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Animal Models |
§§¶
From the Gwen Knapp Center for Lupus and Immunology
Research,*
the Committee on
Immunology,
the Department of
Pathology,
and the Department of
Medicine,§
Howard Hughes Medical
Institute,¶
University of Chicago,
Chicago, Illinois
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, a group of proteins that bind to the cytoplasmic tail of
several TNFR family members has been identified. These proteins are
named TRAFs (for TNFR-associated factors), and to date six family
members have been described. All TRAFs share a ~150-amino acid
C-terminal TRAF domain that mediates the interaction between the TRAF
and its receptor(s)2-4
or other proteins, as well as a
more loosely conserved region of zinc finger repeats. A similar domain
can also be found in the meprin family of mammalian zinc
metalloendopeptidases.5
Among some of the TNF receptors, a
PXQX(T/S) motif has been shown to be a common TRAF domain binding
site.6-8
With the exception of TRAF1, all TRAFs contain
an N-terminal RING finger, which seems to be required for their
downstream signaling potential. Dominant negative (DN) versions of the
TRAFs can be generated by removal of the RING finger
domain.9,10
TRAF2, 5, and 6 share an ability to mediate
NF-
B and JNK activation through their N-terminal
domains.10-12
TRAFs are also able to bind to non-TNFR
family members, as exemplified by TRAF6’s ability to associate with
the interleukin-1 (IL-1) receptor.13
To date, published
reports have been generated on the phenotype of mice that lack
TRAF2,14
TRAF3,15
TRAF5,16
or
TRAF617
or that overexpress TRAF118
or the DN
form of TRAF2.19
The TRAF2-, TRAF3-, or TRAF6-deficient
mice that are born appear normal at birth but die at a very young age
as a result of severe runting. TRAF5-deficient mice appear outwardly
normal.
The immune system of TRAF2-deficient mice is affected, with depletion
of lymphocytes in thymus and spleen and an absence of secondary
germinal centers, perhaps due to a combination of hypersensitivity to
TNF and increased serum TNF levels. B-cell development is impaired, but
T cells develop normally. TNF-mediated NF-B responses are normal,
but JNK activation is deficient. Interestingly, in TRAF2 DN mice with
the transgene being expressed only in lymphocytes, there is an increase
in the number of B cells, splenomegaly, and lymphadenopathy. JNK
activation, but not NF-B responses, is affected just as in
TRAF2-deficient mice.
In mice lacking TRAF3, despite hypotrophy of spleen and thymus similar to what is seen in TRAF2-deficient animals, the immune system is defective when it comes to T-cell-dependent responses to antigen but otherwise is fairly normal.
TRAF5-deficient mice show defects in CD40- and CD27-mediated signaling.
TRAF6-deficient mice have enlarged spleens, despite normal T-cell
function and reduced B-cell proliferation in response to CD40 or
lipopolysaccharide stimulation. IL-1, CD40, or
lipopolysaccharide-mediated NF-B activation is also disrupted, as is
IL-1-mediated JNK activation. In addition, these mice are
osteopetrotic, which is most likely due to the recently described
association between TRAF6 and the receptor activator of NF-B
(RANK),20
which is required for osteoclast differentiation
and lymph node formation.21
TRAF4 was cloned in a differential expression screen, using mRNA from metastatic breast cancer samples.22,23 The 2-kb gene is located on human chromosome 17, q11-q12,22 which is a region that also contains the oncogene c-erbB2, a gene known to be involved in breast cancer. The predicted molecular weight of TRAF4 is 53 kd. TRAF4 was originally found to be expressed in the nucleus of the breast cancer cells, using a polyclonal rabbit anti-serum against a TRAF-C domain synthetic peptide. In mice, using Northern blot analysis, as well as in situ hybridization,24 it was shown that TRAF4 is expressed at high levels during embryogenesis, peaking at embryonic day 13.5. Specifically, transcripts are present in the developing neuroepithelium, neural crest cell condensations, and the first, second, and third branchial arches. Transcripts are also detected in the neuroepithelium of the nasal fossae, as well as in thymus, salivary glands, and intestine of embryos. In the adult, TRAF4 was among the genes induced in postmitotic and undifferentiated cells of the central nervous system.
Recently, a second TRAF4-specific polyclonal antibody was used to investigate expression patterns in normal embryonic and adult human tissue, as well as in tissue from breast and prostate cancers.25 Surprisingly, by using this antibody for immunohistochemistry, TRAF4 was found to be primarily localized to the cytoplasm of the cells, and was not expressed in most breast cancer samples. Most epithelial cells in this study, including thymic epithelial cells and lymph node dendritic cells, expressed TRAF4, whereas the thymocytes and lymphocytes did not demonstrate any TRAF4 staining. In addition, Krajewska et al found evidence that TRAF4 is able to associate with the lymphotoxin-ß receptor, which has an overlapping expression pattern in the scaffolding tissues of thymus and lymph nodes. A weak interaction with the p75 neural growth factor receptor was also detected. The differences in the expression patterns of TRAF4 in this report as compared to the study using in situ hybridization could be explained by posttranscriptional modification of TRAF4 transcripts.24
To better understand the role of TRAF4 in development, we have generated TRAF4-deficient mice through gene targeting. The mice are viable and appear outwardly normal. However, about 20% of TRAF4 knock-out mice present with a high-pitched wheezing noise as they breathe. In addition, there is a decrease in weight of the animals, averaging 10% for adult males and 5% for adult females. Pathological analysis demonstrates that lack of TRAF4 leads to failure to properly form the proximal end of the trachea, at the region where the trachea connects to the larynx, leading to various degrees of constriction in all knock-out mice examined. The air flow is altered, as evidenced by a marked decrease in response to a challenge with aerosolized antigen in immunized mice lacking TRAF4. TRAF4-deficient mice also develop spontaneous inflammation of the lungs, correlating with the occurrence of wheezing. The immune system of TRAF4 knock-out mice appears to respond normally to a variety of stimuli. Taken together, the data suggest that the expression of TRAF4 normally seen in branchial arches 2 and 3 during embryogenesis correlates with a required role for TRAF4 during normal laryngotracheal development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Polymerase chain reaction amplification of a human cDNA library,
using primers derived from the published sequence of human TRAF4, was
used to subclone human TRAF4 cDNA. A 5' 0.6-kb probe,
containing mostly the three zinc finger repeats, was isolated from the
cDNA clone and used to screen a phage library (Lambda FIX II;
Stratagene Cloning Systems, La Jolla, CA) derived from 129/SvJ mice.
The resulting genomic clones were mapped by restriction digest
analysis, Southern blotting, and sequencing. A 2.2-kb
KpnI-XbaI fragment located 3' of the last exon
(exon 7), in combination with either a 7-kb
SalI-BglII fragment containing the first exon
(construct 1, Figure 1A
) or a 4.1-kb
SalI-BamHI fragment containing only sequence 5'
of the gene (construct 2, Figure 1A
), was cut out from the same phage
preparation (the SalI site is derived from the phage) and
subcloned into the pPNT vector.26
On linearization, the
resulting targeting constructs contained the
PGK-neor gene in the opposite
transcriptional orientation as compared to the TRAF4 gene,
between the two subcloned genomic fragments that were flanked on one
side by the PGK-TK gene.
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The two linearized targeting constructs were used to electroporate R1 ES cells, derived from an F1(129/Sv x 129/SvJ) blastocyst.27 Two x 107 ES cells were used for each electroporation at 270 V, 500 µF, using a Bio-Rad Genepulser (Bio-Rad Laboratories, Hercules, CA), and the cells were plated onto eight 10-cm dishes containing mitomycin C-treated neor mouse embryo fibroblasts.28 Culture and selection of neor, TK-negative ES cells were as previously described,29 with a few modifications as described below. High-glucose liquid Dulbecco’s minimum essential medium with L-glutamine (GibcoBRL, Grand Island, NY) was used with the addition of penicillin-streptomycin (GibcoBRL), but without the addition of sodium pyruvate. The fetal bovine serum (HyClone, Logan, Utah) was screened for the optimal growth of undifferentiated ES cells. Selection for targeted ES cells was performed 24 hours after electroporation, using ES cell medium containing 250 µg/ml neomycin and 2 µM gancyclovir. Surviving colonies of ES cells were picked 7 days after electroporation.
The resulting expanded ES cell clones were screened by Southern blot
analysis of HindIII-digested genomic DNA isolated from each
clone. The 1-kb XbaI-HindIII probe used to
identify homologously recombined ES cell clones is shown in Figure 1A
.
ES cells from the selected clones were injected into C57/BL6 blastocysts, and chimeric mice with a large contribution of targeted ES cells could be identified because of their mainly agouti coat color. Largely agouti chimeras were bred to C57/BL6 females, and the resulting F1 progeny were screened by Southern blot analysis of tail DNA to determine their genotype. Mice with germline transmission of a targeted TRAF4 allele were intercrossed to generate TRAF4-deficient mice.
Western and Northern Blot Analysis
Whole-cell extracts of homogenized tissues from wild-type mice and mice derived from all three of the germline transmitted ES cell lines were made using RIPA buffer (50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.1% Triton X-100, 0.5% deoxycholate, 0.1 mmol/L EGTA, 0.1 mmol/L EDTA) with the addition of protease inhibitors. The protein concentration was determined by colorimetric assay (BCA Protein Assay; Pierce, Rockford, IL), and equal amounts of protein were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel. The Western blot was probed with a polyclonal antibody raised against an N-terminal peptide from human TRAF4 (Santa Cruz Biotechnology, Santa Cruz, CA).
Total RNA was purified from wild-type and knock-out mice with the use of TRIzol (GibcoBRL). The RNA was equalized as previously described,30 and the concentration was later determined by spectrophotometric analysis. Approximately 5 µg of total RNA was used for each sample. The samples were loaded on an agarose gel, electrophoresed, and blotted as described.30 TRAF4 transcripts were detected using a probe specific for exons 4–7. (Similar results were obtained with the use of a probe containing the RING domain.) The relative amounts of RNA were visualized with the use of an end-labeled 5S RNA probe.
Examination of Tracheal Morphology
The mice were sacrificed with a lethal dose of ketamine. The tissues in front of the trachea were removed and the chest was opened, but the mice were otherwise left intact. The outer diameter of each cartilage ring was measured, using a dissection microscope equipped with a scale bar with 0.1-mm increments. The average width of either the first three rings below the cricoid cartilage or the last three cartilage rings before the bifurcation of the trachea was used as a measure of proximal and distal tracheal width, respectively. After the tracheal diameter had been documented, tissue containing the entire airway was removed and fixed. The fixed tissue was sectioned in the coronal plane and stained with hematoxylin-eosin. More detailed histological analysis was performed to determine the circumference, width, and density of the cartilage rings, as well as the occurrence of inflammatory cells in the airway tissue.
Ovalbumin Immunization, Nebulization, and Analysis of Lavage Fluid
The method used for ovalbumin (OVA) immunization and nebulization was as described by Krinzman et al,31 with some modifications. Specifically, TRAF4-deficient mice, or littermate controls, were left untreated or were immunized intraperitoneally with 10 µg of OVA (grade IV; Sigma, St. Louis, MO) in 20 µl of 5 mg/ml alum in phosphate-buffered saline (PBS). Each experimental group contained three mice. The immunization was repeated 1 week later. Four days after the second immunization, the mice were challenged with aerosolized 6% OVA in sterile PBS, using a nebulizer, for 3 consecutive days for 30 minutes each time. Five days later the mice were sacrificed with a lethal dose of ketamine. Day 5 was chosen to maximize the presence of eosinophils in the bronchoalveolar lavage (BAL) fluid.32 The airways were rinsed four times with 0.8 ml of sterile PBS, using a syringe with a blunt needle inserted halfway down the trachea, and sealed off with suture thread around the trachea. Similar volumes of BAL fluid were recovered from each mouse. Total white blood cell counts were made directly from the BAL fluid. In addition, samples were centrifuged onto microscope slides with a cytospin (Shandon, Pittsburgh, PA). The cells were stained by standard methods, and the relative contribution of eosinophils versus other white blood cell types was determined.
In Situ Hybridization
Embryos from timed matings (noon of the day of vaginal plug was
considered embryonal day 0.5, E0.5d) were dissected out, fixed in 4%
paraformaldehyde, paraffin-embedded, and sectioned. A 0.6-kb probe from
the human TRAF4 cDNA 5' end was first used on a Northern
blot with total mouse RNA to verify specific recognition of mouse
TRAF4 transcript. The probe was subcloned in pBluescript
(Stratagene Cloning Systems), linearized on either side of the cloning
cassette, and in vitro transcribed and
-35S-labeled with T3 (sense) or T7 (antisense)
polymerase, depending on the site of linearization. The riboprobes were
quantitated and used for in situ hybridization, with washing
and developing conditions as previously described.33
The
sections were dipped in NTB2 emulsion (Kodak, Rochester, NY), and
autoradiography was allowed to proceed for 1 week before the slides
were developed and the cell nuclei were counterstained using Hoechst
33258 (Boehringer Mannheim, Mannheim, Germany). Adjacent sections from
the same embryo probed with either sense or antisense riboprobe were
photographed, using the same exposure time, with a Zeiss Axiophot
microscope for epifluorescence and dark-field microscopy.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The TRAF4 gene was disrupted by homologous gene
targeting. Three independent ES cell lines derived from two different
constructs (Figure 1A)
were used to generate TRAF4-deficient mice.
Construct I eliminated all but part of the RING finger (exon 1),
whereas construct II eliminated the entire gene from the targeted
chromosome. Either of the two constructs resulted in loss of both
detectable TRAF4 protein (Figure 1B)
and TRAF4 mRNA (Figure 1C)
, and
all three lines of the TRAF4-/- mice have the
same phenotype, verifying the successful targeting of TRAF4
in these mice.
TRAF4-deficient mice were born at the expected Mendelian ratio. The
mice bred as well as their wild-type littermates. On gross examination
of the knock-out mice, two findings were evident. First, the mice were
smaller overall than their littermates. Figure 2
shows the weight at 7 weeks of 66
knock-out mice and 89 wild-type mice grouped by sex. There is a
statistically significant reduction in the average weight of mice
lacking TRAF4. Males have undergone a 10% decrease in weight and
females a 5% decrease at 7 weeks of age (Figure 2)
. The weight
reduction is not due to loss of any specific tissue, inasmuch as
overall gross and histopathological examination revealed no significant
differences between TRAF4 wild-type and knock-out mice. Second, a
certain percentage (approximately 20%) of the mice at any one time
displayed inspiratory stridor, as evidenced by a high-pitched wheezing
noise. Mice as young as 2 weeks of age could be heard making the noise.
Affected mice could have severe stridor at one time, but within weeks
could be asymptomatic and make no or very little noise.
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To understand the underlying defect(s) of TRAF4-deficient mice, a
more detailed analysis of the airways was performed. A total of nine
TRAF4-deficient mice and six littermate controls were subjected to
analysis. Tissue samples were delivered to the pathologist in a blinded
fashion to prevent introduction of investigator bias. On studying the
trachea, it became clear that all mice lacking TRAF4 had a narrowing of
the proximal part of the trachea, involving the first few cartilage
rings immediately below the larynx. When the external diameter of the
upper trachea was measured, an average 18% reduction was found in
TRAF4-deficient mice as compared to littermate control mice, resulting
in an estimated decrease of the cross-sectional area of about 32%.
There was no difference in the width of the cartilage rings themselves,
and the U-shaped form of the rings remained intact. However, a
ventrally pointed appearance was often evident. Lack of TRAF4
expression had no effect on the width of the trachea below this region.
The results are summarized in Figure 3A
.
The location of the tracheal constriction just below the larynx in
TRAF4 knock-out mice correlates with the observed inspiratory stridor,
because extrathoracic tracheal stenosis would be expected to give rise
to inspiratory stridor, whereas intrathoracic tracheal stenosis would
result in expiratory stridor.
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shows an example of the tracheal defect seen on dissection of
a TRAF4-deficient mouse as compared to a wild-type mouse. Fixed tissue
containing larynx, trachea, and lungs of another set of mice are shown
in Figure 3C
. There was no evidence of cartilaginous defects in the
trachea of the affected animals, as the tracheal cartilage appearance
in stained tissue sections from TRAF4-deficient mice was
indistinguishable from that in their wild-type littermates (data not
shown). Measuring the length of various bones in the mice, as well as
X-ray imaging, also revealed no genotype-dependent differences (data
not shown), arguing against a generalized defect in tissues of
cartilaginous origin.
In some of the TRAF4 knock-out mice, inflammation of the lungs could be
seen. Figure 3D
shows a section of lung tissue obtained from one of the
affected mice, with arrows pointing to areas of leukocyte infiltration.
In several experiments a total of nine wheezing mice were subjected to
histological analysis, and six were found to have inflammation of the
lungs, whereas none of the asymptomatic mice autopsied displayed
evidence of airway inflammation.
Altered Air Flow to the Lungs of TRAF4-Deficient Mice
To investigate the possibility that the tracheal abnormality could
result in altered air flow to the lungs, perhaps explaining the
susceptibility to lung inflammation and reduced size of mice lacking
TRAF4, the response to an aerosolized challenge was examined.
TRAF4-deficient mice or littermate controls were left untreated or were
immunized intraperitoneally with OVA antigen in adjuvant. The
immunization was repeated 1 week later, and after 4 days the mice were
challenged with aerosolized OVA in a nebulizer on three consecutive
days. Five days later, the mice were sacrificed, and the number of
eosinophils and other white blood cells in BAL fluid from the lungs was
determined. The TRAF4-deficient mice had a significantly lower number
of infiltrating cells in the BAL fluid as compared to immunized control
littermates (Figure 4A)
. However, the BAL
fluid samples from the two groups of immunized mice contained similar
ratios of eosinophils (Figure 4B)
, suggesting that although greatly
diminished, the response to the aerosolized challenge was normal. The
decrease in the response was not due to a generalized T-cell defect,
inasmuch as peripheral T-cells isolated from
TRAF4-/- animals and wild-type littermates
responded equally well to antigen rechallenge in proliferation assays,
as measured by [3H]thymidine uptake and
cytokine production measured in enzyme-linked immunosorbent assays
(data not shown). Taken together, these results suggest that the
tracheal stenosis reduced the amount of aerosolized antigen that
reached the lungs.
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In situ hybridization was used to study the expression
of TRAF4 during embryogenesis. Widespread expression was seen using an
antisense riboprobe (Figure 5A)
, and the
specificity of the staining was confirmed by using the corresponding
sense riboprobe (Figure 5B)
. High expression of TRAF4 can be seen in
the first, second, and third branchial arches during day 10.5 of
embryogenesis, according to the antisense probe (Figure 5C)
as compared
to the sense control probe (Figure 5D)
. These branchial arches are
prominent at this stage of development and later give rise to bones of
the jaw and ear, as well as the hyoid bone and upper pharyngeal
structures. The laryngeal cartilages, including the thyroid and cricoid
cartilages, originate from the subsequent branchial
arches.34
During later stages of development, at embryonal
day 12.5 or 13.5, the region where the laryngotracheal tube is
establishing a connection with the pharynx35,36
shows no
higher level of expression than the surrounding tissue (data not
shown). TRAF4 transcripts can still be found at relatively high levels
in the epithelium of the trachea at this later time, as well as in
most other epithelia analyzed. TRAF4 is also highly expressed in the
developing cerebral cortex as well as in vertebral bodies (Figure 5A
and data not shown). Based on the pathology of TRAF4-deficient mice, it
appears that the absence of TRAF4 expression results in an inability to
properly form the laryngotracheal junction. This correlates with the
high expression of TRAF4 seen in this region during early stages of
development (Figure 5C)
. However, there is no discrete point of TRAF4
expression that can explain the unique defect in TRAF4-deficient mice.
Despite widespread expression of TRAF4, especially in epithelial and
certain neuronal tissues, it appears that the tracheal junction is the
only region where development is critically dependent on TRAF4
expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Laryngotracheal Development
The embryological development of the larynx, trachea, and lungs has been studied mostly through the analysis of serial sections from various stages of development and from wax-plate reconstructions using these sections. These studies established that the larynx and trachea develop as an outpocketing from the ventral side of the foregut (the laryngotracheal groove) (see, for example, ref 34 ). Lung buds are formed at the caudal end, and the trachea grows by extending downward in length.37 The formation of the cranial end of the laryngotracheal tube poses a problem, because initially this end is still connected to the esophagus at the point of entry into the pharynx. The formation of a mature larynx is necessary for separating the two structures. In the last couple of decades, several groups have generated detailed insights into how this process occurs in normal human and mouse embryos.38,39 The mature larynx consists of an infraglottic and a supraglottic cavity. The more cranial supraglottic cavity forms by initial closure of what is termed the sagittal cleft, followed by transverse development of a coronal cleft. This process requires fusion of the two epithelial layers of the sagittal cleft to form the epithelial lamina, which separate the infraglottic cavity from the coronal cleft.39 Eventually, proliferation and rearrangement of the cells in the epithelial lamina allows for establishment of communication between the infraglottic cavity at the cranial end of the trachea and the pharynx, and eventually for development of a complete supraglottic cavity.39
The cartilaginous structures found in adult airways are first seen as clusters of precursor cells that later develop into fully formed cartilage. The origin of the various cartilages can be traced back to the branchial arches that are present before the laryngotracheal groove first forms. The first three branchial arches give rise to structures above the larynx, whereas the laryngeal cartilages originate from the remaining fourth and fifth branchial arches.34
Our data suggest a role for TRAF4 in the establishment of a junction between the trachea and the pharynx at the caudal end of the larynx. The crichoid cartilage is unaffected in TRAF4-deficient mice, but the diameter of the proximal trachea is reduced immediately below the crichoid cartilage. As described above, several complex events occur during laryngotracheal development. TRAF4 could play a role in proliferation and/or reorganization of the involved cells. TRAF4 could also be required in normal innervation of this region of the trachea. The transitional area between the laryngopharynx and esophagus seems to be even more densely innervated than the nasal cavities.39 It is interesting to note that the laryngeal nerve branches stop at the level of the fourth tracheal cartilage, which is precisely the extent of the tracheal defect seen in TRAF4 knock-out mice.
TRAF4 Expression during Development
We and others24 have used in situ hybridization to investigate the expression of TRAF4 during development. For our studies, we focused on the time when the trachea is connecting via the larynx to the pharynx. Despite strong overall expression of TRAF4 in the branchial arches at day 10.5, no localized expression patterns were observed in the region containing the larynx and upper trachea at embryonic day 12.5, which is the time when the connection is starting to form.35,36 E13.5d embryos also were studied, with the same result. TRAF4 is highly expressed in the epithelium of the trachea, but also in other epithelia. We have therefore not been able to implicate a specific cell type in the tracheal deformation of TRAF4-deficient mice based on the embryonic expression pattern of TRAF4.
Wheezing and Pulmonary Inflammation in TRAF4-Deficient Mice
The TRAF4-deficient animals were housed in a barrier facility and were not exposed to any known murine pathogens, but were not kept under sterile conditions. Airborne particles are deposited in the lungs in a size-dependent manner, and failure to clear them can lead to induction of an inflammatory response. Because of the tracheal constriction of TRAF4 knock-out mice, the air flow would be presumed to have a greater element of turbulence. The high-pitched breathing noise heard in some of the TRAF4-deficient mice supports this, because vortices in the trachea are likely to be the cause of this noise.40 The increased turbulence could result in alterations in the deposition of certain airborne particles, pathogens included, depending on their size.41,42 This could contribute to the inflammation observed on autopsy in some mice and the variation in wheezing incidence found. An additional possible explanation for the spontaneous occurrence of airway inflammation is that the mucociliary clearance of larger particles is deficient in these mice, because of tracheal constriction. The reduced weight of TRAF4 knock-out mice may be indicative of the increased effort of respiration in these mice. The energy required for inspiration is dependent on the total airway resistance, and the uppermost part of the airways (including the trachea) is normally responsible for 20–25% of this resistance.43
To investigate whether the tracheal constriction resulted in abnormal air flow to the lungs, the response to aerosolized antigen in immunized mice was determined. Although TRAF4-deficient mice showed a qualitatively normal response, as evidenced by the percentage of eosinophils in the BAL fluid, the total number of white blood cells in the BAL fluid was greatly reduced. These results are consistent with the possibility that the decreased response to OVA in TRAF4 knock-out mice is due to a reduction in the amount of aerosol deposited in the lungs because of abnormal air flow. This further supports the notion of an increase in turbulent air flow as a result of tracheal deformation. Turbulence would result in a greater extent of aerosols being deposited in the oropharynx and upper airways, where they would normally be cleared much more rapidly (about 20-fold faster) than aerosols entering the lungs.41 Even if the tracheobronchial clearance rate is decreased in TRAF4-deficient mice, this presumably would not compensate for the overall reduction in deposited OVA antigen. Although there is spontaneous occurrence of airway inflammation in TRAF4 knock-out mice, the inflammation seen in response to aerosolized antigen is decreased, presumably as a result of differences in deposition patterns of the immunogenic challenge.
TRAF4 Is a Unique Member of the TRAF Family
Despite the importance of other TRAFs in the development and maintenance of a normal immune system, the loss of TRAF4 appears to have no immunological significance. This, as well as the novel role of TRAF4 in tracheal development described here, suggests that TRAF4 plays a unique role, distinct from those of the other TRAF family members. This is consistent with TRAF4 having the most limited sequence homology to the other TRAF family members, and with TRAF4 having yet to be shown to associate with a TNFR family member to a significant extent. The subcellular localization of TRAF4 is also different because, unlike other TRAFs, TRAF4 has been reported to localize to both the nucleus and the cytoplasm.24,25
By generating mice deficient in TRAF4 expression, we have shown that TRAF4 plays an unexpected role in tracheal formation. The TRAF4-deficient mice do not share any of the defects described for the three previously generated TRAF family knock-out mice, which is consistent with the notion that TRAF4 is a functionally distinct member of the family.
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Acknowledgements |
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Footnotes |
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Supported in part by grants by the National Institutes of Health.
Accepted for publication April 27, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J. K. Sax and W. S. El-Deiry Identification and Characterization of the Cytoplasmic Protein TRAF4 as a p53-regulated Proapoptotic Gene J. Biol. Chem., September 19, 2003; 278(38): 36435 - 36444. [Abstract] [Full Text] [PDF]
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 C. J. Zeiss The Apoptosis-Necrosis Continuum: Insights from Genetically Altered Mice Vet. Pathol., September 1, 2003; 40(5): 481 - 495. [Abstract] [Full Text] [PDF]
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F. Henkler, B. Baumann, M. Fotin-Mleczek, M. Weingartner, R. Schwenzer, N. Peters, A. Graness, T. Wirth, P. Scheurich, J. A. Schmid, et al. Caspase-mediated Cleavage Converts the Tumor Necrosis Factor (TNF) Receptor-associated Factor (TRAF)-1 from a Selective Modulator of TNF Receptor Signaling to a General Inhibitor of NF-{kappa}B Activation J. Biol. Chem., August 1, 2003; 278(31): 29216 - 29230. [Abstract] [Full Text] [PDF]
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Y. C. Xu, R. F. Wu, Y. Gu, Y.-S. Yang, M.-C. Yang, F. E. Nwariaku, and L. S. Terada Involvement of TRAF4 in Oxidative Activation of c-Jun N-terminal Kinase J. Biol. Chem., July 26, 2002; 277(31): 28051 - 28057. [Abstract] [Full Text] [PDF]
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 C. H. Regnier, R. Masson, V. Kedinger, J. Textoris, I. Stoll, M.-P. Chenard, A. Dierich, C. Tomasetto, and M.-C. Rio Impaired neural tube closure, axial skeleton malformations, and tracheal ring disruption in TRAF4-deficient mice PNAS, April 16, 2002; 99(8): 5585 - 5590. [Abstract] [Full Text] [PDF]
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J. Y. Chung, Y. C. Park, H. Ye, and H. Wu All TRAFs are not created equal: common and distinct molecular mechanisms of TRAF-mediated signal transduction J. Cell Sci., February 15, 2002; 115(4): 679 - 688. [Abstract] [Full Text] [PDF]
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 H. Harlin, S. B. Reffey, C. S. Duckett, T. Lindsten, and C. B. Thompson Characterization of XIAP-Deficient Mice Mol. Cell. Biol., May 15, 2001; 21(10): 3604 - 3608. [Abstract] [Full Text]
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C. H. Regnier, R. Masson, V. Kedinger, J. Textoris, I. Stoll, M.-P. Chenard, A. Dierich, C. Tomasetto, and M.-C. Rio Impaired neural tube closure, axial skeleton malformations, and tracheal ring disruption in TRAF4-deficient mice PNAS, April 16, 2002; 99(8): 5585 - 5590. [Abstract] [Full Text] [PDF]
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