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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 2142-2150
Behavior and Therapeutic Efficacy of  -Glucuronidase-Positive
Mononuclear Phagocytes in a Murine Model of Mucopolysaccharidosis Type
VII
By
Brian J. Freeman,
Marie S. Roberts,
Carole A. Vogler,
Andrew Nicholes,
A. Alex Hofling, and
Mark S. Sands
From the Department of Internal Medicine, Washington University
School of Medicine, St Louis, MO; and the Department of Pathology, St
Louis University School of Medicine, St Louis, MO.
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ABSTRACT |
Bone marrow transplantation (BMT) is relatively effective for the
treatment of lysosomal storage diseases. To better understand the
contribution of specific hematopoietic lineages to the efficacy of BMT,
we transplanted  -glucuronidase-positive mononuclear phagocytes derived from either the peritoneum or from bone marrow in vitro into
syngeneic recipients with mucopolysaccharidosis type VII (MPS VII).
Cell surface marking studies indicate that the bone marrow-derived
cells are less mature than the peritoneal macrophages. However, both
cell types retain the ability to home to tissues rich in cells of the
reticuloendothelial system after intravenous injection into MPS VII
mice. The half-life of both types of donor macrophages is approximately
7 days, and some cells persist for at least 30 days. In several
tissues, therapeutic levels of -glucuronidase are present, and
histopathologic analysis demonstrates that lysosomal storage is
dramatically reduced in the liver and spleen. Macrophages intravenously
injected into newborn MPS VII mice localize to the same tissues as
adult mice but are also observed in the meninges and parenchyma of the
brain. These data suggest that macrophages play a significant role in
the therapeutic efficacy of BMT for lysosomal storage diseases and may
have implications for treatments such as gene therapy.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
LYSOSOMAL STORAGE diseases are a group of
congenital disorders characterized by the intracellular accumulation of
undegraded catabolites and are usually caused by a single gene defect.
Mucopolysaccharidosis type VII (MPS VII; Sly Syndrome) is caused by the
absence of -glucuronidase activity and results in the accumulation
of undegraded glycosaminoglycans.1-3 MPS VII is a
progressive disease and the clinical features manifest themselves
within the first few months to years of life in humans and include
facial dysmorphism, skeletal deformities, mental retardation, hepatosplenomegaly, corneal clouding, hearing defects, and a shortened life span.
In addition to occurring in humans, murine,3
feline,4 and canine5 models of MPS VII exist.
The mouse model has been extensively characterized and is known to be
caused by a single basepair deletion in exon 10 of the
-glucuronidase gene.6 The MPS VII mouse model shares
most of the clinical and pathologic features of the human disease and
therefore makes an excellent model for the study of lysosomal storage
diseases and for the development of novel therapies.3,7
Bone marrow transplantation (BMT) has been shown to be relatively
effective in treating murine MPS VII.8-10 Adult MPS VII mice receiving BMT demonstrate a marked reduction in lysosomal storage
in many tissues, including the liver, spleen, cornea, and meninges, and
have a dramatically extended life span.8 Because of the
progressive nature of the disease, more effective therapy is achieved
by initiating treatment in the neonatal period. Bone marrow
transplantation in neonatal mice reduces storage in bones, joints, and
periarticular tissue and also reduces the hearing loss associated with
MPS VII.9,10
Although BMT is effective for the treatment of MPS VII, it is unclear
which hematopoietic cell type or cell types are responsible for the
therapeutic response. Our current hypothesis is that donor-derived mononuclear phagocytes account for a large proportion of the
therapeutic enzyme produced in the host. In this study, we examine the
distribution, apparent half-life, enzyme levels, and effect on the
disease of fully differentiated peritoneal or bone marrow-derived
macrophages after intravenous injection into adult and newborn MPS VII mice.
We demonstrate that, although the 2 cell types have different cell
surface markers, both cell types retain the ability to home to the same
tissues and have a distribution similar to that of endogenous
macrophages. Both cell types have similar half-lives in vivo and can
deliver sufficient quantities of enzyme to reduce lysosomal distension
in the liver and spleen. Experiments performed in neonatal MPS VII mice
also demonstrate that the donor macrophages are capable of delivering
enzyme to the brain.
These experiments suggest that both fully differentiated and less
mature macrophages behave similarly in vivo. In addition, macrophages
may be responsible for a significant portion of the therapeutic
efficacy of BMT.
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MATERIALS AND METHODS |
Animals.
Homozygous mutant (mps/mps) mice were obtained from a
B6.C-H-2bm1/ByBir-gusmps/+
colony maintained by M.S.S. at Washington University (St Louis, MO).
Homozygous normal donor mice were obtained either from a separately
bred group of syngeneic normal (+/+) mice or from heterozygote matings
after distinguishing homozygous normal from heterozygotes (+/mps) by polymerase chain reaction (PCR).11
Homozygous mutants were identified at birth by the absence of
-glucuronidase activity using a fluorometric assay on a small sample
of tissue obtained by toe clipping.9
Isolation, differentiation, and injection of mononuclear phagocytes.
Homozygous normal mice were injected intraperitoneally with 1 mL of
thioglycollate (DIFCO Laboratories, Detroit, MI). Peritoneal cells were
harvested 5 days after thioglycollate injection by killing the donor
and performing peritoneal lavage twice, using a total of 10 mL of
Ca2+- and Mg2+-free phosphate-buffered saline
(PBS). The cells were counted and 5 × 105 cells were
frozen for later biochemical analysis. Cytospins of 1 × 105 cells were made and stained with Wright Giemsa stain
(Sigma, St Louis, MO). The cytospins were examined immediately to
determine if the lavage fluid contained a preponderance of macrophages
that appeared to be in an activated state. The presence of large
cytoplasmic vacuoles in 80% to 90% of the mononuclear cells was the
criteria for injection of thyoglycollate-stimulated macrophages into
MPS VII recipients.
Unfractionated bone marrow from +/+ donors was cultured in Dulbecco's
modified Eagles medium supplemented with 10% fetal calf serum
(GIBCO-BRL, Grand Island, NY), 2 mmol/L L-glutamine, 100 U penicillin,
100 µg/mL streptomycin, 250 ng/mL fungizone, 1 µg/mL murine
interleukin-3 (IL-3), and 10 ng/mL murine macrophage colony-stimulating factor (M-CSF; R & D Systems, Minneapolis, MN). Bone marrow cells were
cultured in 100-mm petri dishes at a density of 2.5 × 106 cells/mL. Fresh media was added every 48 hours. After 4 to 6 days in culture, the nonadherent cells were removed and discarded. The adherent cells were washed 3 times in ice-cold PBS and then incubated in 5 mL of ice-cold PBS with 0.02% EDTA (Sigma) for 20 minutes on a rocking platform at 4°C. Cells were resuspended in
PBS, and a sample was saved for cytospins and biochemical analysis.
Adult mps/mps recipients ranging in age from 50 to 123 days
were injected intravenously through the tail vein with 400 µL of
macrophages (1 × 107 cells) from a donor of the same
sex. Twelve adult mutants each were injected with either peritoneal or
progenitor-derived macrophages and 3 of the mice from each group were
killed at each of the following time points: 1, 7, 15, and 30 days
after injection. An additional 5 adult mutants were injected with the
same number of peritoneal macrophages and killed at 15 minutes, 1 hour,
3 hours, 6 hours, and 12 hours after injection.
Neonatal mps/mps mice were injected intravenously with 100 µL
(2.5 × 106 cells) of peritoneal macrophages
from a same-sex donor through the superficial temporal
vein.12 Six neonates were injected on day 1 or 2 of life.
Three were killed 24 hours after injection and 3 were killed 3 weeks
after injection.
Biochemical analysis.
The liver, spleen, lung, thymus, and 1 kidney were harvested from the
injected animals as well as from 3 uninjected positive control (+/+)
and 3 uninjected negative control (mps/mps) animals, and the
total weight of each organ was determined. Fractions for biochemical
analysis were taken from each tissue and also weighed. Samples of
heart, brain, mesenteric lymph node, skeletal muscle, and bone marrow
from 1 femur were also collected. Samples of liver, spleen, kidney,
lung, heart, brain, and thymus were taken from the neonates. The
tissues were homogenized with a motorized pestle in 10 mmol/L Tris (pH
7.5), 150 mmol/L NaCl, 0.2% Triton X-100, and 1 mmol/L dithiothreitol
and centrifuged in a microfuge at 14,000 rpm for 1 minute to remove
debris. -Glucuronidase,  -galactosidase, and -hexoseaminidase
activities were measured fluorometrically using the substrates
4-MU--D-glucuronide, 4-MU--D-galactoside, and
4-MU-N-acetyl--D-glucosaminide (Sigma), respectively.13 A portion of each homogenate was incubated at 37°C for 1 hour with
100 µL substrate and the reactions were stopped with 1 mL 0.1 mol/L
sodium carbonate. The protein concentrations of the homogenates were
determined using the Coomasie dye-binding assay14 (Bio-Rad,
Hercules, CA). The specific activities (1 unit [U] = 1 nmole
substrate cleaved/hr/mg protein) of the lysosomal enzymes in each
tissue at each time point were calculated. The total -glucuronidase activity in the weighed tissues was also calculated. The total -glucuronidase activity in a sample of the injected macrophages (1 × 105 cells) was measured to determine the total
activity introduced into each animal. Statistical significance was
calculated using the Student's t-test.
Histochemistry, immunofluorescence, and immunohistochemistry.
A portion of the liver, spleen, kidney, lung, heart, brain, thymus,
mesenteric lymph node, skeletal muscle, and ribs was obtained from each
experimental adult and frozen in embedding medium for histochemical and
immunohistochemical analysis. Samples of the small intestine, colon,
and foot pad were also taken from a single adult mutant 24 hours after
injection. The same tissues, with the exception of the mesenteric lymph
nodes, were collected from MPS VII mice injected as neonates.
Ten-micron-thick sections of each tissue were obtained. Slides of
peripheral white blood cells were also prepared for histochemical
staining from the 5 adult animals killed from 15 minutes to 12 hours
after injection. Cytospins of donor macrophages were prepared for
histochemical and immunohistochemical analysis. The slides were stored
at  70°C until analyzed.
The staining procedure for enzymatically active -glucuronidase was
performed as previously described using
naphthol-AS-BI--D-glucuronide (Sigma) as a substrate.11
The surface markers Mac-1 (CD11b), Mac-3, F4/80, and macrophage
metalloelastase (MME) on the donor cells were identified by
immunohistochemical and immunofluorescent techniques (Mac-1 and Mac-3
primary antibodies [Pharmingen, San Diego, CA], F4/80 antibody
[Biosource International, Camarillo, CA], and MME antibody [kind
gift of S. Shapiro15]). Sections were fixed
for 20 minutes at 4°C with a solution of 70% acetone, 0.3%
chlorohydrate, and 6% neutral buffered formalin. Endogenous biotin was
blocked with commercially available reagents (Vector Laboratories,
Burlingame, CA). The sections were incubated for 30 minutes at room
temperature in antibody blocking buffer (PBS, 1% bovine serum albumin,
0.2% powdered milk, and 0.03% Triton X-100). The sections were then
incubated overnight at 4°C with primary antibody followed by 30 minutes of incubation in the appropriate secondary antibody. A
biotinylated rat antimouse secondary antibody was used for detection of
Mac-1, Mac-3, and F4/80, and a biotinylated goat antirabbit secondary
antibody was used to detect MME (Vector Laboratories).
Streptavidin-conjugated alkaline phosphatase was used to detect Mac-1,
and fluorescein conjugated to streptavidin was used to detect F4/80 and
Mac-3. Streptavidin-conjugated horseradish peroxidase was used to
detect MME. Control slides were incubated with the appropriate
preimmune sera and then stained as described above.
To perform dual immunohistochemistry for Mac-1 and histochemistry for
-glucuronidase activity on spleen sections, the antibody staining
was performed first. The pH of the sections was then lowered to
approximately 4.5 for histochemical staining.
Histopathology.
Portions of liver, spleen, kidney, lung, heart, thymus, and eye were
taken from each of the 24 adult mice in the 30-day study as well as
from the neonatal mice and preserved in 4% paraformaldehyde and 2%
glutaraldehyde in PBS. These tissue blocks were then embedded in
Spurr's resin, and 0.5-µm sections were stained with toluidine blue.7
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RESULTS |
Donor mononuclear phagocytes.
Cytospins of thioglycollate-stimulated peritoneal cells stained with
Wright's stain demonstrate the characteristic morphology of activated
cells. Approximately 90% of the cells are mononuclear with extensive
cytoplasmic vacuolation (Fig
1A). The population of adherent cells derived from bone marrow in vitro
is composed of approximately 90% mononuclear cells with more
homogeneous cytoplasm (Fig 1B). All of the peritoneal and
progenitor-derived macrophages stain intensely for enzymatically active
-glucuronidase (data not shown). Immunohistochemical and
immunofluorescent staining of the donor cells demonstrates that greater
than 90% of the peritoneal cells express relatively high levels of
Mac-1, Mac-3, and F4/80 and that approximately 5% to 10% express high
levels of MME (Table 1). Bone
marrow-derived macrophages also express relatively high levels of Mac-1
and Mac-3 but express neither F4/80 nor MME. Dual in situ histochemical
and immunohistochemical staining demonstrates the colocalization of
-glucuronidase activity and Mac-1 in individual cells in sections of
spleen obtained 24 hours and 15 days after injection (Fig 1C and 1D).
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| Fig 1.
The morphology of thioglycollate-stimulated
peritoneal macrophages (A) and in vitro-differentiated bone
marrow-derived macrophages (B) injected into MPS VII recipients are
shown after Wright Giemsa staining. Dual histochemical staining for
-glucuronidase activity (diffuse red stain) and immunohistochemical
staining for Mac-1 (black cell surface stain) of spleen sections from
MPS VII mice 24 hours (C) and 15 days (D) after injection of peritoneal
macrophages demonstrates the colocalization of the 2 markers (white
arrows). Mac-1-positive spleen cells that are -glucuronidase
negative (solid arrows) are also seen. ([A] and [B], original
magnification × 1,145; [C] and [D], original magnification × 467).
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The total -glucuronidase activity in 1 × 107
peritoneal and progenitor macrophages was 5,775 and 11,900 U,
respectively. This level represents approximately 10% to 20% of the
total activity measured in liver, spleen, kidneys, lungs, thymus, and
marrow from a normal mouse.
Tissue distribution and kinetics.
The distribution of -glucuronidase positive donor cells in various
tissues is similar after injection with either peritoneal or
progenitor-derived macrophages. The livers of injected adult mice
demonstrate a uniform distribution of cells 24 hours after injection
(Fig 2A). In contrast, the majority of -glucuronidase positive cells are localized in the red pulp of the spleen and appear
to surround the germinal centers (Fig 2B). There are also numerous
-glucuronidase-positive cells observed in the lung and bone marrow
(Fig 2C and D). In the kidney, the introduced cells appear to localize
mainly to the glomeruli (Fig 2E). There are also rare positive cells
present in the thymus, in the lamina propria of the intestine, and in
the myocardium (data not shown). There are no
-glucuronidase-positive cells observed in the peripheral circulation of any of the 5 adult MPS VII mice killed from 15 minutes
to 12 hours after injection (data not shown).
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| Fig 2.
Histochemical staining for -glucuronidase
activity (red) was performed on tissues from adult MPS VII mice 24 hours after injection with progenitor-derived macrophages derived from
normal donors. -Glucuronidase-positive cells assume a relatively
uniform distribution in the liver (A), lung (B), and bone marrow (C).
Enzyme-positive cells in the spleen are present predominantly in the
red pulp, with rare positive cells observed in the germinal centers
(D). In the kidney, the donor cells appear to be present primarily in
the glomeruli (E). The newborn brain contains multiple
-glucuronidase-positive cells that are observed in both the
meninges and the parenchyma (F). ([A], [C], [D], and [F],
original magnification × 188; [B] and [E], original magnification × 300).
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The specific activity observed in the lung of an MPS VII mouse 15 minutes after injection is 277 U (Fig 3A),
which is equal to or greater than the levels seen in the lungs of
normal mice. The activity associated with the lung decreases rapidly to
approximately 83 U by 24 hours after injection. In contrast, the
activity associated with the liver, spleen, and bone marrow increases
during the first 24 hours after injection (Fig 3B).
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| Fig 3.
-Glucuronidase-specific activities (nanomoles of
substrate cleaved per hour per milligram of protein) are shown in the
lung (A), liver, spleen, and bone marrow (B) at 15 minutes, 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours after injection of 1 × 107 donor peritoneal macrophages.
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The percentage of the injected activity that is recovered in the liver,
spleen, lung, kidney, thymus, and bone marrow 24 hours after injection
of peritoneal and progenitor macrophages was 68.1% and 67.3%,
respectively. The total amount of -glucuronidase activity present in
the adult liver, spleen, and lung of recipients decreases as time
progresses (Fig 4A through
C). The rate of decrease is similar for peritoneal and progenitor
macrophages. The half-lives of both types of cells over the entire
30-day period is approximately 7 days. However, the decrease in enzyme
activity does not obey strict first order kinetics, and the approximate
half-lives over the first 15 days after injection is approximately 4 days, and from 15 to 30 days is approximately 20 days.
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| Fig 4.
The decrease in -glucuronidase activity in
the liver (A), spleen (B), and lung (C) of MPS VII mice between 24 hours and 30 days after injection is shown for the peritoneal
macrophages ( ) and the progenitor-derived macrophages ( ). The
error bars represent ±1 standard deviation from the mean.
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Although the liver accounted for the majority of the total activity
(67% of the recovered activity is detected in the liver at all time
points beyond 24 hours), the specific activities of tissues rich in
cells of the reticuloendothelial system are similar. At day 1, the
specific activities in the liver and spleen are 15.2 and 17.9 U,
respectively. By days 7 and 30, the specific activities in the liver
and spleen decrease to 8.1 and 9.7 and to 1.9 and 1.6 U, respectively.
The majority of activity is detected in the liver, spleen, and lung
after injection of either cell type. However, measurable amounts of
-glucuronidase activity are detected in other tissues for a
substantial period of time. At 24 hours after injection, the brain,
kidney, heart, skeletal muscle, thymus, mesenteric lymph nodes, and
bone marrow have measurable enzyme levels. By 1 week after injection,
the lymph nodes and bone marrow still have detectable enzyme levels,
but the remaining tissues have levels that were indistinguishable from
background. However, -glucuronidase-positive cells are detected
histochemically in many of these tissues at 1 week and beyond.
Affect of donor macrophages on MPS VII.
In some lysosomal storage diseases such as MPS VII, the levels of
unaffected lysosomal enzymes increase, in some cases as much as several
hundred percent above normal.8 Results from -galactosidase assays show that the secondary elevation of this enzyme is reduced in liver, spleen, and lung by 30 days after injection
(Fig 5A and B). Furthermore, both
peritoneal and progenitor-derived macrophages result in similar
reductions in enzyme levels. Corresponding reductions in the secondary
elevation of the lysosomal enzyme -hexoseaminidase are also observed
in these tissues 30 days after macrophage injections (data not shown).
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| Fig 5.
Thirty days after a single injection of 1 × 107 -glucuronidase-positive macrophages into adult MPS
VII mice,  -galactosidase levels are significantly decreased in
several tissues. A significant decrease is produced by both
peritoneal-(A) and progenitor-derived (B) cells. An asterisk (*)
indicates a significant (P < .05) decrease in specific
activity compared with untreated MPS VII (MPS) mice.
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Microscopic examination of sections of liver and spleen confirm the
biochemical data, suggesting that the injected macrophages result in
partial correction of the disease (Fig 6).
In the livers of treated animals, lysosomal storage is reduced in
Kupffer cells at 7 days and is virtually eliminated by day 15. However,
by day 30, lysosomal distension is apparent, but at a lower level than that observed in untreated MPS VII mice. In the spleens of injected animals, lysosomal storage is reduced at a slightly slower rate than in
the liver but is virtually eliminated by day 30. There was no
significant reduction in storage observed in the meninges, cornea, or
retinal pigment epithelial cells of treated adult MPS VII mice.
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| Fig 6.
Histopathologic analysis demonstrates no reduction in
lysosomal storage in the liver (A) or spleen (F) 24 hours after
injection of peritoneal macrophages. A dramatic decrease in lysosomal
storage is observed in the liver 7 (B), 15 (C), and 30 (D) days after
injection. A similar reduction in storage is observed in the spleen 7 (G), 15 (H), and 30 (I) days after injection. Progenitor-derived
macrophages provide an equivalent level of correction. The
histopathological appearance of the liver (E) and spleen (J) from an
untreated age-matched MPS VII mouse is shown. (Original magnification × 242.)
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Macrophage injection in newborn MPS VII mice.
Histochemical staining of tissues from newborn mice killed 24 hours
after injection demonstrate -glucuronidase-positive cells in all
tissues examined. The distribution of the cells in the various tissues
is similar to that observed in the adult animals (data not shown). One
difference between newborn and adult injections is the presence of
-glucuronidase-positive cells in the brains of the injected newborn
animals. At 24 hours after injection, there are only rare
-glucuronidase-positive cells observed in the adult brain, and
those appeared to be localized primarily to the meninges or what appear
to be vascular structures. In contrast, the newborn brain has multiple
positive cells per field that are located in both the meninges and
parenchyma (Fig 2F). At 3 weeks after injection, the brains from mice
injected as newborns still have approximately 0.8% normal levels of
activity. The brains of MPS VII mice injected as adults have
undetectable levels of enzyme beyond 24 hours. The histopathologic
correction after macrophage injection in newborns is similar to that
observed in adult animals. There is no apparent reduction in lysosomal
storage in the meninges or neurons of the brain.
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DISCUSSION |
-Glucuronidase-positive macrophages localize in many tissues after
intravenous injection. We believe that these cells are of donor origin.
However, another possible source of the positive cells is from the
release of enzyme from dead donor cells and the subsequent
receptor-mediated uptake by host cells.16-18 Results from
enzyme replacement studies suggest that this is not the case. If enzyme
is released into the circulation and endocytosed by recipient cells,
the distribution of -glucuronidase activity should be more diffuse
and the half-life should be similar to that observed after enzyme
replacement (2 to 4 days rather than 7 days after macrophage
injection).19,20 It is also possible that endogenous
macrophages engulf the donor cells. Although the distribution of
activity should be similar to what we observe in this study, we believe
that this explanation is unlikely for 3 reasons. First, the donor cells
are from syngeneic, sex-matched animals, so there are no
histocompatibility mismatches. Second, it seems likely that the
half-life of the enzyme from an engulfed cell should mimic that of
directly administered enzyme. Finally, a recent report suggests that
mature bone marrow-derived monocytic cells can engraft and repopulate
recipient animals in a pattern similar to endogenous
macrophages.21
Macrophages harvested from the peritoneum or derived from bone marrow
in vitro appear to be at different stages of maturation. Both
populations express relatively high levels of -glucuronidase and are
comprised primarily of mononuclear cells that express the macrophage
markers Mac-1 and Mac-3.22,23 However, the
thioglycollate-stimulated peritoneal cells have the morphology of
activated macrophages and also express the surface antigens F4/80 and
MME that are present on more mature or activated mononuclear
phagocytes.15,24 Interestingly, both cell populations
retain the ability to home to the same tissues when transplanted into a
syngeneic recipient. The distribution of both types of donor cells is
similar to that of endogenous bone marrow-derived macrophages. The
donor cells are localized in a pattern similar to Kupffer cells of the
liver, mesangial cells of the kidney, and phagocytic cells in the red
pulp of the spleen. Donor macrophages also localize in lower numbers to
regions in which antigen-presenting cells are normally found, such as the lung, thymus, white pulp of the spleen, and the lamina propria of
the intestine.
In the case of the thioglycollate-stimulated peritoneal cells, it is
unclear where the cells are recruited from, but it is interesting that
they can home to many different sites in the recipient animal. This
suggests that, once the cells are activated and migrate to a site of
inflammation or irritation, they still retain the signals necessary to
migrate to other sites in the body. It is also interesting that
mononuclear phagocytes differentiated in vitro acquire the correct
signals that mediate migration in vivo to sites where endogenous
macrophages are normally localized.
The half-life of metabolically labeled endogenous macrophages in normal
mice ranges from 20 to 30 days.25,26 The apparent half-life
of the transplanted macrophages in these experiments is considerably
shorter. There may be several reasons for this apparent difference.
First, the half-lives determined in this set of experiments are
indirect measurements based on -glucuronidase activity. The state of
differentiation, activation, or the localization of the macrophages in
the recipient animal may affect the expression of -glucuronidase,
thereby affecting the apparent half-life of the donor cells. Second, it
is possible that a fraction of the donor cells undergo apoptosis and
are eliminated after becoming more mature or activated cells. However,
it seems unlikely that this is the major cause of the shortened
half-life, because the less mature progenitor-derived cells have
essentially the same half-life as the thioglycollate-stimulated cells.
Third, these experiments involve the injection of normal macrophages
into a disease model. It is possible that many of the donor cells die prematurely after being faced with the abnormally large accumulation of
glycosaminoglycans in the recipient animal. Finally, a macrophage that
is manipulated ex vivo and then reintroduced into another animal may
have an intrinsically shorter life span.
Donor macrophages from -glucuronidase-positive animals have a
significant impact on the disease in MPS VII mice. A reduction in the
secondary elevations of other lysosomal enzymes is a relatively sensitive indicator of a positive response to therapy.27
Although the reductions in -galactosidase activity are not
statistically significant in every tissue 30 days after injection, the
levels are consistently decreased in the liver and spleen. As little as
1% of normal levels of -glucuronidase may be sufficient to reduce
lysosomal storage in some tissues in the MPS VII
mouse.27-29 Although enzyme levels sufficient to reduce
lysosomal storage are present in several tissues 24 hours after
injection, no histopathologic improvement is observed at that time. It
appears that several weeks may be required for the relatively low
levels of activity to reduce accumulated lysosomal storage. This can be
seen in the liver and spleen, which have 2% to 5% normal enzyme
levels, where a significant reduction in lysosomal storage is not
observed until 7 to 15 days after injection. In addition, there is
little or no reduction of lysosomal storage in hepatocytes at any time
after injection. This suggests that either the amount of enzyme
transferred from the donor macrophages is insufficient to reduce
storage or the half-life of the donor macrophages in the liver did not
allow for a significant reduction in hepatocytes.
Lysosomal storage diseases are progressive in nature and often there is
little evidence of disease at birth.1 Studies in MPS VII
mice treated with bone marrow transplantation have shown that the
disease can be more effectively treated when the transplants are
performed at birth.9,10 However, in this study, there is
little difference between injecting -glucuronidase-positive macrophages in adult or newborn MPS VII mice. One of the few
differences was observed in the brain. There are more donor cells
present, and higher specific activity in the brains of newborn compared with adult MPS VII mice. In addition, the cells are present in the
meninges as well as in the parenchyma of newborn brains. In contrast,
the positive cells associated with the adult brains appear to be
localized primarily to the meninges or vascular structures. The enzyme
activity also persisted for at least 3 weeks in the newborn brain,
whereas no enzyme activity is detected in the adult brain beyond 24 hours. One explanation for this difference could be that the newborn
MPS VII mice were injected with approximately 5 times more cells per
gram of body weight. Alternatively, it is possible that more
macrophages are able to enter the brain of a newborn mouse before the
blood-brain barrier is fully formed,30 and once there, they
can survive for some time, perhaps becoming resident microglia.
Similarly, the lack of -glucuronidase-positive cells in the adult
brain may be due to the maturity of the blood-brain barrier. Several
studies show that repopulation of bone marrow-derived macrophages in
the brains of adult mice after BMT was dramatically delayed relative to
the repopulation of fixed tissue macrophages in other tissues such as
liver and spleen.31,32 The delay in macrophage repopulation
of the brain after BMT may account for the lack of positive cells in
the adult mice in this study. The life span of the donor macrophages
simply may not be long enough for them to gain access to the brain.
Although enzyme activity persists in the brains of newborn MPS VII
mice, it is insufficient to reduce lysosomal storage in the meninges or neurons.
BMT has been shown to be relatively effective in humans and animal
models of lysosomal storage diseases.8-10,33-37 The
hematopoietic lineage or lineages responsible for the correction in
these diseases are uncertain. These data show that mature peritoneal
macrophages and less mature bone marrow-derived monocytic phagocytes
can localize to many tissues and persist for at least 30 days after
transplantation. Levels of -glucuronidase sufficient to reduce
lysosomal storage in the liver and spleen of MPS VII mice are delivered
after a single intravenous injection of macrophages from normal donor mice. These data suggest that the macrophage lineage is a major source
of corrective enzyme after BMT for lysosomal storage diseases. Because
macrophages are capable of delivering significant amounts of lysosomal
enzymes to many tissues, macrophage-directed therapy38 or
the use of macrophage-specific promoters in a gene transfer setting39 may enhance the efficacy of
hematopoietic-directed therapies for these diseases.
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ACKNOWLEDGMENT |
The authors thank Steve Shapiro (Washington University School of
Medicine, St Louis, MO) for helpful discussions and for performing the
macrophage metalloelastase immunohistochemistry assays.
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FOOTNOTES |
Submitted October 8, 1998; accepted May 13, 1999.
Supported in part by National Institutes of Health Grants No. DK50158
and HD33671 to M.S.S.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Mark S. Sands, PhD, Washington University
School of Medicine, Department of Internal Medicine, Box 8007, 660 S
Euclid Ave, St Louis, MO 63110.
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