|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 41, 32114-32121, October 13, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, June 14, 2000, and in revised form, July 21, 2000
Adenosine deaminase (ADA) deficiency results in a
combined immunodeficiency brought about by the immunotoxic properties
of elevated ADA substrates. Additional non-lymphoid abnormalities are
associated with ADA deficiency, however, little is known about how
these relate to the metabolic consequences of ADA deficiency. ADA-deficient mice develop a combined immunodeficiency as well as
severe pulmonary insufficiency. ADA enzyme therapy was used to examine
the relative impact of ADA substrate elevations on these phenotypes. A
"low-dose" enzyme therapy protocol prevented the pulmonary
phenotype seen in ADA-deficient mice, but did little to improve their
immune status. This treatment protocol reduced metabolic disturbances
in the circulation and lung, but not in the thymus and spleen. A
"high-dose" enzyme therapy protocol resulted in decreased metabolic
disturbances in the thymus and spleen and was associated with
improvement in immune status. These findings suggest that the pulmonary
and immune phenotypes are separable and are related to the severity of
metabolic disturbances in these tissues. This model will be useful in
examining the efficacy of ADA enzyme therapy and studying the
mechanisms underlying the immunodeficiency and pulmonary phenotypes
associated with ADA deficiency.
Adenosine deaminase
(ADA)1 deficiency was the
first of the immunodeficiency diseases for which the underlying
biochemical defect was discovered (1). This unexpected finding revealed
the crucial importance of ADA for the development and function of the
immune system. Subsequent analysis indicated that ADA deficiency
accounted for approximately 20% of the incidence of combined
immunodeficiency disease in humans (2). However, the causal link
between purine metabolic disturbances associated with ADA deficiency
and the resulting immunodeficiency has not been determined. In recent years, ADA deficiency has received added interest because it has served
as a testing ground for the development of novel therapies, especially
enzyme therapy (3) and gene therapy (4, 5).
ADA deficiency is the most severe of the immunodeficiencies in humans
(6). Without intervention, affected individuals die from overwhelming
opportunistic infections within the first few months of life (2, 7).
The immunodeficiency is the most studied feature of ADA deficiency,
however, other abnormalities and disturbances are seen. Additional
features include hepatocellular damage (8), pulmonary insufficiency
(2), neurological disorders (2), renal problems and bony abnormalities
(9). Although these conditions are often thought to be secondary to the
immunodeficiency, some investigators have suggested that they may be a
primary consequence of ADA deficiency and not secondarily related to
infections stemming from the immunodeficiency (8, 10).
In attempting to understand the multitude of abnormalities associated
with ADA deficiency, most attention has focused on the substrates of
the ADA catalyzed reaction, adenosine and 2'-deoxyadenosine. Each
nucleoside is known to be elevated in ADA-deficient patients (11-13),
and may cause developmental or physiological disturbances in a number
of ways. Adenosine functions as an intercellular signaling molecule by
engaging G protein-coupled receptors on the surface of target cells
(14). Mammalian cells possess four different adenosine receptors, A1,
A2a, A2b, and A3, that function in a variety of physiological
circumstances. In this regard, adenosine is involved in T cell receptor
signaling (15), heart rate (16), blood pressure (17), renal function
(18), inflammatory responses (19, 20), and neurotransmission (21). ADA
plays a critical role in controlling the concentration of adenosine,
thereby affecting many areas of intercellular signaling. Thus, in the
absence of ADA, the uncontrolled elevations of adenosine could unleash
a variety of adenosine signaling cascades with detrimental effects on
numerous areas of physiology. The other ADA substrate,
2'-deoxyadenosine, is known to be cytotoxic by interference with a
number of critical metabolic pathways. High levels of 2'-deoxyadenosine
may lead to the interference of transmethylation reactions (22),
reduction of deoxynucleotide synthesis (2), or activation of the
apoptotic caspase cascade (23). We have previously shown that some
tissues in mice, especially the thymus and spleen, produce high levels of 2'-deoxyadenosine (24, 25), making them especially sensitive to
metabolic consequences of 2'-deoxyadenosine-induced metabolic cytotoxicity. We have also shown that the murine fetal liver is sensitive to 2'-deoxyadenosine elevations (26). Thus, ADA deficiency may provoke a variety of consequences, either through metabolic disturbances caused by elevated 2'-deoxyadenosine, or cell signaling disturbances caused by elevated adenosine.
We have recently used a two-stage genetic engineering strategy to
produce a line of ADA-deficient mice (25). These mice develop a
combined immunodeficiency that is associated with profound disturbances
in purine metabolism. Elevations in ADA substrates were greatest in the
thymus and spleen (25), suggesting the metabolic disturbances in these
major immune organs play a role in the ensuing immunodeficiency. In
addition to immunodeficiency, ADA-deficient mice develop severe
abnormalities including rib cage alterations, renal abnormalities, and
pulmonary insufficiency (25). The mice fail to thrive and die from
respiratory insufficiency by 3 weeks of age (25, 27).
ADA-deficient mice serve as a useful model for elucidating the
biochemical basis of the various phenotypic abnormalities associated with the loss of ADA function. In considering the complexity of the
phenotypes associated with ADA deficiency in humans and mice, it is
likely that some features may be attributed to alterations in adenosine
signaling, whereas others are due to 2'-deoxyadenosine-induced metabolic disturbances. Here we report the use of ADA enzyme therapy to
regulate the level of metabolic disturbances associated with ADA
deficiency and show that relatively low levels of ADA enzyme therapy
correct the pulmonary abnormalities. Much higher doses of enzyme
therapy are required to alleviate the immunodeficiency, a finding
relevant to the use of enzyme therapy to treat ADA deficiency in
humans. Available evidence suggests that the pulmonary insufficiency in
ADA-deficient mice results from abnormalities in adenosine signaling
(27), whereas the immunodeficiency stems largely from 2'-deoxyadenosine-induced metabolic cytotoxicity. Thus, ADA enzyme therapy serves as a convenient experimental strategy to regulate the
metabolic consequences of ADA deficiency and differentially correct
abnormalities associated with ADA deficiency.
Transgenic Mice--
Mice heterozygous for the null
Ada allele (m1/+) and carrying a placenta-specific ADA
minigene necessary for prenatal rescue (Tg) were intercrossed (25).
Genotypes were determined by Southern blot analysis of genomic DNA
obtained from tails at weaning (25). Wild type (+/+) and heterozygous
(m1/+) mice with or without the ADA minigene (Tg) were designated as
"ADA+ controls," whereas homozygous mutant mice carrying the ADA
minigene (Tg-m1/m1) were designated ADA-deficient (25). All mice were
housed in cages equipped with microisolator lids and maintained under
strict containment protocols.
PEG-ADA Enzyme Therapy--
Polyethylene glycol-modified-ADA
(PEG-ADA, AdagenTM) was kindly provided by Enzon Inc.
(Piscataway, NJ). A "low-" and "high-dose" protocol was
utilized. For the "low-dose" protocol, mice were injected
intramuscularly with dosages of PEG-ADA designed to deliver 100 to 500 units (1 unit is defined as the amount of enzyme necessary to convert 1 µmol of adenosine to inosine per min at 25 °C) of PEG-ADA per kg
body weight. Injections were started on postnatal day 3 and were given
every 7 days for 6 weeks. For the high-dose protocol, mice were
injected intraperitaneally with dosages of PEG-ADA designed to deliver
1000-5000 units of PEG-ADA per kg body weight. Injections were started
on postnatal day 10 and were given every 3-4 days.
Zymogram Analysis and Determination of PEG-ADA Trough
Values--
Whole blood was collected from the tail vein or from the
heart at the time of euthanasia. One volume of homogenizing buffer (10 mM Tris, pH 8.0, 1 mM Na2EDTA, and
1 mM Histological Analysis--
Animals were sacrificed and the lungs
infused with 0.5 ml of fixative (4% paraformaldehyde in
phosphate-buffered saline) prior to fixation overnight at 4 °C.
Fixed lung tissues were rinsed in phosphate-buffered saline,
dehydrated, and embedded in paraffin according to standard techniques.
Sections (5 µm) were stained with hematoxylin and eosin (H&E) using a
Rapid Chrome staining kit (Shandon Lipshaw).
Flow Cytometry of Leukocyte Populations--
The following
monoclonal antibodies used for flow cytometric analysis were obtained
from Phar-Mingen, Inc. as fluorescein isothiocyanate or
phycoerthrin conjugates: anti-CD3- Analysis of Adenine Nucleosides and Nucleotides and Determination
of AdoHcy Hydrolase Enzymatic Activity--
Tissues were removed from
mice and quickly frozen in liquid nitrogen. Nucleosides and nucleotides
were extracted and analyzed by HPLC according to established procedures
(24, 29). AdoHcy hydrolase enzymatic activity was determined in freshly
prepared tissue extracts according to established procedures (24,
30).
Stability and Distribution of PEG-ADA in Mice--
Before
attempting to use PEG-ADA to treat ADA-deficient mice, it was necessary
to conduct experiments to determine adequate PEG-ADA treatment
protocols. ADA+ control mice were treated with PEG-ADA in order to
monitor the consistency of drug delivery. Mice were given an
intramuscular injection of PEG-ADA (2.5 to 10 µl, depending on age)
once weekly for 6 weeks. This dose was designed to deliver between 100 and 500 units of PEG-ADA/kg body weight. PEG-ADA levels were estimated
semiquantitatively in whole blood 2 days following the last injection
and were found to be similar in all samples (Fig.
1A), suggesting that the
treatment protocol was capable of consistently delivering enzyme to the circulation of mice. To determine the stability of PEG-ADA in the
circulation, ADA+ control mice were treated with PEG-ADA as described
above, and blood PEG-ADA levels were estimated at various time points
following the last treatment (Fig. 1B). At 2 days following
the last injection, PEG-ADA levels were approximately three times
greater than endogenous ADA. By 4 days following the last injection,
blood levels had dropped, and were equivalent to endogenous ADA levels.
At 8 days post-PEG-ADA, only trace amounts were seen, and by 16 days
post-treatment PEG-ADA was not detectable. From these results it was
estimated that the half-life of PEG-ADA in the murine circulatory
system was 3-4 days. Trough values of PEG-ADA activity achieved with
this protocol are shown in Table I. This
regimen was referred to as a low-dose treatment protocol, in comparison
with the higher dose regimen used in later studies (see below).
PEG-ADA Enzyme Therapy Rescues ADA-deficient
Mice--
ADA-deficient mice die by 3 weeks of age in association with
respiratory distress, lymphopenia, and severe metabolic
disturbances (25). To determine if PEG-ADA enzyme therapy could
rescue ADA-deficient mice, 3-day-old pups were treated intramusculary
with PEG-ADA according to the low-dose regimen. At 3 weeks of age,
blood was collected and analyzed for endogenous ADA or PEG-ADA (Fig.
2A). As expected, blood
collected from an untreated ADA-deficient animal (Fig. 2A,
lane 2) did not exhibit ADA or PEG-ADA enzyme activity. This
animal was smaller than control littermates, exhibited severe respiratory distress, and died late on day 21. In contrast, blood collected from a treated ADA-deficient animal showed only PEG-ADA enzyme activity (Fig. 2A, lane 4). Growth of this
animal was indistinguishable from that of ADA+ control littermates and
it survived more than 6 months on this treatment protocol with no sign
of morbidity. This result demonstrated that a low-dose of PEG-ADA
resulted in circulating PEG-ADA activity sufficient to prevent
lethality in ADA-deficient mice.
PEG-ADA Is Found Only in the Circulatory System of ADA-deficient
Mice--
PEG-ADA does not enter erythrocytes or blood lymphocytes of
human ADA-deficient patients, however, the distribution of PEG-ADA in
other tissues is not known (2, 28). ADA-deficient mice provided the
opportunity to examine the levels of PEG-ADA present in various
non-hematopoietic tissues. Shown in Fig. 2B is the analysis
of PEG-ADA activity in extracts from various tissues taken from an
ADA-deficient animal 2 days following its last PEG-ADA injection (low
dose regimen). Whereas PEG-ADA was detectable in the blood of this
animal, it was not detected in any of the tissues examined, including
the thymus, spleen, liver, kidney, and lung. These data suggested that
most if not all of the PEG-ADA in ADA-deficient mice was found in the
circulatory system, as has been assumed to be the case in humans (2,
28).
Low-dose PEG-ADA Treatments Prevent Lung Inflammation and
Damage--
ADA-deficient mice succumb to severe respiratory distress
by 3 weeks of age (25, 27). Lungs of these animals show defects in
alveogenesis and severe lung inflammation and damage is present (Fig.
3B) (27). The ability of the
low-dose PEG-ADA regimen to rescue ADA-deficient mice from lethality
(Fig. 2A) was associated with significant reduction in the
severe lung inflammation and damage seen in untreated ADA-deficient
mice (Fig. 3). These findings suggested that low-dose PEG-ADA provided
adequate metabolic protection to prevent the pulmonary phenotype and
allowed for the survival of these animals.
High Doses of PEG-ADA Are Required to Establish ADA Trough Values
in ADA-deficient Mice Similar to Those Seen in ADA-deficient
Humans--
The initial treatment for seriously ill, newly diagnosed
human infants with ADA-deficient severe combined immunodeficiency disease, is a twice weekly injection of 30 units/kg PEG-ADA (31, 32). This treatment protocol typically leads to trough plasma ADA
activity levels that range from approximately 50 to 120 µmol/h/ml (2). Based on body weight, the low-dose treatment protocol used in
ADA-deficient mice was considerably higher than this initial dosage
used in ADA-deficient humans. However, the mean trough plasma ADA
activity in mice treated with this regimen was considerably lower than
the trough levels observed in ADA-deficient humans (Table I, (2)).
Therefore, we also treated mice with 10-fold greater amounts of
PEG-ADA. This high-dose protocol was sufficient to maintain trough
plasma PEG-ADA activity at levels equivalent to that observed with a 60 unit/kg/week regimen in ADA-deficient humans (Table I (2)). These data
demonstrated that much higher doses of PEG-ADA, on a per kg body weight
basis, are required in the mouse to establish plasma PEG-ADA levels
that are therapeutically effective in humans.
High Doses of PEG-ADA Overcome the Block in Thymocyte Development
Seen in ADA-deficient Mice--
ADA deficiency in humans (2) and mice
(25) is associated with pronounced lymphopenia. Treatment of
ADA-deficient humans with PEG-ADA results in varying degrees of
restoration of mature lymphoid cells (2, 31). To begin to assess the
impact of PEG-ADA enzyme therapy on lymphoid development in
ADA-deficient mice, total lymphoid counts were examined in the thymuses
of ADA+ control and ADA-deficient mice given either a low- or high-dose of PEG-ADA (Fig. 4, A and
B). Lymphoid counts in ADA-deficient thymuses were greatly
reduced on postnatal day 17 (Fig. 4A). Low doses of PEG-ADA
did not have a significant impact on the lymphopenia seen in the thymus
(Fig. 4B), however, the high-dose treatment allowed for an
improvement in thymocyte counts to near control values (Fig.
4B).
Immature thymocytes are organized into two major groups based on the
cell surface expression of CD4 and CD8 (33). The most immature
thymocytes express neither CD4 nor CD8 and are termed double negative
(DN), while more mature thymocytes express both CD4 and CD8 and are
termed double positive (DP). Flow cytometry using antibodies to
specific markers of thymocyte differentiation were used to determine
the effect of ADA enzyme therapy on thymocyte differentiation. The
percentage of DP cells was diminished from 80% in ADA+ control
thymuses to 20% in ADA-deficient thymuses on postnatal day 17 (Fig.
4C). In addition, there was an increase in DN cells in
ADA-deficient thymuses, verifying a potential block in thymocyte
differentiation before the DP stage. The distribution of DP and DN
cells in ADA-deficient thymuses improved slightly following low-dose
PEG-ADA treatment, while a high-dose treatment improved the
distribution of cells in ADA-deficient thymuses to near control values
(Fig. 4D). These data suggested that high doses of PEG-ADA
are needed to see an improvement in thymocyte differentiation in
ADA-deficient mice.
Improvement of Peripheral T, B, and NK Cell Lymphopenia in
ADA-deficient Mice Treated with PEG-ADA--
ADA deficiency in humans
leads to a combined immunodeficiency that is characterized by a
pronounced T, B, and NK cell lymphopenia (6). PEG-ADA enzyme therapy
has been shown to be effective in improving the status of these cell
types (2, 31, 34). ADA-deficient mice are known to exhibit T and B cell
lymphopenia (25), however, the status of NK cells in these mice was not known. Antibodies against cell surface markers specific for mature T,
B, and NK cells were used to monitor these cell populations in
ADA-deficient mice and ADA-deficient mice treated with PEG-ADA. Similar
to what is seen in ADA-deficient humans, all three lymphoid cell
populations were greatly diminished in ADA-deficient mice on postnatal
day 17 (Fig. 5B). There was a
slight improvement in the absolute numbers of these cells in the
spleens of ADA-deficient mice treated with a low-dose of PEG-ADA, and
an even greater improvement in ADA-deficient mice treated with a
high-dose regimen (Fig. 5C). These results suggested that
the immune phenotype seen in ADA-deficient mice resembled that seen in
ADA-deficient humans. Furthermore, a high-dose of ADA enzyme therapy
was capable of providing a substantial improvement in T, B, and NK
cells numbers.
The Improvement of Metabolic Disturbances in Tissues of
ADA-deficient Mice Varies with the Amount of PEG-ADA
Given--
Elevated levels of the ADA substrates adenosine and
2'-deoxyadenosine in plasma and urine, resulting in markedly elevated pools of dATP and reduced AdoHcy hydrolase activity in red blood cells,
are the biochemical hallmarks of ADA deficiency (2). PEG-ADA enzyme
therapy has been shown to correct these metabolic disturbances in
erythrocytes of ADA-deficient humans (2, 3, 31). However, it has not
been possible to quantitate the levels of substrate elevations in their
tissues. The generation of ADA-deficient mice has made assessment of
tissue substrate elevations possible, and it has been shown that both
adenosine and 2'-deoxyadenosine are highly elevated in circulation and
in tissues of ADA-deficient mice (25, 35). To determine what effect
PEG-ADA enzyme therapy has on ADA substrates in tissues, adenine
nucleosides were extracted from tissues of ADA-deficient mice treated
with PEG-ADA, and were quantitated by reversed-phase HPLC (Fig.
6). Treatment of ADA-deficient mice with
a low-dose regimen of PEG-ADA prevented elevations of adenosine and
2'-deoxyadenosine in the serum and lung, but not in the thymus and
spleen (Fig. 6). In contrast, high doses of PEG-ADA were able to
markedly improve the metabolic disturbances found in serum and lung as
well as the thymus and spleen (Fig. 6). These results demonstrated that
PEG-ADA provided varying degrees of metabolic protection in the whole
animal.
AdoHcy Hydrolase Enzyme Activity and Elevations in dATP Vary in
Tissues of ADA-deficient Mice Following PEG-ADA Enzyme
Therapy--
Decreased AdoHcy hydrolase enzymatic activity and the
accumulation of dATP in RBCs are commonly noted features in
ADA-deficient patients (2, 36, 37). In humans, PEG-ADA enzyme therapy readily prevents the inhibition of AdoHcy hydrolase and dATP
accumulation in RBCs (2, 3, 31). However, as with the assessment of substrate elevations, it has not been possible to examine the effects
of PEG-ADA enzyme therapy on AdoHcy hydrolase and dATP in other
tissues. To address this, we determined the levels of AdoHcy hydrolase
enzyme activity and dATP levels in various tissues of ADA-deficient
mice following PEG-ADA treatment. Previous examination of AdoHcy
hydrolase enzymatic activity in ADA-deficient mice demonstrated that
the greatest decrease in activity was seen in RBCs and in the thymus
and spleen (25, 35). In ADA-deficient mice treated with a low-dose
regimen of PEG-ADA, a substantial decrease in AdoHcy hydrolase activity
was still noted in the thymus and spleen, and to a lesser extent in
RBCs and lung (Fig. 7A). A
high-dose regimen restored AdoHcy hydrolase activity in RBCs and lung
and provided partial improvement in the thymus and spleen (Fig.
7A). The levels of dATP that accumulate in RBCs of
ADA-deficient mice are 7-8-fold higher than those measured in the
thymus (25, 35). In contrast, the levels of dATP that accumulated in
the RBCs of ADA-deficient mice treated with a low-dose regimen of
PEG-ADA were similar to those measured in other tissues from the same animals (Fig. 7B). Elevations in dATP were noted in all
tissues following a low-dose regimen, with the greatest relative
accumulation occurring in the thymus (Fig. 7B). This
suggested that a low-dose of PEG-ADA was capable of preventing much of
the accumulation of dATP in erythrocytes and lungs, but not dATP
accumulation in other tissues. Treatment of ADA-deficient mice with a
high-dose of PEG-ADA completely prevented the accumulation of dATP in
both erythrocytes and tissues (Fig. 7B). Improvements in
these metabolic end points correlated with the degree of immune
reconstitution, adding support to the hypothesis that metabolic
abnormalities are mechanistically involved in the immune phenotypes
associated with ADA deficiency.
ADA deficiency in humans is most often associated with a severe
combined immunodeficiency (2, 6). However, additional non-lymphoid
abnormalities have been noted, including bone, kidney, and adrenal
abnormalities (9), liver damage (8), and problems associated with the
nervous system (10). Pulmonary insufficiency in ADA-deficient patients
is similar to pulmonary insufficiency seen in patients with other forms
of severe combined immunodeficiency disease. It is not present at
birth, and is most commonly found in association with known pathogens.
The lung injury in ADA-deficient mice is present early in life and is
not associated with infection (27). In the current study, PEG-ADA
enzyme therapy was used to study the impact of tissue metabolic
disturbances on the pulmonary and immune phenotypes seen in
ADA-deficient mice (25, 27). ADA-deficient mice were initially given a
low-dose of PEG-ADA that resulted in circulating enzyme activities
below plasma PEG-ADA levels achieved in patients with ADA-deficient
severe combined immunodeficiency disease (2, 31, 32).
Nonetheless, this low-dose of PEG-ADA was capable of rescuing
ADA-deficient mice from lethality due to pulmonary injury. This
low-dose treatment corrected systemic and lung accumulations of
adenosine and 2'-deoxyadenosine, but did not prevent elevations of
adenosine and 2'-deoxyadenosine in the thymus and spleen. Consequently,
defects in lymphoid development persisted. A PEG-ADA treatment protocol
designed to maintain circulating PEG-ADA activity at concentrations
that are in the effective range seen in humans (31, 32) resulted in
substantial improvements in immune status as well as a major reduction
in the degree of substrate elevations in the thymus and spleen. These
findings suggest that the pulmonary and immune phenotypes seen in
ADA-deficient mice are separable and are mediated by the different
degrees of metabolic disturbances associated with ADA deficiency.
The pulmonary phenotype seen in ADA-deficient mice is characterized by
a developmental defect in alveogenesis that results in enlarged
alveolar airways (27). In addition, these animals develop severe lung
inflammation and damage that kills these animals by 3 weeks of age.
Lung damage and inflammation was associated with the elevation of
adenosine and to a lesser extent, 2'-deoxyadenosine in the lungs (25).
We show that maintaining ADA-deficient mice on a low-dose of PEG-ADA
resulted in normal lung development, and lung inflammation and damage
was not seen. This was associated with prevention of adenosine and
2'-deoxyadenosine elevations in the lungs, suggesting ADA substrates
are mediating the abnormal lung development and inflammation. Both
adenosine (14) and 2'-deoxyadenosine (2) have potent physiological
effects on cells. Adenosine elicits its actions on cells by engaging G
protein-coupled receptors on the cell surface (14). This signaling
pathway plays important roles in many aspects of lung inflammation and
damage (20), and adenosine elevations in the lung could influence
normal adenosine signaling during lung development, and may promote
inflammation and damage in ADA-deficient lungs. Efforts to identify the
adenosine receptor subtypes expressed in normal and ADA-deficient
lungs, and the use of genetic and pharmacological approaches to study their function will help us to understand the impact of adenosine signaling in this model. 2'-Deoxyadenosine elevation has been implicated to lead to the disruption of cell growth and development (38, 39) and influence apoptosis (23). Although 2'-deoxyadenosine elevations in ADA-deficient lungs were relatively low, the potential impact of 2'-deoxyadenosine cytotoxicity on the pulmonary phenotype cannot be ruled out at this time.
A major finding of this study was the observation that the pulmonary
and immune phenotypes could be separated. This was made possible by the
finding that a low-dose of PEG-ADA was sufficient to normalize
adenosine and 2'-deoxyadenosine levels in the circulation and the well
vascularized lung, but was not sufficient to prevent substrate
elevations in the thymus and spleen. Elevated thymic nucleosides are
likely due to high levels of adenosine and 2'-deoxyadenosine generated
in the thymus due to the large amount of apoptosis naturally occurring
in this organ as a part of thymocyte
development2 (24, 25). This
idea is supported by the observation that enzyme treatments that
maintained high PEG-ADA trough values were needed to achieve metabolic
and phenotypic improvement in the thymus and spleen. The benefits of
PEG-ADA enzyme therapy in humans have focused predominantly on the
immune system; however, some studies show a rapid improvement of
non-immune phenotypes following PEG-ADA treatment or red cell
transfusion (8, 10). Pulmonary insufficiency is common in ADA-deficient
patients, and these insufficiencies are most often attributed to
bacterial or viral pneumonia that arises from a compromised immune
system. However, in many cases of interstitial pneumonia an organism
cannot be isolated (2). Our observations in ADA-deficient mice suggest
that it is possible that the metabolic disturbances may directly
contribute to the pulmonary insufficiency occurring in ADA-deficient patients.
ADA deficiency in humans results in a severe combined immunodeficiency
characterized by a depletion of T, B, and NK cells (6). All three of
these cell populations were reduced in ADA-deficient mice, making them
a valuable model for studying mechanisms governing the immunodeficiency
seen as a result of ADA deficiency. In this model we were able to
conduct experiments not permissible in humans, including the removal of
lymphoid organs for the examination of specific lymphoid cells, and the
investigation of metabolic disturbances in various tissues. Results
showed an apparent block in T cell development at the transition from
DN to DP stage in ADA-deficient thymuses, which was in agreement with
previously observed findings2 (24, 25). High doses of
PEG-ADA were required to see an improvement in lymphocyte
differentiation and an increase in the number of mature T, B, and NK
cells. T cells have been demonstrated to be sensitive in
vitro to the metabolic consequences of ADA deficiency (reviewed in
Ref. 2). Toxicity has been demonstrated for adenosine (40), however;
more notable are the effects of 2'-deoxyadenosine cytotoxicity on
lymphoid cells (2, 30, 41). Less is known about the impact of ADA
substrate accumulation on B and NK cells. However, results presented
here and results in ADA-deficient patients during PEG-ADA therapy and
after its discontinuation (8, 34), suggest that B and NK cells are
directly impacted by ADA substrate accumulation. More research into the
mechanisms of substrate actions on B and NK cells are needed to improve
our understanding of the immunodeficiency associated with ADA deficiency.
These studies lay the groundwork for the use of ADA-deficient mice to
advance the treatment of ADA deficiency in humans. Rescuing ADA-deficient mice from lethality using PEG-ADA enzyme therapy will
make possible the examination of bone marrow transplantation and gene
therapy interventions in these animals. Furthermore, this model will be
useful in the analysis of the metabolic and immunologic consequences
associated with the cessation of PEG-ADA enzyme therapy. This
information will be important for fully understanding the potential
benefits of ADA gene therapy, which is currently performed in
conjunction with PEG-ADA therapy. In addition to therapeutic advances,
the ability to manipulate substrate accumulations in ADA-deficient mice
using various doses of ADA enzyme therapy will allow for the
examination of the impact of these substrates or their metabolites on
the immune system and other phenotypes associated with ADA deficiency.
In particular, the use of PEG-ADA in ADA-deficient mice provides a
means to manipulate the levels of adenosine in tissues and cells
in vivo. This may provide an opportunity to study a vast
array of physiological systems that are influenced by adenosine signaling.
We thank Enzon Inc., Piscataway, NJ, for
providing AdagenTM.
*
This work was supported by National Institutes of Health
Grants AI43572 and HL61888 (to M. R. B.), DK46207 and DK54443
(to R. E. K.), and DK20902 (to M. S. H.), Texas
Higher Education Coordinating Board Applied Technology Grant 011618-060 (to M. R. B.), and a grant from Enzon Inc. (to M. S. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 713-500-6087;
Fax: 713-500-0652; E-mail: Michael.R.Blackburn@uth.tmc.edu.
Published, JBC Papers in Press, July 24, 2000, DOI 10.1074/jbc.M005153200
2
L. F. Thompson, unpublished observations.
The abbreviations used are:
ADA, adenosine
deaminase;
AdoHcy, S-adenosylhomocysteine;
RBCs, red blood
cells;
PEG-ADA, polyethylene glycol-ADA;
NK cells, natural killer
cells;
DN, double negative;
DP, double positive;
HPLC, high performance
liquid chromatography.
The Use of Enzyme Therapy to Regulate the Metabolic and
Phenotypic Consequences of Adenosine Deaminase Deficiency in
Mice
DIFFERENTIAL IMPACT ON PULMONARY AND IMMUNOLOGIC
ABNORMALITIES*
§,,,,,, and
Department of Biochemistry and Molecular
Biology, University of Texas Health Science Center at Houston Medical
School, Houston, Texas 77030 and the ¶ Departments of Medicine and
Biochemistry, Duke University Medical Center,
Durham, North Carolina 27707
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol) was added and the samples were
lysed by several rounds of freeze-thawing. Tissues were dissected,
rinsed in cold phosphate-buffered saline, and sonicated in 3 volumes of homogenizing buffer. Samples were then centrifuged at
14,000 × g at 4 °C and protein concentration determined in the supernatants (Bio-Rad). Samples were run on thin
agarose gels (Innovative Chemistry) in the cold, and were then overlaid
with enzyme substrate and colorimetric detection mixture (24). For
determination of ADA trough values, heparinized blood samples were
collected from the tail vein of mice just prior to the next injection
of PEG-ADA, and plasma isolated by low-speed centrifugation. ADA
specific activity in plasma was determined using established protocols
(28).
, clone 145-2C11; anti-CD4, clone
RM4-5; anti-CD8a, clone 53-6.7; anti-CD45R/B220, clone RA3-6B2; clone
M1/70; anti-TCR chain, clone H57-597; anti-pan-NK cells, clone DX5;
anti-IgM, clone R6-60.2; Fc control anti-CD16/CD32 (Fc
III/IIR),
clone 2.4G2. Phenotyping of leukocyte populations in thymuses and
spleens was conducted by direct two-color analysis on an EPIC Profile
Analyzer (PharMingen, Inc.). Thymuses and spleens were excised from
control and ADA-deficient age-matched littermates or mice treated with
PEG-ADA and processed for flow cytometry (24). Two-color flow cytometry
analysis was conducted on 1 × 106 viable leukocytes,
and data were obtained on two-color dot plot profiles.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (50K):
[in a new window]
Fig. 1.
Equal delivery and half-life determination of
PEG-ADA. ADA+ control mice were treated with a low-dose regimen of
PEG-ADA for 6 weeks. Panel A, levels of endogenous ADA
(mADA) and PEG-ADA were examined by zymogram analysis. Hemoglobin
(Hg) was used as an internal control for equal loading of
samples. The genotypes of the mice were determined by Southern blot
analysis and are shown above each sample. Panel
B, blood was isolated at 2, 4, 8, and 16 days following the last
low-dose PEG-ADA treatment.
Plasma ADA activity
View larger version (64K):
[in a new window]
Fig. 2.
Zymogram analysis demonstrating the rescue of
ADA-deficient mice, and the tissue distribution of PEG-ADA.
Panel A, zymogram analysis of blood from untreated mice
(lanes 1 and 2) or mice treated (lanes
3-5) with a low-dose of PEG-ADA. Panel B, zymogram
analysis of equal amounts of protein from ADA+ control blood or blood
and various tissues from an ADA-deficient mouse 2 days after a low-dose
regimen of PEG-ADA. Purine nucleoside phosphorylase (PNP)
was used as a positive control. Protein concentrations for ADA were 5 µg/lane, while protein concentrations for PNP were 1 µg/lane.
View larger version (111K):
[in a new window]
Fig. 3.
Prevention of the pulmonary phenotype in
ADA-deficient mice treated with a low-dose of PEG-ADA. Lungs were
collected from ADA+ control mice and processed for histological
analysis. Panel A, H&E stained lung section from an
18-day-old ADA+ control mouse. Panel B, H&E stained section
from an 18-day-old ADA-deficient mouse demonstrating severe alveolar
abnormalities (*), inflammation (arrow), and thickening of
pulmonary blood vessel walls (bv). H&E section through
18-day-old (panel C) or 6-week-old (panel D)
ADA-deficient mouse maintained on a low-dose regimen of PEG-ADA. Notice
that the pulmonary abnormalities described in B are
prevented in ADA-deficient mice at both stages. The scale
bar in each panel is equal to 50 µm. Day 18 analysis was
repeated four times, and 6-week analysis was conducted twice, all with
similar results.
View larger version (20K):
[in a new window]
Fig. 4.
Lymphoid cell counts and thymocyte
distributions. Panel A, lymphoid cell counts from ADA+
control (open bar, n = 6) and ADA-deficient
(black bar, n = 6) thymuses at postnatal day
17. Panel B, cell counts from thymuses of 7-8-week-old ADA+
control mice (open bar, n = 5), 6-week-old
ADA-deficient mice 2 days after a "low-dose" (shaded
bar, n = 6) or high-dose (black bar,
n = 3) regimen of PEG-ADA. All mean cell counts are
given in millions ± S.E., and percentages represent percent of
control values. DP and DN cells were determined
using anti-CD4 and anti-CD8. Panel C, relative percentage of
DN and DP thymocytes found in the thymus of 17-day-old ADA+ control
(open bar) and ADA-deficient (black bar) mice.
Panel D, relative percentage of DN and DP thymocytes found
in the thymus of 7-8-week-old ADA+ control mice (open bar)
and ADA-deficient mice 2 days after a low-dose (shaded bar)
and high-dose (black bar) regimen of PEG-ADA. Values are
presented as mean percentages ± S.E., and the n for
each sample is the same as earlier in this legend.
View larger version (23K):
[in a new window]
Fig. 5.
Analysis of T, B, and NK cell
populations. Spleenocytes were collected and subjected to flow
cytometric analysis. Specific antibodies to cell surface markers were
used to identify T cells (anti-CD3 and anti-TCR ), B cells
(anti-CD45R and anti-IgM), and NK cells (anti-DX5). Total cells counts
for each were determined in spleens from 17-day-old ADA+ control
(panel A, n = 6) and ADA-deficient
(panel B, n = 6) mice, and from
7-8-week-old ADA+ control mice and ADA-deficient mice treated with a
low- (n = 6) or high-dose (n = 3)
regimen of PEG-ADA (panel C). Values are presented as mean
cell counts ± S.E., and percentages represent percent of control
values.
View larger version (20K):
[in a new window]
Fig. 6.
Metabolic disturbances in tissues.
Adenine nucleosides were extracted from various tissues of
7-8-week-old ADA+ control mice (open bars) or ADA-deficient
mice 2 days following a low-dose (shaded bar) or high-dose
(black bar) regimen of PEG-ADA, and quantitated using HPLC.
Panel A, adenosine levels. Panel B,
2'-deoxyadenosine levels. Values are given as mean nanomoles per mg of
protein ± S.E., n = 3 for each. ND,
not detectable at a minimal detection value of 
0.001 nmol/mg of
protein.
View larger version (23K):
[in a new window]
Fig. 7.
AdoHcy hydrolase enzymatic activity and dATP
accumulation. Panel A, AdoHcy hydrolase enzymatic
activity in various tissues collected from 7-8-week-old ADA+ control
mice (open bars) and ADA-deficient mice 2 days following a
low-dose (shaded bars) or high-dose (black bars)
regimen of PEG-ADA. Mean AdoHcy hydrolase specific activities are given
as nanomoles of adenosine converted to AdoHcy/min/mg of protein ± S.E., n = 5 for each. Panel B, dATP was
quantitated in various tissues of 7-8-week-old ADA+ control mice
(open bars) and ADA-deficient mice 2 days following a
low-dose (shaded bars) and high-dose (black bars)
regimen of PEG-ADA. Mean dATP values are presented as nanomoles/mg of
protein ± S.E., n = 4 for each.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Giblett, E. R.,
Anderson, J. E.,
Cohen, F.,
Pollara, B.,
and Meuwissen, H. J.
(1972)
Lancet
2,
1067-1069
2.
Hershfield, M. S.,
and Mitchell, B. S.
(1995)
in
The Metabolic and Molecular Basis of Inherited Disease
(Scriver, C. R.
, Beaudet, A. L.
, Sly, W. S.
, and Valle, D., eds), Vol. 1
, pp. 1725-1768, McGraw-Hill, Inc., New York
3.
Hershfield, M. S.
(1995)
Clin. Immunol. Immunopathol.
76,
S228-232
4.
Blaese, R. M.,
Culver, K. W.,
Miller, A. D.,
Carter, C. S.,
Fleisher, T.,
Clerici, M.,
Shearer, G.,
Chang, L.,
Chiang, Y.,
Tolstoshev, P.,
et al..
(1995)
Science
270,
475-480
5.
Bordignon, C.,
Notarangelo, L. D.,
Nobili, N.,
Ferrari, G.,
Casorati, G.,
Panina, P.,
Mazzolari, E.,
Maggioni, D.,
Rossi, C.,
Servida, P.,
et al..
(1995)
Science
270,
470-475
6.
Buckley, R. H.,
Schiff, R. I.,
Schiff, S. E.,
Markert, M. L.,
Williams, L. W.,
Harville, T. O.,
Roberts, J. L.,
and Puck, J. M.
(1997)
J. Pediatr.
130,
378-387
7.
Hirschhorn.
(1999)
in
Primary Immunodeficiency Disease: A Molecular and Genetic Approach
(Ochs, H. D.
, Smith, C. I. E.
, and Puck, J. M., eds)
, pp. 121-138, Oxford University Press, New York
8.
Bollinger, M. E.,
Arredondo-Vega, F. X.,
Santisteban, I.,
Schwarz, K.,
Hershfield, M. S.,
and Lederman, H. M.
(1996)
N. Engl. J. Med.
334,
1367-1371
9.
Ratech, H.,
Greco, M. A.,
Gallo, G.,
Rimoin, D. L.,
Kamino, H.,
and Hirschhorn, R.
(1985)
Am. J. Pathol.
120,
157-169
10.
Hirschhorn, R.,
Paageorgiou, P. S.,
Kesarwala, H. H.,
and Taft, L. T.
(1980)
N. Engl. J. Med.
303,
377-380
11.
Mitchell, B. S.,
Sidi, Y.,
Hershfield, M.,
and Koller, C. A.
(1985)
Ann. N. Y. Acad. Sci.
451,
129-137
12.
Donofrio, J.,
Coleman, M. S.,
Hutton, J. J.,
Daoud, A.,
Lampkin, B.,
and Dyminski, J.
(1978)
J. Clin. Invest.
62,
884-887
13.
Morgan, G.,
Levinsky, R. J.,
Hugh-Jones, K.,
Fairbanks, L. D.,
Morris, G. S.,
and Simmonds, H. A.
(1987)
Clin. Exp. Immunol.
70,
491-499
14.
Olah, M. E.,
and Stiles, G. L.
(1995)
Annu. Rev. Pharmacol. Toxicol.
35,
581-606
15.
Huang, S.,
Apasov, S.,
Koshiba, M.,
and Sitkovsky, M.
(1997)
Blood
90,
1600-1610
16.
Belardinelli, L.,
Linden, J.,
and Berne, R. M.
(1989)
Prog. Cardiovasc. Dis.
32,
73-97
17.
Fukunaga, A. F.,
Flacke, W. E.,
and Bloor, B. C.
(1982)
Anesth. Analg.
61,
273-278
18.
Churchill, P. C.
(1982)
J. Pharmacol. Exp. Ther.
222,
319-323
19.
Cronstein, B. N.
(1997)
in
Purinergic Approaches in Experimental Therapeutics
(Jacobson, K. A.
, and Jarvis, M. F., eds)
, pp. 285-299, Wiley-Liss, New York
20.
Jacobson, M. A.,
and Bai, T. R.
(1997)
in
Purinergic Approaches in Experimental Therapeutics
(Jacobson, K. A.
, and Jarvis, M. F., eds)
, pp. 585-591, Wiley-Liss, Inc., Danvers, MA
21.
Fredholm, B. B.,
and Dunwiddie, T. V.
(1988)
Trends Pharmacol. Sci.
9,
130-134
22.
Hershfield, M. S.,
Kurtzberg, J.,
Chaffee, S.,
and Greenberg, M. L.
(1985)
Prog. Clin. Biol. Res.
198,
305-312
23.
Liu, X.,
Kim, C. N.,
Yang, J.,
Jemmerson, R.,
and Wang, X.
(1996)
Cell
86,
147-157
24.
Blackburn, M. R.,
Datta, S. K.,
Wakamiya, M.,
Vartabedian, B. S.,
and Kellems, R. E.
(1996)
J. Biol. Chem.
271,
15203-15210
25.
Blackburn, M. R.,
Datta, S. K.,
and Kellems, R. E.
(1998)
J. Biol. Chem.
273,
5093-5100
26.
Wakamiya, M.,
Blackburn, M. R.,
Jurecic, R.,
McArthur, M. J.,
Geske, R. S.,
Cartwright, J., Jr.,
Mitani, K.,
Vaishnav, S.,
Belmont, J. W.,
Kellems, R. E.,
et al..
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
3673-3677
27.
Blackburn, M. R.,
Volmer, J. B.,
Thrasher, J. L.,
Zhong, H.,
Crosby, J. R.,
Lee, J. J.,
and Kellems, R. E.
(2000)
J. Exp. Med.
129,
159-170
28.
Hershfield, M. S.,
Buckley, R. H.,
Greenberg, M. L.,
Melton, A. L.,
Schiff, R.,
Hatem, C.,
Kurtzberg, J.,
Markert, M. L.,
Kobayashi, R. H.,
Kobayashi, A. L.,
et al..
(1987)
N. Engl. J. Med.
316,
589-596
29.
Knudsen, T. B.,
Winters, R. S.,
Otey, S. K.,
Blackburn, M. R.,
Airhart, M. J.,
Church, J. K.,
and Skalko, R. G.
(1992)
Teratology
45,
91-103
30.
Hershfield, M. S.
(1979)
J. Biol. Chem.
254,
22-25
31.
Hershfield, M. S.,
Chaffee, S.,
and Sorensen, R. U.
(1993)
Pediatr. Res.
33,
S42-47
32.
Weinberg, K.,
Hershfield, M. S.,
Bastian, J.,
Kohn, D.,
Sender, L.,
Parkman, R.,
and Lenarsky, C.
(1993)
J. Clin. Invest.
92,
596-602
33.
Tourigny, M. R.,
Mazel, S.,
Burtrum, D. B.,
and Petrie, H. T.
(1997)
J. Exp. Med.
185,
1549-1556
34.
Kohn, D. B.,
Hershfield, M. S.,
Carbonaro, D.,
Shigeoka, A.,
Brooks, J.,
Smogorzewska, E. M.,
Barsky, L. W.,
Chan, R.,
Burotto, F.,
Annett, G.,
Nolta, J. A.,
Crooks, G.,
Kapoor, N.,
Elder, M.,
Wara, D.,
Bowen, T.,
Madsen, E.,
Snyder, F. F.,
Bastian, J.,
Muul, L.,
Blaese, R. M.,
Weinberg, K.,
and Parkman, R.
(1998)
Nat. Med.
4,
775-780
35.
Migchielsen, A. A.,
Breuer, M. L.,
van Roon, M. A.,
te Riele, H.,
Zurcher, C.,
Ossendorp, F.,
Toutain, S.,
Hershfield, M. S.,
Berns, A.,
and Valerio, D.
(1995)
Nat. Genet.
10,
279-287
36.
Hershfield, M. S.,
Kredich, N. M.,
Ownby, D. R.,
Ownby, H.,
and Buckley, R.
(1979)
J. Clin. Invest.
63,
807-811
37.
Coleman, M. S.,
Donofrio, J.,
Hutton, J. J.,
Hahn, L.,
Daoud, A.,
Lampkin, B.,
and Dyminski, J.
(1978)
J. Biol. Chem.
253,
1619-1626
38.
Benveniste, P.,
Zhu, W.,
and Cohen, A.
(1995)
J. Immunol.
155,
536-544
39.
Carson, D. A.,
Seto, S.,
Wasson, D. B.,
and Carrera, C. J.
(1986)
Exp. Cell Res.
164,
273-281
40.
Kizaki, H.,
Suzuki, K.,
Tadakuma, T.,
and Ishimura, Y.
(1990)
J. Biol. Chem.
265,
5280-5284
41.
Ullman, B.,
Gudas, L. J.,
Cohen, A.,
and Martin, D. W., Jr.
(1978)
Cell
14,
365-375
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  D. A. Carbonaro, X. Jin, D. Cotoi, T. Mi, X.-J. Yu, D. C. Skelton, F. Dorey, R. E. Kellems, M. R. Blackburn, and D. B. Kohn Neonatal bone marrow transplantation of ADA-deficient SCID mice results in immunologic reconstitution despite low levels of engraftment and an absence of selective donor T lymphoid expansion Blood, June 15, 2008; 111(12): 5745 - 5754. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Raskovalova, A. Lokshin, X. Huang, Y. Su, M. Mandic, H. M. Zarour, E. K. Jackson, and E. Gorelik Inhibition of Cytokine Production and Cytotoxic Activity of Human Antimelanoma Specific CD8+ and CD4+ T Lymphocytes by Adenosine-Protein Kinase A Type I Signaling Cancer Res., June 15, 2007; 67(12): 5949 - 5956. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Mortellaro, R. J. Hernandez, M. M. Guerrini, F. Carlucci, A. Tabucchi, M. Ponzoni, F. Sanvito, C. Doglioni, C. D. Serio, L. Biasco, et al. Ex vivo gene therapy with lentiviral vectors rescues adenosine deaminase (ADA)-deficient mice and corrects their immune and metabolic defects Blood, November 1, 2006; 108(9): 2979 - 2988. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Willems, M. E. Reichelt, J. G. Molina, C.-X. Sun, J. L. Chunn, K. J. Ashton, J. Schnermann, M. R. Blackburn, and J. P. Headrick Effects of adenosine deaminase and A1 receptor deficiency in normoxic and ischaemic mouse hearts Cardiovasc Res, July 1, 2006; 71(1): 79 - 87. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Ozdemir, E. Lainka, T. Niehues, and M. Hershfield Increasing importance of stem cell gene therapy in adenosine deaminase deficiency? Clin. Vaccine Immunol., March 1, 2006; 13(3): 433 - 435. [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Chunn, A. Mohsenin, H. W. J. Young, C. G. Lee, J. A. Elias, R. E. Kellems, and M. R. Blackburn Partially adenosine deaminase-deficient mice develop pulmonary fibrosis in association with adenosine elevations Am J Physiol Lung Cell Mol Physiol, March 1, 2006; 290(3): L579 - L587. [Abstract] |