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Blood, 15 July 2002, Vol. 100, No. 2, pp. 560-568
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
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.
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Abstract |
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 (TRII) gene to normal recipient animals. The TRII-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 TRII gene is disrupted upon induction with
interferon- or polyI:polyC. We show that induction of TRII
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.
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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 TRI and TRII. Complex formation
leads to phosphorylation of TRI 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 mouse models for
T-cell-specific TGF- type II receptor (TRII) deficiency: abrogation of TGF- signaling in CD4- and CD8-expressing cells generated a phenotype reminiscent of the inflammation of TGF-1-null animals27 whereas CD2-specific TRII 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 TRII 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 TRII-null animals22,23,32
and a total loss of TGF- signaling. In addition, the mutation causes
a cell-autonomous 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 TRII
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.
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Materials and methods |
Targeting of the TRII genomic locus
A cDNA probe encompassing 580 base pair (bp) of the sequence
encoding the extracellular domain of mouse TRII 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 TRII 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
electroporated into the embryonic stem (ES) cell line RI which was
subsequently grown under selection (300 µg/mL neomycin G418 and 4 µM 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 TRII 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 TRII 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 µM cell strainer (Falcon, BD,
Franklin Lakes, NJ) and 2 × 106 cells were injected in a
volume of 500 µL 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-µm nylon cell
strainer (Falcon). For selection of donor 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 µg/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 µg/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 µg/mL histone (unfractionated whole histone type II-A from calf
thymus; Sigma, St Louis, MO) and thereafter with 50 µg/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-[3-ethylbenzthiazoline 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).
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Results |
Generation of ES cells and mouse strains carrying "floxed"
or null TRII alleles
TRII 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 TRII (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" TRII alleles, ES cells were
subjected to homologous recombination with a gene construct containing
TRII 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 TRII-null allele (TRII+/ ) (ie, clones lacking
both neo and exon 4), to be used for functional verification
of gene targeting. "Floxed" and TRII-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).
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| Figure 1.
Conditional targeting of the TRII gene.
(A) The wild-type TRII 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 TRII+/flox ES-cell DNA; R2,
TRII+/ ES-cell DNA; R3, wild-type DNA.
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In order to functionally confirm successful targeting of exon 4, TRII+/ mice were mated. Genotyping of the litters
showed that TRII+/+ and TRII+/ pups
were born in the expected 1:2 ratio, whereas no pups with the
TRII/ (TRII null) genotype were found, indicative
of an embryonic lethal TRII/ 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
TRII 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 TRII/ 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 TRII +/flox × Mx1-Cre mice
treated 3 times at 2-day intervals with polyI:polyC (250 µg). Bone
marrow from these mice was, in addition, tested separately by seeding
into 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 TRII-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 TRII 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 TRII 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 TRII flox/flox × Mx1-Cre 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 TRII disruption causes multifocal inflammation in
multiple organs
We tested the pathologic consequences of induced TRII gene
disruption by treating 4 TRII flox/flox × Mx1-Cre
and 3 TRII+/flox × Mx1-Cre mice with polyI:polyC at
an age of 8 weeks. The mice were injected 3 times intraperitoneally
with polyI:polyC (250 µg) at 2-day intervals and all 4 TRII
flox/flox × Mx1-Cre mice developed a wasting syndrome
that was fatal by 8 to 10 weeks after induction, whereas the
TRII+/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 TRII flox/flox × Mx1-Cre mice was reduced in size (see results from
transplanted mice below) in agreement with previous observations of
TGF--signaling-deficient mice.27,39 In conclusion,
TRII 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 TRII
flox/flox × Mx1-Cre mice followed by transfer of
their bone marrow (TRII/) to normal and lethally
irradiated (950 cGy) C57BL/6 recipient mice. In the first series of
transplantations, 2 TRII flox/flox × Mx1-Cre mice
were induced at 7 weeks of age to serve as donors of
TRII/ bone marrow for a total of 10 C57BL/6
recipients aged 12 weeks (5 recipients for each donor). Another 5 C57BL/6 animals received TRII+/ control bone marrow
from 2 induced TRII+/flox × Mx1-Cre mice (3 and 2 recipients, respectively, for each donor). All 10 recipients of
TRII/ 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 TRII-deficient tissues.
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| Figure 2.
Clinical progression of the inflammatory disease.
Animal weight change following transplantation with
TRII/ (filled line) and TRII+/
control bone marrow (dotted line) is shown. The initial numbers of
transplanted animals were 10 (TRII/ donor bone
marrow) and 5 (TRII+/ donor bone marrow). The mean
weights of recipients of TRII/ bone marrow and
TRII+/ 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 TRII/
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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The symptomatic profile and histopathology of TRII/
bone marrow recipients was reminiscent of the phenotype of induced
TRII 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), 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 TRII/ 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 × 103 and
3.02 ± 0.73 × 103 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.
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| Figure 3.
Pathologic changes in symptomatic animals at 6 to 9 weeks after
transplantation.
Animals transplanted with TRII/ donor bone marrow
are compared with control animals (TRII+/ 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. TRII/ 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.
TRII/ 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). TRII/ donor bone marrow. (G)
Normal liver of control animal. (H) Perivenular infiltrates of
lymphocytes in the liver with extension to the liver parenchyma.
TRII/ donor bone marrow. (A) and (B): × 40
magnification. (C) through (H): × 20 magnification. Lu indicates
lumen; Br, bronchioli. Sections were stained with Erlich
eosin.
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| 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
TRII/ donor bone marrow. × 40
magnification.
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In contrast to the symptomatic animals described above, a series
of 8 recipients of TRII/ 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
TRII-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.
TRII/ bone marrow recipients show an activated
T-cell phenotype
Further investigation of peripheral lymphoid tissues from B6SJL
recipients (Ly5.1+) of induced bone marrow were done in
order to determine the influence of TRII 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 TRII/ and
TRII+/, respectively. In the lymph nodes,
TRII/ and TRII+/ 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 TRII/ recipients
showed increased numbers of CD69-expressing 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
TRII dominant-negative mice.27
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[in a new window]
| Figure 5.
T-cell activation in the lymph nodes.
(A) Flow cytometric analysis of lymph nodes (mesenteric and
inguinal) from 2 representative recipients of TRII/
and TRII+/ 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 (× 104).
(B) Average fractions of CD4+ or CD8+ cells, respectively, expressing
CD69. The numbers of animals examined were 6 (TRII/
recipients) and 3 (TRII+/ recipients), respectively.
P < .02 for CD4+ cells and < .001 for
CD8+ cells. At least 10 000 counts were collected for each
sample.
|
|
Autoimmune manifestations in recipients of TRII/
bone marrow
We analyzed the levels of autoantibodies against nuclear antigens
(histone/dsDNA) in serum of lethally irradiated C57BL/6 recipient mice
transplanted with TRII/ 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 TRII+/ 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
TRII+/ 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 TRII-deficient bone
marrow.
View larger version (12K):
[in this window]
[in a new window]
| Figure 6.
ELISA titers of autoantibodies against nuclear antigens
(histone/dsDNA) in transplanted mice.
(A) Sera from C57BL/6 recipients of TRII/ 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 TRII+/ bone marrow derived from induced
TRII+/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  .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."
|
|
| |
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 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 recipient-derived 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 TRII-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.
TRII/ 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 TRII in
mice, preserving normal genetic function until the gene is disrupted by
induction with polyI:polyC.
The conditional knockout strategy of TRII 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 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 TRII+/ animals. The ratio of
TRII+/+ and TRII+/ pups born from
heterozygous crossings was as expected 1:2, demonstrating lethality caused by the TRII/ but not the
TRII+/ genotype. Furthermore, TRII+/
embryos at 9.5 to 10.5 days after coitus appeared morphologically indistinguishable from wild-type embryos (data not shown) and TRII+/ pups develop and reproduce normally and show a
normal life span.
The induction of TRII deficiency in adult TRII flox/flox × Mx1-Cre animals leads to a lethal inflammatory disorder by 8 to 10 weeks after induction. TRII/ 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
TRII/ 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 TRII-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 TRII/ 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 TRII-null bone marrow cells. The further
block of signaling by the other 2 TGF- members, TGF-2 and
TGF-3, in the TRII-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.
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 TRII/ 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 TRII-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
up-regulated 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 TRII/ 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 TRII
dominant-negative transgenic mice showing that TRII 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 TRII 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 TRII 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.
| |
Footnotes |
Submitted June 22, 2001; accepted March 5, 2002.
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 Foundation, New York, NY.
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.
Reprints: Stefan Karlsson, Molecular Medicine and Gene
Therapy, Lund University, Sölvegatan 17, S-22184, BMC A12, Lund,
Sweden; e-mail: stefan.karlsson{at}molmed.lu.se.
| |
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Top
Abstract
Introduction