1 Neural Development Group, Mouse Cancer Genetics Program, NCI, NIH, Frederick,
MD 21702-1201, USA
2 Experimental Immunology Branch, NCI, NIH, Bethesda, MD 20892, USA
3 Aventis Pharmaceuticals, Bridgewater, NJ 08807-0800, USA
* Author for correspondence (e-mail: tessarol{at}ncifcrf.gov)
Accepted 2 August 2004
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SUMMARY |
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Key words: TrkA, NGF, Immune system, Mouse
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Introduction |
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It has long been hypothesized that neurotrophic factors, particularly NGF,
play a role in the development and function of structures outside the nervous
system (Levi-Montalcini,
1987). Indeed, neurotrophins and their receptors are widely
expressed in a variety of non-neuronal tissues throughout development,
including the cardiovascular, endocrine, reproductive, and immune systems
(reviewed by Aloe et al., 1999
;
Tessarollo, 1998
;
Vega et al., 2003
). However,
our knowledge of the function of neurotrophins in non-neuronal tissues is
still fragmented. Evidence for such role was provided by mice lacking NT3
(Ntf3 Mouse Genome Informatics) and BDNF, which revealed the
requirement of these ligands and their receptors for the normal development of
the cardiovascular system (Donovan et al.,
1996
; Donovan et al.,
2000
; Tessarollo et al.,
1997
). In addition, neurotrophin functions outside the nervous
system have been suggested from phenotypes identified in hair follicles and
thymus of neurotrophin and Trk receptor mutant mice
(Botchkarev et al., 2004
;
Garcia-Suarez et al., 2002
;
Garcia-Suarez et al., 2000
).
However, one can not exclude that some of these deficits were indirect effects
of the severe nervous system defects. Moreover, mice lacking neurotrophin
functions, with the exception of p75 and NT5 null mice, die soon after birth
thus precluding analysis of phenotypes in the adult animal.
Within the immune system, p75 and TrkA receptors are widely distributed and
there is an increasing body of evidence suggesting that NGF can regulate
immune cell functions including inflammatory responses
(Aloe et al., 1999;
Micera et al., 2003
;
Tessarollo, 1998
;
Vega et al., 2003
). For
example, NGF blood levels are increased in several autoimmune and allergic
human diseases (Aloe et al.,
1997
; Aloe et al.,
1994
). Furthermore, NGF can accelerate wound healing by modulating
the inflammatory response in mice (Matsuda
et al., 1998
; Micera et al.,
2001
). However, the most intriguing data concern the effects of
NGF on B-cell (Brodie and Gelfand,
1994
; Brodie et al.,
1996
; Otten et al.,
1989
) and mast cell function
(Leon et al., 1994
). NGF can
regulate immunoglobulin (Ig) production in vitro and serves as a survival
factor for memory B cells (Brodie and
Gelfand, 1994
; Brodie et al.,
1996
; Torcia et al.,
1996
). Furthermore, NGF is synthesized, stored and released by
mast cells (Leon et al.,
1994
). Yet, the physiological rather than pharmacological
relevance of these effects is not clear
(Tessarollo, 1998
).
P75 mutant mice are viable and therefore amenable to investigation of
non-neuronal structures including the immune system in the adult
(Lee et al., 1992). However, a
recent investigation has not found major deficits in the spleen and thymus of
p75 mutant mice (Garcia-Suarez et al.,
2001
) and our own preliminary analysis has failed to unveil any
overt immunological deficits (V.C. and L.T., unpublished). Therefore, we
hypothesized that TrkA mediates most of the NGF functions described in the
immune system likewise in the nervous system. To test this hypothesis in vivo,
we decided to generate and characterize mice with a conditional null allele of
the TrkA gene.
The classic approach to study the function of a gene in a specific organ
includes the generation of a mouse strain with loxP sites flanking the gene of
interest (floxed) and another mouse strain with the Cre recombinase under the
transcriptional control of a promoter expressed in the organ of interest. By
crossing these two strains, deletion of the gene flanked by loxP sequences
should be achieved in an organ site-specific manner. However, one major
limitation of this approach is the fact that deletion of the floxed gene is
not always complete in all cells where Cre should be active
(Rickert et al., 1997). Thus,
the residual activity of the gene can potentially confound the analysis. This
is particularly critical for the immune system in which clonal expansion of
cells with a functional floxed allele may compensate for certain phenotypes.
To obviate this problem we have reversed the strategy and activated TrkA
specifically in neuronal structures within a null mutation background. This
approach provides a means to distinguish between an intrinsic role of TrkA in
the immune system versus defects that are caused by nervous system
deficiencies in the complete knockout mouse model.
Using this strategy, we found that on the whole, the TrkA-deficient immune system develops normally. This result demonstrates that many of the dramatic effects previously reported by pharmacological or immuno-depletion approaches do not reflect physiological roles of TrkA in the immune system during homeostasis. However, we also identified disregulation of Ig production and accumulation of a B-cell subset. These data are the first demonstration that NGF/TrkA indeed modulates immunological functions not mediated by the nervous system in vivo.
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Materials and methods |
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The Rosa-26 reporter mouse strain was kindly provided by Phil Soriano
(Soriano, 1999). Adult
C57Bl/6-SCID mice (Bosma et al.,
1983
) used in reconstitution experiments were purchased from
Charles River (Frederick, MD, USA) and reconstituted by tail-vein injection
with 5x106 E14.5 fetal liver cells.
All animals were treated in accordance with the guidelines provided by the Animal Care and Use Committee of the National Cancer Institute at Frederick (MD, USA).
Immunization protocols
To test T-cell-dependent (TD) humoral responses, 3-6-month-old mice were
subjected to retro-ocular bleeding to determine basal Ig levels and then were
injected intraperitoneally with 100 µg KLH-TNP (keyhole limpet
hemocyanin-2,4,6,-trinitrophenyl; Biosearch Technologies, Novato, CA, USA) in
PBS (day 0). After 40 days, mice were given a boosting dose of the same Ag and
blood was drawn again after 1 week (day 47) and 2 weeks (day 54) from the
boost. To test T-cell-independent (TI) type I and type II responses, mice were
prebled and immunized with 20 µg of lipolysaccaride (LPS)-TNP or 25 µg
of Ficoll-TNP (Biosearch Technologies), respectively. Blood was collected one
and two weeks after challenge.
Northern blot and RT-PCR
Total RNA was isolated from different tissues by a single-step method (RNA
STAT-60, Tel-Test `B', Friendswood, TX, USA) and analyzed by classical
northern blot analysis. TrkA transcripts were detected by using a rat
kinase-specific TrkA probe (spanning exon 12 to exon 17). RT-PCR was performed
using SuperScriptTM RT-PCR System (GIBCO-Invitrogen, Grand Island, NY,
USA) according to manufacturer instructions with the following primers: TrkA
Exon10-Exon14 fragment: F primer: 5'-ACG GTA ACA GCA CAT CAA
GAG-3'; R primer: 5'-CGG AGG AAA CGG TTG AGG TC-3'.
ß-actin (F primer: 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3'; R
primer: 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3').
Histological procedures
Sections (5 µm) from paraffin-embedded tissues were stained with
anti-CD45R, 1:200 (B220, RA3-6B2 BD-Pharmingen, San Jose, CA, USA), anti-CD3,
1:600 (Dako, Carpinteria, CA, USA), and anti-mouse F4/80, 1:25 (Caltag,
Burlingame, CA, USA) for B-cell, T-cell and macrophage-specific
immunostaining, respectively. Apoptotic cells were detected with the Apoptag
kit (Intergen, Purchase, NY, USA) following the manufacturer's
recommendations. For ß-galactosidase staining, E11.5 embryos were fixed
in PBS without Mg2+/Ca2+, 1% formaldehyde, 0.2%
glutaraldehyde, 0.02% NP-40 for 30 minutes at 4°C, washed twice in PBS
without Mg2+/Ca2+ at RT for 20 minutes, and stained
overnight at 30°C with PBS without Mg2+/Ca2+, 5 mM
K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM
MgCl2, and 1 mg/ml X-gal. For adult tissue staining, 20 µm
frozen sections were post-fixed with 4% phosphate-buffered paraformaldehyde
for 5 minutes, washed three times for 5 minutes with 100 mM sodium phosphate
buffer, 20 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, then
stained overnight as above, rinsed in dH2O and counterstained with
neutral red for 40 seconds. After three rinses in 100% ethanol, cover slips
were mounted in xylene.
Automated blood cell counts were performed using the HEMAVET® 850 apparatus (CDC Technologies, Oxford, CT, USA).
FACS and ELISA Ig analysis
For FACS analysis, samples were prepared as previously described
(Coppola et al., 1998). All
the unlabelled antibodies, phycoerythrin (PE), fluorescein isothiocyanate
(FITC) or PerCP-conjugated were purchased from BD Pharmingen (San Jose, CA,
USA). Elisa Ig analysis was performed using a modified protocol from BD
Pharmingen. Briefly, plates (Dynex-Immulon®) were coated
overnight with capture anti-Ig-specific Ab in PBS, washed once with PBS-0.05%
Tween® 20, blocked for 1 hour at RT with PBS-3% BSA and
incubated with serial dilutions of sera overnight at 4°C. The following
day, the plates were washed five times and incubated with a secondary Ab Ig
conjugated to HRP diluted 1:1000 for 1 hour at RT. After six further washes,
substrate (ABTS Microwell Peroxidase Substrate System, Gaithersburg, MD, USA)
was added. Capture mouse anti-IgM, anti-IgG1, anti-IgG2a, anti-IgG2b,
anti-IgG3, anti-IgA, and the HRP-conjugated Abs were purchased from Southern
Biotech (Birmingham, AL, USA). All standards for the different Ig classes,
capture anti-IgE, and biotin-conjugated anti-IgE were purchased from BD
Pharmingen. For the IgE Elisa, HRP-streptavidin was purchased from Jackson
ImmunoResearch (West Grove, PA, USA). For anti-TNP-specific Ig dosage, the
plates were coated overnight at 4°C with OVA-TNP (Biosearch Technologies)
at 10 µg/ml in carbonate/bicarbonate buffer pH 9.6. The following steps
were similar to the dosage of total sera Ig. Plates were read using a
Benchmark Bio-Rad Elisa reader at 405 nm wavelength (Software: Microplate
Manager III®, Bio-Rad, Hercules, CA, USA). Data were analyzed
and plotted using GraphPad PRISM® 3.0 (GraphPad Software, San
Diego, CA, USA).
Passive cutaneous anaphylaxis
IgE-dependent passive cutaneous reaction was generated by sensitizing the
skin with an intradermal injection of anti-dinitrophenyl (DNP) IgE, followed
18-20 hours later by an injection of DNP-albumin (100 µl of a 1 µg/µl
solution in saline) into the mouse tail vein. The right ear was injected
intradermally with 25 ng of anti-DNP IgE and the left ear received 25 µl of
saline to serve as control. Ear thickness was measured with a caliper (Dyer
Company, Lancaster, PA, USA) 15 minutes before and 15 minutes after the
antigen challenge.
Generation of bone marrow derived mast cells and degranulation assay
Bone marrow derived mast cells (BMMC) were generated by culturing femoral
bone marrow cells from 6- to 8-week-old mice. Mice were sacrificed by
CO2 asphyxiation and intact femurs were removed. Bone marrow cells
were harvested by flushing the bone shaft with DMEM (GIBCO-Invitrogen) medium
with 2% FBS. Red blood cells were lysed by incubation with 0.8% ammonium
chloride with 0.1 mM EDTA. Bone marrow cell cultures were established at a
density of 1x106 cells/ml in DMEM containing 20% FBS, 100
U/ml penicillin, 100 µg/ml streptomycin, 50 ng/ml recombinant mouse stem
cell factor (SCF), 10 ng/ml recombinant mouse IL-3. Non-adherent cells were
transferred to fresh culture plates once a week and fed by replacing 50% of
the medium twice a week. After 4 to 6 weeks of culture, the purity of BMMC
culture was greater that 95% as confirmed by FACS analysis for FcRI and
c-kit expression. BMMC were activated via Fc
RI stimulation by
first sensitizing overnight with anti-DNP IgE antibody (Sigma Aldrich, St
Louis, MO, USA) at 1 µg/ml in culture medium. Sensitized cells were washed
and stimulated for 30 minutes at 37°C with 100 ng/ml DNP conjugate of
human serum albumin (DNP-HSA) (Sigma) in Hanks' Balance Salt Solution
supplemented with 2 mg/ml glucose and 0.03% BSA. Cell supernatants were
harvested and the cell pellets were lysed by freeze- and-thaw cycle.
ß-hexosaminidase was quantified in the supernatants and cell lysates by
spectrophotometric analysis of hydrolysis of p-NAG
(p-hitrophenyl-N-acetyl-D-glucosaminidine) (Sigma). ß-hexosaminidase
release was calculated as the percentage of ß-hexosaminidase presented in
the supernatants relative to the total amount of ß-hexosaminidase present
in the supernatant and cell pellet.
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Results |
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TrkA is dispensable for the development of immune system organs
Over the years, the NGF/TrkA ligand/receptor system has been extensively
implicated in regulating the development and function of the immune system.
However, the lack of suitable in vivo models has hampered the investigation
toward the dissection of a direct role of NGF/TrkA in this system. For
example, systemic knockouts or models based on immunodepletion of endogenous
NGF affect all organs including the nervous system, which may be required for
normal development of the lymphoid organs. Our model inactivates the NGF
receptor TrkA only in non-neuronal tissues and thus allows us to investigate
the intrinsic function of this receptor in structures including the organs of
the immune system. Anatomical and histological analysis of thymus, spleen and
lymph nodes did not reveal any significant difference between TrkA-deficient
and control tissues. For example, cellularity and morphology of the spleen of
mutant mice were similar to control littermates (not shown). In addition,
lymphoid follicles (Fig. 2A,B)
and distribution of apoptotic cells (Fig.
2C,D) were also present in similar numbers. Spleen sections
immunostained with the B-cell-specific B220 and T-cell-specific anti-CD3
antibodies did not detect any difference in number or distribution of B- and
T-cells in the T-1-Cre;TrkAneo/neo mutants compared
with control littermates. (Fig.
2E-H). Similarly, lymph nodes, thymus and bone marrow seemed
unaffected by TrkA loss at the anatomical and histological level (data not
shown). To assess the major cell populations of the immune system organs, we
performed cytofluorimetric analysis on thymus, spleen and bone marrow cells
with anti-B220, -IgM, -IgD, -CD19, -CD3, -CD4, -CD8, -Mac-1, -Gr1 and -CD5
antibodies. No quantitative or qualitative abnormalities in the B- and T-cell
populations, macrophages and granulocytes (not shown) were detected in mutant
mice. Furthermore, blood cell counts of mutant mice were also comparable to
littermate controls (Table
1).
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Mast cells develop and function in TrkA mutant mice
Mast cells constitute another cell population for which there is ample
literature suggesting a direct role of NGF in their development and function
(Aloe et al., 1999;
Tessarollo, 1998
;
Vega et al., 2003
). Toluidine
blue and alcian blue/safranin staining of histological preparations from skin,
spleen and lymph node of T
1-Cre;TrkAneo/neo mice
showed that mast cells were present in comparable numbers and distribution to
that of tissues from control littermates
(Fig. 2L,M and data not shown).
Thus, TrkA signaling is not required for mast cell differentiation in vivo. To
begin to address whether mast cells lacking TrkA displayed functional
abnormalities in vivo, we employed a wheal and flare paradigm. The skin of
ears from mutant and control mice was sensitized with an intradermal injection
of anti-DNP IgE followed by a systemic injection of DNP-albumin, as described.
Compared with saline challenged skin, IgE/DNP intradermal injections caused
prominent mast cell degranulation and mixed inflammatory cell infiltrates in
both mutant and wild-type mice (Fig.
3A-F). However, no significant differences were observed in mast
cell degranulation activity in the two genotypes
(Fig. 3 and data not shown). It
should be noted that this result does not preclude the presence of subtle
defects caused by TrkA loss because we have observed great variability in the
response among individuals of both control and mutant group. In addition, on
the current genetic background, the response to DNP by mutant and control mice
was approximately 20-fold lower than that of mice on a pure Balb/c background,
making it difficult to assess the presence of subtle phenotypes in vivo (data
not shown). Nevertheless, the in vivo degranulation response of mast cells
lacking TrkA would suggest that this gene is dispensable for mast cells'
activity. Because in vivo mast cells develop normally and can be activated by
passive cutaneous reaction, we then derived mast cells from bone marrow (BMMC)
of TrkA mutant and control mice to investigate whether loss of TrkA had any
effect at all on the differentiation and functional potential of this cell
population. No apparent differences were observed in their capacity to
differentiate in vitro. However, when BMMC were induced to degranulate by IgE
cross-linking, the TrkA-deficient BMMC showed a modest but significant
reduction in ß-hexoseaminidase release in comparison with BMMC derived
from control mice (P<0.01)
(Fig. 3G). This result suggests
that, although TrkA is not required for mast cell development in vivo, its
loss may nevertheless impact their normal activity.
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Discussion |
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The phenotype observed in the mutant mouse used in our study is less severe
than the one observed in the complete TrkA-deficient mice which have thymus
abnormalities consistent with organogenesis defects
(Garcia-Suarez et al., 2000).
Because the difference between these two mutants is mainly the presence (this
study) or the absence (Garcia-Suarez et
al., 2000
) of a fully developed nervous system, it is tempting to
speculate that a cross-talk between the immune system and the nervous system
is required not only for normal function but also development of the immune
system. Similarly, less severe phenotypes were observed in our mutant compared
with those of a transgenic mouse model producing anti-NGF neutralizing
antibodies (Ruberti et al.,
2000
) which develops massive cell death in the spleen, skeletal
dystrophy and body weight reduction resulting in mouse lethality. Although
that model is of value for other studies
(Capsoni et al., 2000
), our
data suggest that the non-neuronal phenotypes are most likely caused by
abnormal innervation or a toxic effect of the anti-NGF antibodies on different
tissues. Indeed, the massive cell death observed in the spleen is not easily
reconcilable with the limited expression of NGF receptors in this organ.
NGF has been particularly studied for its effects on B lymphocytes. Several
reports have suggested that in vitro NGF can modulate Ig production as well as
B-cell proliferation and survival. However, these data often lack consistency
and depending on the model used, somewhat contrasting results were described
(Brodie et al., 1996;
Kimata et al., 1991a
;
Kimata et al., 1991b
;
Kimata et al., 1991c
). Thus,
to date it is still unclear what role NGF exerts in vivo. To a certain degree,
the lack of consistency of those results may reflect differences between cell
lines. In this study, we have found that although B lymphocytes appear to
develop normally in the absence of TrkA, Ig production is affected. Some Ig
classes are significantly increased. These increases were subtle, but could be
confirmed in a `classical reconstitution model'. Mice reconstituted with
TrkA-deficient cells showed the same B- and T-cell numbers compared with
animals reconstituted with littermate wild-type cells (not shown). As observed
in the `reverse conditional TrkA mouse model', we found increased Ig levels in
mice reconstituted with TrkA-deficient fetal cells. Importantly, these data
validate our genetic model.
It has been reported that antibody neutralization of NGF in vitro and in
vivo affects the viability of B memory lymphocytes, suggesting an autocrine
role for NGF on the survival of memory B lymphocytes
(Torcia et al., 1996).
Although the mechanism for this effect is still unknown, it has recently been
suggested that inactivation of p38 MAPK, possibly through a TrkA-activated
phosphatase, may be required to prevent B memory lymphocyte apoptosis
(Torcia et al., 2001
). In
contrast, we have found that TrkA-deficient mice can mount a robust B memory
response (Fig. 5A). We tested
the response one and two weeks after the boost dose of antigen, whereas Torcia
et al. (Torcia et al., 1996
)
analyzed the response after only four days from the recall-dose of immunogen
(six days after anti-NGF treatment). Thus, we cannot exclude that the
discrepancy is caused by different timing of the analysis. Alternatively, we
have exclusively ablated TrkA function while anti-NGF antibodies were used in
the previous study. Therefore, it is also possible that other players such as
p75 (or other yet unknown NGF receptors) may be involved in the development of
a B memory response. However, our preliminary data suggest that p75-deficient
mice can also mount a humoral response.
Following immunization with a T-dependent antigen (KLH-TNP), TrkA-deficient
mice are able to produce specific Ig at levels comparable to those of control
littermates. Notably, following immunization the mutants produce more IgG2b, a
class that is overall similarly represented in basal conditions. These data
provide definitive evidence that NGF/TrkA does not regulate Ig class switch as
previously suggested (Torcia et al.,
1996).
In addition to disregulation of Ig levels, we have also found that TrkA
deficiency causes an increase in peritoneal B1 cells with aging. Several
studies have shown that B1 cells are increased in mouse models deficient for
molecules that negatively regulate BCR signaling, whereas B1 are decreased
when molecules positively regulating BCR signaling are inactivated
(Hardy et al., 2000;
Hayakawa and Hardy, 2000
).
Therefore, it appears that TrkA may play an inhibitory role on BCR signaling.
A direct link between BCR- and TrkA-signaling has not yet been described.
However, both receptors can independently affect vav, a Rac guanine nucleotide
exchange factor expressed exclusively in hematopoietic cells
(Melamed et al., 1999
). Thus,
it is tempting to speculate that TrkA may influence BCR signaling by acting on
one of its downstream players such as vav. This effect on BCR signaling could
ultimately be involved in dictating B-cell commitment to proliferation or
differentiation.
Collectively, our data suggest that TrkA, although modulating immune system
functions, is not required for its normal development. B- and T-cell number
and distribution in spleen, thymus, bone marrow and blood appear unaffected by
TrkA deletion. Similar results have been obtained for mast cells in skin,
spleen and lymph nodes despite reports that NGF injections cause their
increase in various tissues of neonatal rats
(Aloe and Levi-Montalcini,
1977; Bienenstock et al.,
1987
; Bullock and Johnson, Jr,
1996
). This is a striking result because many studies suggested
that disruption of the NGF/TrkA system in the immune system would cause
dramatic deficits. In our initial in vivo studies of TrkA-deficient mast
cells, we have been unable to detect a clear phenotype probably because of
compensatory mechanisms or vagaries associated with the mixed genetic
background typical of targeted mutation mouse models
(Fig. 3A-F). However, we have
detected some abnormalities in their degranulation potential in vitro,
suggesting that TrkA may have a role in mast cell function
(Fig. 3G). Mast cell
degranulation followed by mediator release is a well-described contributor to
airway hyper-reactivity. It has been reported that NGF augments the allergic
early-phase reaction in the lung (Path et
al., 2002
). In vivo experiments using this TrkA mutant mouse model
in a pure genetic background will help elucidate the role and contributions of
NGF/TrkA in mast cell function and their net contribution to mast cell-driven
pathophysiology. It has been reported that plasma and/or serum levels of NGF
are increased in several allergic disorders including asthma,
urticaria-angioedema, allergic rhinoconjunctivitis and vernal
keratoconjunctivitis (Aloe and Tuveri,
1997
; Bonini et al.,
1996
; Lambiase et al.,
1995
). Because mast cells express exclusively TrkA and not p75, it
is tempting to suggest that this receptor may provide a target for the
management of inflammatory diseases
(Micera et al., 2003
). Yet,
efforts toward this goal have not been vigorously pursued because the
available data had suggested that tampering with the NGF/TrkA pathway would
cause severe immunological deficits. However, the relatively limited impact on
immune system development reported here indicates that TrkA could provide a
useful target for the control of inflammatory diseases. Thus, this mouse model
should provide a useful system to test the contribution of NGF/TrkA in the
pathology of these disorders.
Finally, this novel approach by `reverse conditional gene targeting', could be applied to many other systems in which complete inactivation of a gene in specific organs/tissues is required but cannot be achieved with the currently available recombinase and transgenic systems.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Aloe, L. and Levi-Montalcini, R. (1977). Mast cells increase in tissues of neonatal rats injected with the nerve growth factor. Brain Res. 133,358 -366.[CrossRef][Medline]
Aloe, L. and Tuveri, M. A. (1997). Nerve growth factor and autoimmune rheumatic diseases. Clin. Exp. Rheumatol. 15,433 -438.[Medline]
Aloe, L., Bracci-Laudiero, L., Bonini, S. and Manni, L. (1997). The expanding role of nerve growth factor: from neurotrophic activity to immunologic diseases. Allergy 52,883 -894.[Medline]
Aloe, L., Simone, M. D. and Properzi, F. (1999). Nerve growth factor: a neurotrophin with activity on cells of the immune system. Microsc. Res. Tech. 45,285 -291.[CrossRef][Medline]
Aloe, L., Skaper, S. D., Leon, A. and Levi-Montalcini, R. (1994). Nerve growth factor and autoimmune diseases. Autoimmunity 19,141 -150.[Medline]
Bibel, M. and Barde, Y. A. (2000).
Neurotrophins: key regulators of cell fate and cell shape in the vertebrate
nervous system. Genes Dev.
14,2919
-2937.
Bienenstock, J., Tomioka, M., Matsuda, H., Stead, R. H., Quinonez, G., Simon, G. T., Coughlin, M. D. and Denburg, J. A. (1987). The role of mast cells in inflammatory processes: evidence for nerve/mast cell interactions. Int. Arch. Allergy Appl. Immunol. 82,238 -243.[Medline]
Bonin, A., Reid, S. W. and Tessarollo, L. (2001). Isolation, microinjection, and transfer of mouse blastocysts. Methods Mol. Biol. 158,121 -134.[Medline]
Bonini, S., Lambiase, A., Angelucci, F., Magrini, L., Manni, L.
and Aloe, L. (1996). Circulating nerve growth factor levels
are increased in humans with allergic diseases and asthma. Proc.
Natl. Acad. Sci. USA 93,10955
-10960.
Bosma, G. C., Custer, R. P. and Bosma, M. J. (1983). A severe combined immunodeficiency mutation in the mouse. Nature 301,527 -530.[Medline]
Botchkarev, V. A., Botchkareva, N. V., Peters, E. M. and Paus, R. (2004). Epithelial growth control by neurotrophins: leads and lessons from the hair follicle. Prog. Brain Res. 146,493 -513.[Medline]
Brodie, C. and Gelfand, E. W. (1994). Regulation of immunoglobulin production by nerve growth factor: comparison with anti-CD40. J. Neuroimmunol. 52, 87-96.[CrossRef][Medline]
Brodie, C., Oshiba, A., Renz, H., Bradley, K. and Gelfand, E. W. (1996). Nerve growth-factor and anti-CD40 provide opposite signals for the production of IgE in interleukin-4-treated lymphocytes. Eur. J. Immunol. 26,171 -178.[Medline]
Bullock, E. D. and Johnson, E. M., Jr (1996).
Nerve growth factor induces the expression of certain cytokine genes and bcl-2
in mast cells. Potential role in survival promotion. J. Biol.
Chem. 271,27500
-27508.
Capsoni, S., Ugolini, G., Comparini, A., Ruberti, F., Berardi,
N. and Cattaneo, A. (2000). Alzheimer-like neurodegeneration
in aged antinerve growth factor transgenic mice. Proc. Natl. Acad.
Sci. USA 97,6826
-6831.
Coppola, V., Veronesi, A., Indraccolo, S., Calderazzo, F., Mion,
M., Minuzzo, S., Esposito, G., Mauro, D., Silvestri, B., Gallo, P. et al.
(1998). Lymphoproliferative disease in human peripheral blood
mononuclear cell-injected SCID mice. IV. Differential activation of human Th1
and Th2 lymphocytes and influence of the atopic status on lymphoma
development. J. Immunol.
160,2514
-2522.
Crowley, C., Spencer, S. D., Nishimura, M. C., Chen, K. S., Pitts-Meek, S., Armanini, M. P., Ling, L. H., MacMahon, S. B., Shelton, D. L., Levinson, A. D. et al. (1994). Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons. Cell 76,1001 -1011.[Medline]
Donovan, M. J., Hahn, R., Tessarollo, L. and Hempstead, B. L. (1996). Identification of an essential nonneuronal function of neurotrophin-3 in mammalian cardiac development. Nat. Genet. 14,210 -213.[Medline]
Donovan, M. J., Lin, M. I., Wiegn, P., Ringstedt, T., Kraemer,
R., Hahn, R., Wang, S., Ibanez, C. F., Rafii, S. and Hempstead, B. L.
(2000). Brain derived neurotrophic factor is an endothelial cell
survival factor required for intramyocardial vessel stabilization.
Development 127,4531
-4540.
Garcia-Suarez, O., Blanco-Gelaz, M. A., Lopez, M. L., Germana, A., Cabo, R., Diaz-Esnal, B., Silos-Santiago, I., Ciriaco, E. and Vega, J. A. (2002). Massive lymphocyte apoptosis in the thymus of functionally deficient TrkB mice. J. Neuroimmunol. 129, 25-34.[CrossRef][Medline]
Garcia-Suarez, O., Germana, A., Hannestad, J., Ciriaco, E., Laura, R., Naves, J., Esteban, I., Silos-Santiago, I. and Vega, J. A. (2000). TrkA is necessary for the normal development of the murine thymus. J. Neuroimmunol. 108, 11-21.[CrossRef][Medline]
Garcia-Suarez, O., Germana, A., Hannestad, J., Ciriaco, E., Silos-Santiago, I., Germana, G. and Vega, J. A. (2001). Involvement of the NGF receptors (Trka and p75lngfr) in the development and maintenance of the thymus. Ital. J. Anat. Embryol. 106,279 -285.
Gloster, A., Wu, W., Speelman, A., Weiss, S., Causing, C., Pozniak, C., Reynolds, B., Chang, E., Toma, J. G. and Miller, F. D. (1994). The T alpha 1 alpha-tubulin promoter specifies gene expression as a function of neuronal growth and regeneration in transgenic mice. J. Neurosci. 14,7319 -7330.[Abstract]
Hardy, R. R. and Hayakawa, K. (2001). B cell development pathways. Annu. Rev. Immunol. 19,595 -621.[CrossRef][Medline]
Hardy, R. R., Li, Y. S., Allman, D., Asano, M., Gui, M. and Hayakawa, K. (2000). B-cell commitment, development and selection. Immunol. Rev. 175, 23-32.[CrossRef][Medline]
Hasan, M., Polic, B., Bralic, M., Jonjic, S. and Rajewsky, K. (2002). Incomplete block of B cell development and immunoglobulin production in mice carrying the muMT mutation on the BALB/c background. Eur. J. Immunol. 32,3463 -3471.[CrossRef][Medline]
Hayakawa, K. and Hardy, R. R. (2000). Development and function of B-1 cells. Curr. Opin. Immunol. 12,346 -353.[CrossRef][Medline]
Hempstead, B. L. (2002). The many faces of p75NTR. Curr. Opin. Neurobiol. 12,260 -267.[CrossRef][Medline]
Huang, E. J. and Reichardt, L. F. (2001). Neurotrophins: roles in neuronal development and function. Annu. Rev. Neurosci. 24,677 -736.[CrossRef][Medline]
Kaplan, D. R. and Miller, F. D. (2000). Neurotrophin signal transduction in the nervous system. Curr. Opin. Neurobiol. 10,381 -391.[CrossRef][Medline]
Kimata, H., Yoshida, A., Ishioka, C., Kusunoki, T., Hosoi, S. and Mikawa, H. (1991a). Nerve growth factor specifically induces human IgG4 production. Eur. J. Immunol. 21,137 -141.[Medline]
Kimata, H., Yoshida, A., Ishioka, C. and Mikawa, H. (1991b). Nerve growth factor inhibits immunoglobulin production by but not proliferation of human plasma cell lines. Clin. Immunol. Immunopathol. 60,145 -151.[CrossRef][Medline]
Kimata, H., Yoshida, A., Ishioka, C. and Mikawa, H. (1991c). Stimulation of Ig production and growth of human lymphoblastoid B-cell lines by nerve growth factor. Immunology 72,451 -452.[Medline]
Lambiase, A., Bonini, S., Bonini, S., Micera, A., Magrini, L., Bracci-Laudiero, L. and Aloe, L. (1995). Increased plasma levels of nerve growth factor in vernal keratoconjunctivitis and relationship to conjunctival mast cells. Invest. Ophthalmol. Vis. Sci. 36,2127 -2132.[Abstract]
Lee, K. F., Li, E., Huber, L. J., Landis, S. C., Sharpe, A. H., Chao, M. V. and Jaenisch, R. (1992). Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system. Cell 69,737 -749.[Medline]
Leon, A., Buriani, A., Dal Toso, R., Fabris, M., Romanello, S., Aloe, L. and Levi-Montalcini, R. (1994). Mast cells synthesize, store, and release nerve growth factor. Proc. Natl. Acad. Sci. USA 91,3739 -3743.[Abstract]
Levi-Montalcini, R. (1987). The nerve growth factor 35 years later. Science 237,1154 -1162.[Medline]
Liebl, D. J., Klesse, L. J., Tessarollo, L., Wohlman, T. and
Parada, L. F. (2000). Loss of brain-derived neurotrophic
factor-dependent neural crest-derived sensory neurons in neurotrophin-4 mutant
mice. Proc. Natl. Acad. Sci. USA
97,2297
-2302.
Ma, W., Tessarollo, L., Hong, S. B., Baba, M., Southon, E.,
Back, T. C., Spence, S., Lobe, C. G., Sharma, N., Maher, G. W. et al.
(2003). Hepatic vascular tumors, angiectasis in multiple organs,
and impaired spermatogenesis in mice with conditional inactivation of the VHL
gene. Cancer Res. 63,5320
-5328.
Matsuda, H., Koyama, H., Sato, H., Sawada, J., Itakura, A.,
Tanaka, A., Matsumoto, M., Konno, K., Ushio, H. and Matsuda, K.
(1998). Role of nerve growth factor in cutaneous wound healing:
accelerating effects in normal and healing-impaired diabetic mice.
J. Exp. Med. 187,297
-306.
Melamed, I., Patel, H., Brodie, C. and Gelfand, E. W. (1999). Activation of Vav and Ras through the nerve growth factor and B cell receptors by different kinases. Cell Immunol. 191,83 -89.[CrossRef][Medline]
Micera, A., Puxeddu, I., Aloe, L. and Levi-Schaffer, F. (2003). New insights on the involvement of Nerve Growth Factor in allergic inflammation and fibrosis. Cytokine Growth Factor Rev. 14,369 -374.[CrossRef][Medline]
Micera, A., Vigneti, E., Pickholtz, D., Reich, R., Pappo, O.,
Bonini, S., Maquart, F. X., Aloe, L. and Levi-Schaffer, F.
(2001). Nerve growth factor displays stimulatory effects on human
skin and lung fibroblasts, demonstrating a direct role for this factor in
tissue repair. Proc. Natl. Acad. Sci. USA
98,6162
-6167.
Osborn, L., Rosenberg, M. P., Keller, S. A. and Meisler, M. H. (1987). Tissue-specific and insulin-dependent expression of a pancreatic amylase gene in transgenic mice. Mol. Cell. Biol. 7,326 -334.[Medline]
Otten, U., Ehrhard, P. and Peck, R. (1989). Nerve growth factor induces growth and differentiation of human B lymphocytes. Proc. Natl. Acad. Sci. USA 86,10059 -10063.[Abstract]
Path, G., Braun, A., Meents, N., Kerzel, S., Quarcoo, D., Raap,
U., Hoyle, G. W., Nockher, W. A. and Renz, H. (2002).
Augmentation of allergic early-phase reaction by nerve growth factor.
Am. J. Respir. Crit. Care Med.
166,818
-826.
Rickert, R. C., Roes, J. and Rajewsky, K.
(1997). B lymphocyte-specific, Cre-mediated mutagenesis in mice.
Nucleic Acids Res. 25,1317
-1318.
Ruberti, F., Capsoni, S., Comparini, A., Di Daniel, E., Franzot,
J., Gonfloni, S., Rossi, G., Berardi, N. and Cattaneo, A.
(2000). Phenotypic knockout of nerve growth factor in adult
transgenic mice reveals severe deficits in basal forebrain cholinergic
neurons, cell death in the spleen, and skeletal muscle dystrophy.
J. Neurosci. 20,2589
-2601.
Schlesinger, M., Boyse, E. A. and Old, L. J. (1965). Thymus cells of radiation-chimeras: TL phenotype, sensitivity to guinea-pig serum, and origin from donor cells. Nature 206,1119 -1121.[Medline]
Smeyne, R. J., Klein, R., Schnapp, A., Long, L. K., Bryant, S., Lewin, A., Lira, S. A. and Barbacid, M. (1994). Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 368,246 -249.[CrossRef][Medline]
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21,70 -71.[CrossRef][Medline]
Tessarollo, L. (1998). Pleiotropic functions of neurotrophins in development. Cytokine Growth Factor Rev. 9,125 -137.[CrossRef][Medline]
Tessarollo, L. (2001). Manipulating mouse embryonic stem cells. Methods Mol. Biol. 158, 47-63.[Medline]
Tessarollo, L., Tsoulfas, P., Donovan, M. J., Palko, M. E.,
Blair-Flynn, J., Hempstead, B. L. and Parada, L. F. (1997).
Targeted deletion of all isoforms of the trkC gene suggests the use of
alternate receptors by its ligand neurotrophin-3 in neuronal development and
implicates trkC in normal cardiogenesis. Proc. Natl. Acad. Sci.
USA 94,14776
-14781.
Torcia, M., Bracci-Laudiero, L., Lucibello, M., Nencioni, L., Labardi, D., Rubartelli, A., Cozzolino, F., Aloe, L. and Garaci, E. (1996). Nerve growth factor is an autocrine survival factor for memory B lymphocytes. Cell 85,345 -356.[Medline]
Torcia, M., De Chiara, G., Nencioni, L., Ammendola, S., Labardi,
D., Lucibello, M., Rosini, P., Marlier, L. N., Bonini, P., Dello Sbarba, P. et
al. (2001). Nerve growth factor inhibits apoptosis in memory
B lymphocytes via inactivation of p38 MAPK, prevention of Bcl-2
phosphorylation, and cytochrome c release. J. Biol.
Chem. 276,39027
-39036.
Vega, J. A., Garcia-Suarez, O., Hannestad, J., Perez-Perez, M. and Germana, A. (2003). Neurotrophins and the immune system. J. Anat. 203,1 -19.[CrossRef][Medline]
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