⫹/⫺ donor bone marrow, respectively; P ⬍ .002), but not in
the spleen, compared with control recipients. Accordingly, the T-cell
fraction was reduced in the lymph nodes (41 ⫾ 8% and 71 ⫾ 5% for
⫺/⫺ and ⫹/⫺ donor bone marrow, respectively; P ⬍ .003).
The activation status of T cells in peripheral lymphoid organs
of symptomatic animals was investigated by flow cytometric
analysis of the fraction of CD4⫹ and CD8⫹ cells expressing the
activation markers CD25⫹, CD69⫹, and CD62L. CD25 and
CD69 are expressed at low levels and CD62L at high levels on
normal naive T cells. The fractions of CD4⫹ and CD8⫹ cells,
respectively, expressing CD69⫹ were significantly elevated in
the lymph nodes of symptomatic animals whereas the other
activation markers were unchanged in the spleen and lymph
nodes (Figure 5 and data not shown). In addition, 2 of the
T␤RII⫺/⫺ recipients showed increased numbers of CD69expressing cells among the CD4⫹ fraction in spleen (34% and
36%, respectively, compared with the mean value of 9.2 ⫾ 3.6%
among control animals). Spontaneous activation leading to a
CD69⫹ phenotype is consistent with T-cell studies of TGF-␤1–
null39 and T␤RII dominant-negative mice.27
Autoimmune manifestations in recipients of T␤RIIⴚ/ⴚ
bone marrow
We analyzed the levels of autoantibodies against nuclear antigens
(histone/dsDNA) in serum of lethally irradiated C57BL/6 recipient
mice transplanted with T␤RII⫺/⫺ marrow (129SV ⫻ C57BL/6
genetic background) by enzyme-linked immunosorbent assay
(ELISA). Significantly elevated titers were found at 4 weeks after
transplantation compared with nontransplanted C57BL/6 controls
or recipients of T␤RII⫹/⫺ bone marrow (Figure 6). There was,
however, no further increase in autoantibody levels in the same
mice when analyzed at the terminal stage of disease. The similarity
of autoantibody titers in nontransplanted C57BL/6 controls and
recipients of T␤RII⫹/⫺ bone marrow indicates that the transplantation procedure or immunohistologic incompatibility does not
contribute to the elevated titers of experimental animals. As in the
TGF-␤1–null mice, the increases were, in general, moderate and
showed large interindividual variations. In addition to these results,
immunohistochemical analysis of glomerular IgG deposits showed
positive stainings above control levels in 2 of 5 animals examined
(data not shown). In conclusion, these results indicate the occurrence of autoimmune manifestations in mice with T␤RII-deficient
bone marrow.
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
INDUCED T␤RII GENE DISRUPTION
565
Figure 3. Pathologic changes in symptomatic animals at 6 to 9 weeks after transplantation. Animals
transplanted with T␤RII⫺/⫺ donor bone marrow are compared with control animals (T␤RII⫹/⫺ donor bone marrow)
analyzed by 7 to 15 weeks after transplantation. (A)
Normal colon of control animal. (B) Pronounced inflammation of the colon mucosa indicated by extensive infiltration
of lymphocytes, plasma cells, and granulocytes, and
tissue destruction in lamina propria. Note the few remaining abnormal glands (arrow) invaded by inflammatory
cells. T␤RII⫺/⫺ donor bone marrow. (C) Normal lung of
control animal. (D) Lymphocytic infiltration of the lung
parenchyma (arrow) surrounding vessels and bronchioli.
The lymphocytes are infiltrating the wall of a venule. They
are also seen in the epithelium of a bronchiolus and in the
walls of the alveoli. T␤RII⫺/⫺ donor bone marrow. (E)
Normal pancreas of control animal. (F) Extensive pancreatitis with massive infiltration of lymphocytes destroying
large parts of the exocrine pancreas with insulitis (arrow).
T␤RII⫺/⫺ donor bone marrow. (G) Normal liver of control
animal. (H) Perivenular infiltrates of lymphocytes in the
liver with extension to the liver parenchyma. T␤RII⫺/⫺
donor bone marrow. (A) and (B): ⫻ 40 magnification. (C)
through (H): ⫻ 20 magnification. Lu indicates lumen; Br,
bronchioli. Sections were stained with Erlich eosin.
Discussion
Previous studies on animal models of TGF-␤ signaling deficiency
have clearly substantiated the importance of TGF-␤ signaling for
immune functions and inflammation.9,22,27,28 The TGF-␤1–null
mutation in mice led to an autoimmune inflammatory condition
involving nuclear autoantibodies and autoreactive T cells. In light
of the multifunctional nature of TGF-␤1 and the early lethality of
TGF-␤1–null mice, this animal model was not adequate to study
the pathogenesis and the specific role of TGF-␤ in immune
function in terms of molecular and cellular mechanisms. Furthermore, accurate in vivo studies on TGF-␤1 function in cells of the
immune system require complete loss of TGF-␤ signaling as well
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566
LEVÉEN et al
BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
Figure 5. T-cell activation in the lymph nodes. (A) Flow cytometric analysis of lymph
nodes (mesenteric and inguinal) from 2 representative recipients of T␤RII⫺/⫺ and T␤RII⫹/⫺
bone marrow, respectively, at 8 weeks after transplantation. The samples were gated for
FSC, SSC, Ly5.2, and CD4 or CD8, respectively. The percentages indicate the fraction of
CD4⫹ or CD8⫹ cells that expresses CD69. Absolute numbers are shown in paranthesis
(⫻ 10⫺4). (B) Average fractions of CD4⫹ or CD8⫹ cells, respectively, expressing CD69.
The numbers of animals examined were 6 (T␤RII⫺/⫺ recipients) and 3 (T␤RII⫹/⫺
recipients), respectively. P ⬍ .02 for CD4⫹ cells and ⬍ .001 for CD8⫹ cells. At least 10 000
counts were collected for each sample.
Figure 4. Immunostainings of B- and T-cell infiltrates in the stomach. (A) T cells,
seen as dark stainings, are mainly located at the base of the lamina propria, close to
the muscularis mucosae whereas (B) the B cells are more evenly distributed
throughout the entire thickness of the lamina propria. Both stainings derive from
recipients of T␤RII⫺/⫺ donor bone marrow. ⫻ 40 magnification.
as the absence of secondary phenotypes resulting from interference
of the systemic illness with the cells to be investigated. These
requirements could not be fulfilled by the TGF-␤1–null mice
because the first signs of inflammatory lesions took place by 8 days
of age, while the contribution of maternally transferred TGF-␤1
through lactation persisted at least until 14 days of age.40 At this
stage functional and developmental studies on leukocytes would be
hampered by the influence of systemic illness (eg, release of
cytokines and corticosteroids). Such shortcomings could potentially be bypassed by transplantation of TGF-␤1–null bone marrow
from asymptomatic donors to normal recipient mice. However,
attempts to develop the inflammatory phenotype by transplanting
TGF-␤1–null bone marrow to normal recipient mice only led to
minor inflammatory lesions, predominantly in the esophagus.25
Mechanistically, this could be explained by substitution for the lack
of TGF-␤1 expression in donor bone marrow cells by recipientderived paracrine or endocrine TGF-␤1 sources. Alternatively, all
the cellular components necessary for the fully developed inflammatory phenotype may not be present within the bone marrow.
These questions could be addressed by using T␤RII-null donor
bone marrow instead, which, in contrast to TGF-␤1–null marrow,
would produce a cell autonomous phenotype (ie, mutant cells
would be affected by intrinsic genetic dysfunction and not respond
to any source or isoform of TGF-␤). Autocrine as well as endocrine
and paracrine TGF-␤ signaling in bone marrow cells would, thus,
be blocked. T␤RII⫺/⫺ bone marrow is, however, not available from
the conventional knockout model because the phenotype is embryonic lethal by days 9.5 to 10.5 after coitus.32 This was circumvented
in the present study by using the Cre/lox gene targeting approach to
generate a conditional knockout of T␤RII in mice, preserving
normal genetic function until the gene is disrupted by induction
with polyI:polyC.
The conditional knockout strategy of T␤RII included insertions
of loxP sequences at the flanks of exon 4, which then will be
deleted when exposed to transgenic Cre-recombinase. This deletion results in a frameshift mutation that permits only the coding
Figure 6. ELISA titers of autoantibodies against nuclear antigens (histone/
dsDNA) in transplanted mice. (A) Sera from C57BL/6 recipients of T␤RII⫺/⫺ bone
marrow were analyzed by ELISA at 4 weeks after transplantation. The 10 recipients (5 ⫹ 5)
received bone marrow from 2 donors, as distinguished by filled and empty circles. We
analyzed 9 animals. (B) All animals except one were, in addition, analyzed at the terminal
stage of inflammatory disease (ie, 6 to 9 weeks after transplantation). Values obtained from
the same animal at the 2 time points are connected with a line. (C) As controls, sera were
taken from recipients of T␤RII⫹/⫺ bone marrow derived from induced T␤RII⫹/flox ⫻
Mx1-Cre donor mice. (D) In addition, 3 nontransplanted C57BL/6 mice were included as
controls for transplantation artifacts. A positive control (serum from the MRL-lpr strain; a
mouse model of the human autoimmune disease lupus erythematosus) showed a
reference value of 1.8. Mean values are indicated by horizontal lines. Comparisons
between experimental animals at 4 weeks after transplantation and transplanted controls
were statistically significant (P ⱕ .022) using the 2-tailed Student t test. The higher titers at
the terminal stage of disease compared with controls were, however, not statistically
significant. For titer definition, see “Materials and methods.”
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BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
sequences of the extracellular domain of the receptor to be
synthesized. Such a receptor remnant could, if translated and
processed correctly, potentially have a toxic or dominant-negative
impact on cells. However, we have not obtained any evidence for
embryonic abnormalities or lethality in T␤RII⫹/⫺ animals. The
ratio of T␤RII⫹/⫹ and T␤RII⫹/⫺ pups born from heterozygous
crossings was as expected 1:2, demonstrating lethality caused
by the T␤RII⫺/⫺ but not the T␤RII⫹/⫺ genotype. Furthermore,
T␤RII⫹/⫺ embryos at 9.5 to 10.5 days after coitus appeared
morphologically indistinguishable from wild-type embryos (data
not shown) and T␤RII⫹/⫺ pups develop and reproduce normally
and show a normal life span.
The induction of T␤RII deficiency in adult T␤RII flox/flox ⫻
Mx1-Cre animals leads to a lethal inflammatory disorder by 8 to 10
weeks after induction. T␤RII⫺/⫺ bone marrow from these animals
taken immediately after induction caused inflammation and death
when transferred to normal C57BL/6 recipients by 6 to 9 weeks
after transplantation. As judged from the weight-reduction profile
and histopathologic findings, the apparent onset of disease occurred
at 3 to 4 weeks after transplantation. The pathogenic T␤RII⫺/⫺ cell
populations that were transplanted are likely to have developed as a
result of their intrinsic deficiency of TGF-␤ signaling and not due
to interactions with other mutated tissues since bone marrow that
was induced in the recipient mice after transplantation caused the
same phenotype. These results suggest that abnormal and pathogenic cell population(s) located within the bone marrow of donor
animals are sufficient to cause a lethal inflammatory disease. Thus,
TGF-␤ signaling deficiency of the endothelium or the parenchyma
of peripheral or central lymphoid organs is not required for the
pathogenesis. However, these data do not rule out an immunoregulatory role for TGF-␤ in the endothelium since studies have shown
inhibitory effects of TGF-␤ and the reverse action of anti–TGF-␤
antibodies on lymphocyte adherence to vascular endothelial cells
following treatment of the latter cell type with TGF-␤.41,42
The phenotype of mice transplanted with T␤RII-null bone
marrow showed both similarities and dissimilarities to the one
reported from nontransplanted TGF-␤1–null animals.22,43 Both
models generated a similar clinical picture and a multifocal
inflammatory disease affecting a multitude of organs. Moreover,
lymphocytes were the most frequently observed inflammatory cell
type in both models. However, some differences in tissue distribution of inflammatory lesions between the 2 models were noticed. In
particular, the heart was affected in 87% of the TGF-␤1⫺/⫺ mice,
mostly by macrophage infiltrates, whereas this organ was unaffected in all recipients of T␤RII⫺/⫺ bone marrow. Differences in the
genetic backgrounds of these outbred mouse models are likely to
account for some of the dissimilarities of tissue distribution and
severity of inflammation. Similar differences were seen in a
comparison of the TGF-␤1 models reported.22,43
As previously discussed, TGF-␤1–null bone marrow only
caused a mild phenotype when transferred to normal recipient
mice.25 Nevertheless, our transplantation data show that TGF-␤
signaling deficiency in bone marrow cells is sufficient to cause
lethal inflammation. Thus, the discrepancy between the 2 phenotypes is likely to be explained by the additional elimination of
endocrine or paracrine signaling possibilities in T␤RII-null bone
marrow cells. The further block of signaling by the other 2 TGF-␤
members, TGF-␤2 and TGF-␤3, in the T␤RII-null transplantation
model is, however, not likely to contribute to the greater severity of
disease as these isoforms did not rescue nontransplanted TGF-␤1–
null mice.
INDUCED T␤RII GENE DISRUPTION
567
The TGF-␤1–null model provided support for the immunosuppressive role of TGF-␤ by the occurrence of elevated levels of autoantibodies to the nuclear antigens single-strand DNA (ssDNA), dsDNA, Smith
(SM), and ribonucleoprotein (RNP).25 In addition, autoimmune IgG
deposits were detected in renal glomeruli. We showed a similar
elevation of nuclear autoantibodies in serum of C57BL/6 recipients
transplanted with T␤RII⫺/⫺ bone marrow. The peak levels were reached
already at 4 weeks after transplantation, when no symptoms were
apparent, and remained stable or decreased slightly until the mice were
severely ill by 6 to 9 weeks after transplantation. Immunohistochemical
analysis of kidneys from animals with T␤RII-deficient bone marrow
provided evidence for glomerular IgG deposits in some of the mice. The
clinical significance of immunoglobulin-mediated autoimmunity is,
however, unclear as the findings were not obligatory and it remains to be
shown whether it contributes to the fatal course of disease progression.
In studies of TGF-␤1–null animals, multiple tissues showed upregulated expression of major histocompatibility complex (MHC)
molecules that might cause improper antigen presentation that triggers
infiltration of lymphocytes and, therefore, may play a role in the
initiation of autoimmune disease.26,44,45 However, it could not be
deduced whether the abnormal MHC expression was a primary
consequence of TGF-␤1 deficiency or caused by the systemic illness.
Primary dysregulation of MHC expression in the recipient cannot
precede inflammation in normal animals transplanted with T␤RII⫺/⫺
bone marrow and is thus not required for the initiation of inflammation
caused by TGF-␤ signaling deficiency. This conclusion is consistent
with a study using T-cell–specific T␤RII dominant-negative transgenic
mice showing that T␤RII deficiency in T cells alone is sufficient to cause
inflammation.27 These mice, with a specific TGF-␤ signaling block in
CD4⫹ and CD8⫹ T cells, developed a multifocal inflammatory disease
but showed less severe inflammation and slower disease progression
compared with our conditional T␤RII knockout and the TGF-␤1
knockout model. The less prominent signs of inflammation in the
dominant-negative model of TGF-␤ deficiency suggests the requirement of TGF-␤ in populations other than T cells to control the immune
response. Alternatively, these transgenic mouse models do not generate
absolute TGF-␤ signaling deficiency or do not affect all developmental
stages of T cells and their precursors, as is the case with our gene
deletion approach. A similar study of transgenic mice expressing
dominant-negative T␤RII in T cells under the control of the CD2
promoter showed no inflammation but a lymphoproliferative disorder
involving peripheral expansion of CD8⫹ T cells.28 The phenotypic
discrepancy between these transgenic approaches further suggests
incomplete receptor inactivation in one or both models. We conclude
that the animal model presented in this paper gives promise to serve as a
unique and powerful tool to clarify the pathogenic mechanisms in
animals deficient for TGF-␤ signaling and to elucidate the specific
cellular and molecular mechanisms of TGF-␤ to maintain homeostasis
within the immune system.
Acknowledgments
We thank Dr Werner Muller for advice and professor Reinhard
Fässler for kindly providing us with the Mx1-Cre transgenic mouse
strain; Marianne Ahmad and Linda Hellborg for participating in the
recombinant DNA work; Lilian Wittman for help with animals;
Anna-Karin Lindqvist for assistance with autoantibody analysis;
and Anna Makowska for participation in the flow cytometric
analysis. Finally, we would like to thank the members of The
Molecular Medicine and Gene Therapy and Stem Cell Biology
departments, Lund University, for helpful discussions.
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568
BLOOD, 15 JULY 2002 䡠 VOLUME 100, NUMBER 2
LEVÉEN et al
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2002 100: 560-568
doi:10.1182/blood.V100.2.560
Induced disruption of the transforming growth factor beta type II
receptor gene in mice causes a lethal inflammatory disorder that is
transplantable
Per Levéen, Jonas Larsson, Mats Ehinger, Corrado M. Cilio, Martin Sundler, Lottie Jansson
Sjöstrand, Rikard Holmdahl and Stefan Karlsson
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