From the Department of Medicine and Cardiovascular
Research Institute, University of California, San Francisco, California
94143-0911 and the ¶ Departments of Internal Medicine,
Pathology and Immunology, and ** Genetics, Washington University
School of Medicine, St. Louis, Missouri 63110-1093
Received for publication, January 10, 2001, and in revised form, February 23, 2001
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ABSTRACT |
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Dipeptidyl peptidase I (DPPI) is the sole
activator in vivo of several granule-associated serine
proteases of cytotoxic lymphocytes. In vitro, DPPI also
activates mast cell chymases and tryptases. To determine whether DPPI
is essential for their activation in vivo, we used enzyme
histochemical and immunohistochemical approaches and solution-based
activity assays to study these enzymes in tissues and bone
marrow-derived mast cells (BMMCs) from DPPI +/+ and DPPI Granule-associated serine proteases of cytotoxic lymphocytes,
neutrophils, and mast cells (i.e. granzymes, elastase,
cathepsin G, and chymases) are structurally related (1). They have a two-amino acid propeptide (also referred to as the "activation dipeptide") and an isoleucine at the NH2 terminus of the
activated enzyme (2). The activation dipeptide maintains the protease in an inactive state. After removal of the dipeptide, the new NH2-terminal isoleucine moves from its position on the
surface of the protease to a region in the interior of the enzyme,
where it interacts with an aspartate residue near the catalytic center (3). These movements are thought to change the enzyme substrate binding
cleft and catalytic apparatus to a catalytically competent conformation.
Initial studies investigating the activation of the granule-associated
serine proteases focused on the cysteine protease dipeptidyl peptidase
I (DPPI), 1 also known as
cathepsin C (2, 4-8). DPPI was a logical candidate activator of these
proteases because it is found in the same cells and is a promiscuous
exopeptidase that hydrolyzes most NH2-terminal dipeptides
(9). Studies using the DPPI inhibitor Gly-Phe-diazomethyl ketone
reported that DPPI inhibition reduces granzyme A, neutrophil elastase,
and cathepsin G activity in the cells (2). However, because the
inhibitor is not entirely specific for DPPI and does not eliminate the
activity of the proteases in question, the possibility remained that
another protease was responsible for their activation. This possibility
was tested in a DPPI knockout mouse (10). Cytotoxic lymphocytes of
these animals produce normal amounts of granzymes A and B, but both
enzymes are inactive and present in their pro-forms. These findings
suggest that DPPI is the sole activator of granzymes A and B in
vivo.
In mast cells, the two major types of serine proteases are chymases and
tryptases. Chymases are grouped into To establish whether DPPI is essential for activating mast cell
chymases and tryptases in vivo, we examined these enzymes in
DPPI-null mice. We found that mast cells from these mice contain normal
amounts of at least two chymases (mMCP-4 and -5), but they and other
chymases are completely inactive as determined by enzyme histochemistry
and solution-based assays. In contrast, the mast cells contain reduced
amounts of normally processed, active tryptase (mMCP-6). Thus, DPPI is
required for the processing and activation of chymases, but not
tryptases, in mouse mast cells. These findings improve our
understanding of the regulation of serine protease activity in mast
cells and have important implications for understanding their
biological functions in vivo.
Materials--
All chemicals were from Sigma unless otherwise
noted. Rabbit anti-rat mast cell protease I (rMCP-I) was from Moredun
Scientific Limited, Penicuik, Scotland, rabbit anti-mMCP-5 and rabbit
anti-mMCP-6 were gifts from Richard Stevens, Harvard University,
Cambridge, MA, and monoclonal anti-dinitrophenyl IgE and anti- Experimental Animals--
DPPI Tissue Preparation--
Harvested tissues were washed in PBS
before being fixed for 6-18 h in PBS containing 4% paraformaldehyde.
Tissues were then embedded directly in paraffin or incubated in PBS
containing 30% sucrose for 18 h at 4 °C before freezing at
Enzyme Histochemistry--
To detect active chymase in tissue
sections, naphthol AS-D chloroacetate esterase (CAE)
histochemistry was performed as described (16). Slides were then washed
with water, counterstained in 0.5% eosin Y, dehydrated in 100%
ethanol, and mounted with Histomount. Active tryptase was detected
using the method of Valchanov and Proctor (17). Briefly, tissue
sections were incubated at 20 °C in a 0.1 M phosphate
buffer (pH 6.5) containing 0.25 mg/ml benzyloxycarbonyl-Gly-L-Pro-Arg-4-methoxy-2-naphthylamide
(Enzyme Systems Products, Dublin, CA) and 3 mM Fast
Blue B salt. After incubation for 1-5 min, the slides were rinsed in
water and photographed.
Immunohistochemistry--
Tissues were equilibrated in PBS,
incubated in blocking solution (PBS containing 5% dehydrated milk, 3%
nonimmune goat serum, 0.1% Triton X-100, and 1% glycine) at 20 °C
for 15 min, then incubated overnight at 4 °C with a 1:200 dilution
of rabbit anti-rMCP-I. Tissues were then washed in PBS-Tween, and the
bound antibody was detected with fluorescein isothiocyanate-conjugated
goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA).
Culture of Mast Cells from Bone Marrow--
Mouse BMMCs were
cultured in WEHI-3B-conditioned medium as described (18). The cells
were used after 4 weeks in culture, at which time the cell populations
consisted of >95% mast cells as assessed by the presence of
metachromatic granules in toluidine blue-stained cells.
Protease Activity Assays--
BMMC were harvested by
centrifugation, washed in PBS, and resuspended in 10 mM
bis-Tris (pH 6.1) containing 2 M NaCl at 40 × 106 cells/ml. Cells were then lysed by sonication, debris
was pelleted by centrifugation, and the supernatant was recovered. DPPI
activity was measured spectrophotometrically by monitoring hydrolysis
of L-Ala-Ala-p-nitroanilide. 20-µl aliquots of
cell lysates were incubated for 5 min in 500 µl of activation buffer
(100 mM Na2HPO4 buffer, 20 mM NaCl, 1 mM EDTA, 4 mM cysteine
(pH 6)) followed by the addition of 400 µl of substrate buffer (100 mM Na2HPO4, 20 mM NaCl,
1 mM EDTA, 125 µM
L-Ala-Ala-p-nitroanilide, 1% dimethylsulfoxide (pH 6)). Tryptase activity was measured using
benzoyl-Gly-L-Pro-Arg-p-nitroanilide. 1 µl of
enzyme solution was incubated in 1 ml of 0.06 M Tris-HCl (pH 7.8) containing 0.4% dimethylsulfoxide, 30 µg/ml heparin, and 80 µg/ml substrate at 37 °C. Chymase activity was measured by the
addition of 20 µl of enzyme solution to 1 ml of 0.45 M Tris-HCl (pH 8.0) containing 1 mM
succinyl-L-Ala-Ala-Pro-Phe-p-nitroanilide, 1.8 mM NaCl, and 1% dimethylsulfoxide. Release of free
nitroaniline was measured spectrophotometrically at 410 nm for 5-10
min in all assays.
Immunoblotting--
Cell lysates were subjected to
SDS-polyacrylamide gel electrophoresis under reducing conditions and
transferred to polyvinylidene difluoride membranes (PerkinElmer Life
Sciences Products, Boston, MA) in transfer buffer containing 25 mM Tris base, 200 mM glycine, and 15% methanol
for 1 h at 4 °C. The membrane was washed with 50 mM
Tris-HCl containing 0.5 M NaCl, 0.01% Tween 20 (TBS; pH 7.5) and incubated for 1 h in TBS containing a 1:1,000 dilution of
antibody. The membrane was then washed with TBS, incubated in TBS for
30 min containing a 1:2,000 dilution of horse radish peroxidase-conjugated goat anti-rabbit IgG (New England Biolabs, Beverly, MA), and washed again. Immunoreactivity was detected using the
phototope horseradish peroxidase detection kit (New England Biolabs). After detection of the mast cell protease signal, the
immunoblots were probed a second time with monoclonal anti- Mast Cell Degranulation--
5 × 106 BMMCs
were cultured overnight in the presence of monoclonal
anti-dinitrophenyl IgE (25 µg). These sensitized cells were harvested
by centrifugation, washed twice in Ca2+- and
Mg2+-free PBS, and resuspended in RPMI 1640 at 20 × 106 cells/ml. Cells were then incubated alone or with 400 ng/ml dinitrophenyl-conjugated bovine serum albumin for 60 min.
Aliquots were removed at 0 and 60 min then centrifuged immediately.
Degranulation supernatants were separated from cell pellets, which were
resuspended in the same volume as the supernatant and lysed by repeated
cycles of freeze-thawing. Cellular debris were pelleted by centrifuging at 15,000 × g. Recovered supernatants were assayed for
histamine content using an enzyme-linked immunosorbent assay kit
(Research Diagnostics, Flanders, NJ) according to the manufacturer's
instructions. The net percent of histamine released was calculated as
previously described (19).
Activation of Mouse Pro-chymase--
5 µl of DPPI Tryptase Purification and Sequencing--
Cell lysates from
40 × 106 DPPI +/ RNA Blotting--
Total RNA was isolated from DPPI +/ Identification of Active Chymase in Mouse Tissues--
We detected
active chymase in tissue sections via esterase activity using CAE
enzyme histochemistry. In tissue sections from DPPI +/+ mice, we
identified CAE-positive cells in ear, tongue, stomach, and trachea.
CAE-positive cells had a distribution, number, and morphology typical
of mast cells. In contrast, DPPI Chymase Activity in BMMCs--
To confirm the enzyme histochemical
results, we measured protease activities in mast cells cultured from
DPPI +/ Mast Cell Degranulation--
Prior studies using chymase
(Fab)2 fragments (23), nonspecific inhibitors (24), or
added chymase (25) suggested that chymase plays a role in mast cell
degranulation. To determine if endogenous chymase regulates this
process, we compared IgE-mediated degranulation of DPPI +/ Identification of Active Tryptase in Mouse Tissues--
The enzyme
histochemical substrate
benzyloxycarbonyl-Gly-L-Pro-Arg-4-methoxy-2-naphthylamide
detected active tryptase in situ in mouse tissues. Ear,
tongue, and stomach tissue sections obtained from DPPI +/+ mice possess
multiple, red-brown, tryptase-positive cells (Fig.
4). A comparable number of
tryptase-positive cells were seen in tissue sections from DPPI Tryptase Activity in BMMCs--
Because tryptase enzyme
histochemistry is qualitative, not quantitative, the possibility
remained that levels of active tryptase differ between DPPI +/ In this study, DPPI-deficient mice reveal that DPPI is essential
for the in vivo activation of mast cell chymases.
Surprisingly, DPPI is not essential for the activation of the tryptase
mMCP-6, although it does influence the amount of active mMCP-6 in mast cells. These alterations of protease activity do not affect mast cell
growth, maturation, tissue migration, or degranulation, indicating that
DPPI and chymase are not essential for these processes. These findings
suggest a mechanism for the intracellular activation and regulation of
a major class of mast cell preformed mediators.
Prior studies report that purified DPPI activates human pro-chymase
in vitro (6, 26). Mice have five chymases, mMCP-1, -2, -4, -5, and -9 (11), all with similar measured or predicted activity and
primary structure. MMCP-1, -2, -4, and -9 are predicted to have
identical propeptides (EE) and mature NH2 termini (IIGG) (11). MMCP-5 also has a similar propeptide (GE) and an identical NH2 terminus (IIGG). Our results show that DPPI is
essential for the activation of mouse chymases in BMMCs and mucosal and
connective tissue mast cells in vivo. Furthermore, in the
absence of DPPI, the chymases exist in an activable pro-form. In our
immunodetection studies we focused on mMCP-4 and -5 because the former
is the major isoform extracted from mouse skin (21), and the latter is
the major product of mature BMMCs (27, 28). Because of the high degree
of structural similarity of the chymases, it is likely that our
findings are true for all chymases. Furthermore, the absence of
CAE-positive cells in DPPI Our observation that DPPI Recombinant human Our observation that DPPI Most proteases are synthesized as inactive zymogens that are activated
by proteolytic removal of a propeptide. Some proteases autoactivate
(e.g. cathepsins L and K), whereas others are activated by a
different protease hydrolyzing a specific site (e.g. trypsin activation by enteropeptidase). In some instances, a series of proteases must be activated sequentially before activation of a
protease. The best known example of this is the clotting cascade, where
sequential activation of proteases culminates in conversion of
prothrombin to thrombin. This activation cascade serves two purposes.
First, because each active protease activates more than one molecule of
zymogen at each stage of the cascade, the final signal (in this
example, formation of a fibrin clot by thrombin) is greatly amplified.
Second, the sequential activation generates multiple branch points at
which the cascade can be regulated. Our finding that DPPI is the sole
chymase activator establishes a cascade in mast cells in which DPPI
activates chymase, which in turn activates gelatinase B (32). What
remains to be determined is how DPPI itself is activated. Two
possibilities are that it is auto-activated in a manner similar to that
of the related cysteine proteases, cathepsin L and K (33, 34), or that
it is activated by one or more other proteases. Defining a cascade of
protease activation in mast cells is important because it provides an
understanding of how the proteolytic activity and subsequent biological
effects of the cascade are regulated.
Mutations in the DPPI gene resulting in a loss of DPPI activity are
linked to the Papillon-Lefèvre syndrome (35, 36), a disease
characterized by early periodontitis, palmoplantar hyperkeratosis, and
a predisposition to bacterial infections (37). How the absence of DPPI
activity produces the manifestations of the Papillon-Lefèvre syndrome is currently unknown. However, past studies in knockout mice
suggest that the predisposition to bacterial infections may be due in
part to altered amounts of serine protease activity (10, 38, 39). The
current studies allow us to predict that patients with
Papillon-Lefèvre syndrome will have no active mast cell chymase
and reduced levels of tryptase.
In summary, this report establishes that DPPI is essential for the
activation of mouse mast cell chymases in vivo. Furthermore, DPPI regulates the total amount of active tryptase within mast cells,
although it is not essential for tryptase activation. These findings
further our understanding of the biogenesis of the major preformed
mediator proteins of mast cells and suggest a means of regulating their
biologic functions in vivo.
/
mice. We
find that DPPI
/
mast cells contain normal amounts of
immunoreactive chymases but no chymase activity, indicating that DPPI
is essential for chymase activation and suggesting that DPPI
/
mice
are functional chymase knockouts. The absence of DPPI and chymase
activity does not affect the growth, granularity, or staining
characteristics of BMMCs and, despite prior predictions, does not alter
IgE-mediated exocytosis of histamine. In contrast, the level of active
tryptase (mMCP-6) in DPPI
/
BMMCs is 25% that of DPPI +/
BMMCs.
These findings indicate that DPPI is not essential for mMCP-6
activation but does influence the total amount of active mMCP-6 in mast
cells and therefore may be an important, but not exclusive mechanism
for tryptase activation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
types based on
structural and functional differences. Humans express
-chymase only.
In contrast, mice express five known chymases: the
-chymase
mouse mast cell protease-5 (mMCP-5) and the
-chymases mMCP-1,
-2, -4, and -9 (11). Chymases possess an acidic activation dipeptide
similar to that of most granule-associated serine proteases. Studies
using recombinant human pro-
-chymase suggest that DPPI can activate
prochymase in vitro by removing the activation dipeptide (6). Tryptases also are a diverse group of structurally related proteases that include
,
, and
isoforms in humans (12, 13) and two or three tryptases (mMCP-6, mMCP-7, and transmembrane/
) in
mice (14). MMCP-7 is not expressed in C57BL/6 mice, and thus, its
expression is strain-dependent (15). Available evidence suggests that activation of tryptases is more complicated than that of
chymases and other granule-associated serine proteases. In
vitro studies indicate that, after removal of a signal peptide by
signal peptidase, human
-tryptase requires two additional, sequential cleavages of its propeptide for activation (7). The first
such cleavage is thought to be by tryptase itself, which leaves a
two-amino acid activation dipeptide to be removed by DPPI.
Whether this sequence of events occurs in vivo is not known. Furthermore, the mechanism of activation of
-tryptase, which appears
to lack an activation dipeptide, may differ from that of the other tryptases.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-actin
were from Sigma.
/
mice were originally
developed by homologous recombination in the 129/C57BL/6 background
(10). For our studies, we used DPPI
/
mice and DPPI +/+ or DPPI
+/
littermates as controls. All experimental procedures were
performed in mice that were >8 weeks of age and were approved by the
University of California San Francisco Committee on Animal Research.
70 °C in Tissue-Tek OCT compound (Miles, Elkhart, IN), then
sectioned (5 µm). Before use, paraffin-embedded sections were
de-paraffinized in xylene, hydrated through graded alcohols, and
equilibrated in PBS. Cryosections were washed in PBS. To visualize mast
cells, tissue sections were incubated in 0.1% methylene blue for
10 s, rinsed with water, dehydrated in 100% ethanol, mounted
under coverslips with Histomount (Zymed Laboratories
Inc. Laboratories, South San Francisco, CA), and photographed.
-actin as
a loading control.
/
BMMC
lysate (equivalent to 2 × 106 BMMCs) was added to 50 µl of the DPPI activation buffer containing 0.3 µg of
pre-activated, purified, dog DPPI (20). The mixture was incubated at
37 °C for 2 h, then assayed for chymase activity using the
method noted.
BMMCs were loaded onto a 1 × 1-cm column of benzamidine-agarose (Sigma) equilibrated in high salt
buffer (10 mM bis-Tris (pH 6.1) containing 2 M
NaCl), washed extensively in high salt buffer, and then eluted with
high salt buffer containing 150 mM benzamidine. Fractions
containing eluted protein were checked for the presence of mMCP-6 by
immunoblotting. Purity was checked by SDS-polyacrylamide gel
electrophoresis. A separate column was used to purify tryptase from
40 × 106 DPPI
/
BMMCs. For sequencing, 5 µg of
tryptase purified from DPPI
/
BMMCs were subjected to
SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene
difluoride membrane in 10 mM CAPS buffer containing 10%
methanol for 1 h at 4 °C, then stained with Coomassie Blue.
Protein bands at Mr 35,000 were cut from the
membrane and subjected to Edman degradation using a 470A gas phase
sequencer with an on-line 120A phenylthiohydantoin analyzer
(Applied Biosystems, Foster City, CA) by the Biomolecular Resource
Center at the University of California at San Francisco.
and
/
BMMCs using Tri-reagent (Invitrogen, Carlsbad, CA). Denatured RNA
was size-fractionated by agarose gel electrophoresis and transferred to
Nytran Plus nylon membrane (Schleicher and Schuell). Vacuum-baked
membranes were prehybridized at 42 °C for 2 h and hybridized
with an [
-32P]dCTP (Amersham Pharmacia Biotech) random
prime-labeled, mMCP-6 probe (obtained by cloning a 310-base pair
cDNA polymerase chain reaction product corresponding to base pairs
235-545 of the mMCP-6 open reading frame) at 42 °C overnight. After
two washes at room temperature and two at 37 °C, the membrane was
exposed to film for 48 h, then developed. After removal of
previously bound probe, the membrane was hybridized with an
-actin-labeled probe to control for possible variations in signal
intensity due to differing amounts of mRNA loaded per lane.
RESULTS
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ABSTRACT
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RESULTS
DISCUSSION
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/
mice had no CAE-positive
staining cells (Fig. 1). Nonetheless,
DPPI
/
tissues contained mononuclear cells staining
metachromatically with methylene blue (Fig. 1), indicating that the
absence of CAE activity is not due to a lack of mast cells.
Furthermore, mast cells in sections of ear and tongue from DPPI
/
mice contain chymase because they are immunoreactive when stained with
anti-rMCP-I, which recognizes its mouse orthologue mMCP-4 (Fig.
2) (21). These findings suggest that the
mast cells of DPPI
/
mice contain inactive mMCP-4, which is the
principal extractable chymase isoform in mouse skin (21).
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Fig. 1.
Enzyme histochemical and histological
detection of mast cells in mouse tissues. Sections of mouse ear,
tongue, stomach, and trachea obtained from wild type (+/+) or
DPPI knockout ( /
) mice were stained for CAE activity using the
substrate naphthol AS-D chloroacetate and counterstained with
Eosin-Y. Note the blue staining CAE-positive mast cells in
tissue sections obtained from DPPI +/+ animals (top row). In
contrast, tissues sections obtained from DPPI
/
mice have no
CAE-positive cells (middle row). DPPI
/
tissue sections
stained with methylene blue demonstrate the presence of mast cells
(bottom row). Representative mast cells are indicated by
arrowheads. Images were obtained using a 40× objective.
These results were confirmed in tissues obtained from four
animals.
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Fig. 2.
Immunolocalization of chymase in mouse
tissues. 5-µm cryosections of mouse tongue (A,
B, E) and ears (C, D,
F) obtained from DPPI +/+ (A, C,
E, F) or DPPI /
mice (B and
D) were immunostained using either rabbit anti-rMCP-I
(detects mMCP-4) (A-D) or rabbit nonimmune IgG
(E and F). rMCP-I-immunoreactive cells fluoresce
green when detected with an fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody
(A-D). Representative immunoreactive cells are indicated by
arrowheads. Tissues stained with rabbit nonimmune IgG have
no immunoreactive cells (E and F). Images were
obtained using a 40× objective. These results were confirmed in
tissues from two animals.
and
/
marrow. We used DPPI +/
animals as controls for
these and all subsequent experiments because pilot studies revealed
that protease activities are similar in extracts of DPPI +/+ and DPPI
+/
tissues. DPPI
/
BMMCs develop normally and are similar to DPPI
+/
BMMCs in gross morphology and granularity (Fig.
3A). Cell lysates of DPPI +/
BMMCs contain DPPI and chymase activity (Table
I). In contrast, DPPI
/
BMMC lysates
lack detectable DPPI or chymase activity. The relative amount of
-chymase in DPPI +/
and
/
BMMCs was determined by
immunoblotting using an anti-mMCP-5 antibody. Immunoblots of DPPI +/
and DPPI
/
BMMC lysates, normalized for total protein, demonstrate
equal immunoreactive bands (Fig. 3B). The higher apparent
size of immunoreactive mMCP-5 in DPPI
/
cell extracts is consistent
with the pro-form of the enzyme (22). At least some of the pro-chymase
in DPPI
/
cell extracts remains activable, as suggested by our
ability to generate active chymase (0.38
A410/h/106 cells) in DPPI
/
extracts incubated with DPPI.
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Fig. 3.
Characterization of chymase in BMMCs.
A, DPPI +/ and DPPI
/
BMMCs cultured in
WEHI-3-conditioned medium and stained with toluidine blue. Note the
similar size, shape, and granularity of DPPI +/
and
/
BMMCs.
Images were obtained using a 100× objective. B,
quantification of relative amounts of chymase. Shown is an immunoblot
of cell lysates of DPPI +/
and
/
BMMCs (5 µg of total
protein/lane) probed sequentially with anti-mMCP-5 and
anti-
-actin antibodies as a loading control. These results were
confirmed in BMMCs obtained from three animals. C,
IgE-mediated degranulation of BMMCs. BMMCs were cultured for 24 h
in the presence of monoclonal anti-dinitrophenyl IgE. These sensitized
cells (20 × 106 cells/ml) were then incubated with
400 ng/ml dinitrophenyl-conjugated bovine serum albumin. At 0 and 60 min, an aliquot of medium was removed and separated from cells by
centrifugation. Cell pellets were resuspended in an equal volume of
medium and lysed. Supernatants were assayed for histamine by
enzyme-linked immunosorbent assay. Values represent mean net percentage
release ± S.E. for three experiments.
Protease activity in BMMC lysates
and
/
mast cells, finding that DPPI +/
and
/
BMMC histamine release is
indistinguishable after IgE receptor activation (Fig. 3C).
These findings suggest that endogenous chymase does not play a major
role in the degranulation of mouse BMMCs.
/
mice, indicating that active tryptase is present in these animals as
well. No red-brown staining cells are present in control sections
treated in the absence of substrate (data not shown).
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Fig. 4.
Identification of active tryptase in mouse
tissues. Tryptase enzyme histochemistry was performed using the
substrate
benzyloxycarbonyl-Gly-L-Pro-Arg-4-methoxy-2-naphthylamide
to identify tryptase activity in cryosections of mouse ear
(A and B), tongue (C and
D), and stomach (E and F).
Red-brown tryptase-positive cells appear in tissues from
both DPPI +/+ (A, C, and E) and DPPI
/
(B, D, and F) animals. In all
sections, representative tryptase-positive mast cells are indicated by
arrowheads. Images were obtained using a 40× objective.
These results were confirmed in tissues from four animals.
and
/
mast cells. To test this possibility, we measured tryptase
activity in DPPI +/
and
/
BMMC lysates normalized for total
protein. Although tryptase activity is present in both lysates, the
activity in DPPI
/
mast cell lysates is only 25% that of DPPI +/
lysates (Table I). Seeking an explanation for the 75% reduction of
active tryptase in DPPI
/
BMMCs, we quantified the relative amounts
of mouse tryptase (mMCP-6) protein and mRNA in DPPI +/
and
/
BMMCs. We found reduced mMCP-6 immunoreactivity in the DPPI
/
cell
lysates compared with +/
controls (Fig.
5A). Pro-mMCP-6 or mMCP-6
degradation fragments were not detected in either lysate. The relative
amounts of mRNA are equal in DPPI +/
and
/
mast cells (Fig.
5B). MMCP-6 purified from DPPI
/
mast cells possesses an
NH2-terminal amino acid sequence (IVGGHEAS) corresponding
to normally processed, active mMCP-6. No sequences corresponding to
protryptase were found. Furthermore, no mMCP-6 immunoreactivity was
detected in the benzamidine column flow-through fractions. These
findings suggest that the 75% reduction of active mMCP-6 in DPPI
/
mast cell extracts stems from decreased amounts of active protein,
suggesting that the reduction is due to post-transcriptional events.
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Fig. 5.
Characterization of mMCP-6 protein and
mRNA in DPPI +/ and
/
BMMCs. A, quantification of relative amounts of
tryptase. Immunoblotted cell lysates of DPPI +/
and
/
BMMCs (5 µg of total protein/lane) were probed sequentially with
anti-mMCP-6 and anti-
-actin antibodies. Results were confirmed in
BMMCs from three animals, and cells were lysed in the presence of
protease inhibitors. B, quantification of relative amounts
of mMCP-6 mRNA. 10 µg of total RNA isolated from DPPI +/
and
/
BMMCs were electrophoresed and blotted, then probed sequentially
for mMCP-6 and actin with the corresponding
[
-32P]CTP-labeled cDNA probes.
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mouse tissues containing mMCP-1, 2, 4, and 5 (28, 29) supports this assertion, since the CAE procedure is an
activity-based histochemical approach that detects chymase activity in
all known chymase-containing subsets of mast cells in a broad spectrum
of mammalian tissues.
/
mast cells degranulate as well as DPPI
+/
mast cells indicates that endogenous DPPI and chymase play little
or no role in the IgE-mediated release of histamine from BMMCs. These
findings are contrary to expectations generated by reports that chymase
directly degranulates or potentiates IgE-mediated degranulation
(23-25, 30). These reports are based largely on experiments conducted
in vitro using inhibitors that are not chymase-specific and
potentially non-physiologic quantities of chymase. Also, in vitro findings may fail to predict in vivo behavior of
chymases when conducted in the absence of natural chymase inhibitors or using a chymase that is unassociated with the proteoglycans to which
chymases are bound to upon release from mast cell granules. Our studies
overcome some of these limitations by comparing degranulation using
mast cells with and without endogenously synthesized and packaged
chymase. However, our findings in mouse BMMCs do not rule out the
possibility that chymases modulate mast cell exocytosis in humans or
other mammals or in subsets of mouse mast cells expressing a different
profile of chymases.
-protryptase can be activated in vitro
by two sequential steps of propeptide processing (7). First,
-protryptase is autoprocessed to the inactive intermediate
pro'-tryptase, which has a residual pro-dipeptide. The pro'-tryptase
intermediate can then be fully activated by DPPI, which removes the
dipeptide. Because of sequence similarities of the pro-region of mMCP-6
to
-tryptase, we expected that mMCP-6 would be inactive in DPPI
/
mouse mast cells and that any immunoreactive protein would be in
the pro' form. To our surprise, enzyme histochemistry showed active
tryptase in mast cells of tissues obtained from DPPI
/
mice. This
activity is due in part to mMCP-6 because it exists entirely in its
normally processed, active form in DPPI
/
BMMCs. This finding is
similar to that of granzyme C in DPPI
/
mice. In these animals,
50% of granzyme C is found in the pro-form, and 50% is found in the
active form (10). These observations indicate that DPPI is not
essential for the activation of all mast cell tryptases or granzyme C. How mMCP-6 becomes activated and whether tryptase and granzyme C share
a mechanism of activation in the absence of DPPI are unclear. One
possibility is that mMCP-6 undergoes two-step processing by tryptase
and a second protease, possibly an aminopeptidase (31), which
compensates for DPPI. Alternatively, mMCP-6 may be activated directly
by an endoprotease. Candidates include cathepsins B, L, and S, which
are present in BMMCs.2
/
BMMCs contain less mMCP-6 than DPPI
+/
control cells indicates that DPPI influences the total amount of
active tryptase in BMMCs. Furthermore, this influence probably is
exerted post-transcriptionally, given the equivalence of steady state
mRNA levels in the two types of cells. One explanation for our
findings is that DPPI normally plays a role in two-step processing of
protryptase to an active, protease-resistant form. In the absence of
DPPI, tryptase exists in its pro- or pro'-form longer because the
compensating protease is less efficient, thereby increasing the time of
exposure to degradative enzymes as pro-tryptases traffic from the
endoplasmic reticulum to the secretory granules. This explanation is
consistent with prior observations that unprocessed granzymes are
susceptible to degradation in DPPI
/
mice (10). Alternative, less
likely explanations are that DPPI processes proteins that regulate the
quantity of degradative enzymes or that DPPI directly stabilizes
tryptase molecules during biogenesis.
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ACKNOWLEDGEMENT |
---|
We thank Jon Mallen-St. Clair for excellent technical support.
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FOOTNOTES |
---|
* This work was supported in part by the American Lung Association of California and National Institutes of Health Grants HL-04055 (to P. J. W.), HL-03774 (to C. T. N. P.), DK-49786 (to T. J. L.), and HL-24136 (to G. H. C.).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: University of California, San Francisco, Box 0911, San Francisco, CA 94143-0911. Tel.: 415-514-2601; Fax: 415-476-9749; E-mail: pjwolt@itsa.ucsf.edu.
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M100223200
2 P. J. Wolters, C. T. N. Pham, D. J. Muilenburg, T. J. Ley, and G. H. Caughey, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: DPPI, dipeptidyl peptidase I; mMCP, mouse mast cell protease; rMCP, rat mast cell protease; BMMC, bone marrow-derived mast cell; CAE, naphthol AS-D chloroacetate esterase; CAP, 3-(cyclohexylamino)propanesulfonic acid; PBS, phosphate-buffered saline; TBS, Tris-buffered saline.
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