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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 849-856
A Secreted Proform of Neutrophil Proteinase 3 Regulates the
Proliferation of Granulopoietic Progenitor Cells
By
Stefan Sköld,
Bodil Rosberg,
Urban Gullberg, and
Tor Olofsson
From the Department of Hematology, Research Department 2, University
Hospital, Lund, Sweden.
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ABSTRACT |
Myeloid leukemia cells, the human promyelocytic cell line HL-60, and
a subpopulation of normal marrow cells produce a leukemia-associated inhibitor (LAI) that reversibly downmodulates DNA synthesis of normal
granulopoietic progenitor cells colony-forming unit
granulocyte-macrophage (CFU-GM). We isolated an active 125-kD
component of LAI from HL-60 conditioned medium (CM), subjected it to
cyanogen bromide cleavage and show by amino acid
sequencing of the resulting peptides that it consists of a complex of
the serine proteinase inhibitor  1-antitrypsin and a 31-kD fragment
that retained the S-phase inhibitory activity, but resisted sequencing.
This finding suggested that the 31-kD fragment originated from one of
the neutrophil serine proteases (ie, elastase, proteinase 3, or
cathepsin G) produced by normal promyelocytes, as well as HL-60 cells,
for storage in primary granules and partly secreted during synthesis as
enzymatically inactive proforms. Immunoblot analysis showed that the
125-kD complex contained proteinase 3 (PR3), and immunoprecipitation of
PR3 from HL-60 CM abrogated the S-phase inhibitory activity, whereas immunoprecipitation of cathepsin G or elastase did not. Immunoprecipitation of PR3 from CM of a subpopulation of normal marrow
cells also abrogated the S-phase inhibitory effect. Furthermore, CM
from rat RBL and murine 32D cell lines transfected with human PR3 both
reduced the fraction of CFU-GM in S-phase with 30% to 80% at 1 to 35 ng/mL PR3, whereas CM of the same cells transfected with cathepsin G or
elastase did not. Also, an enzymatically silent mutant of PR3 exerted
full activity, showing that the S-phase modulatory effect is not
dependent on proteolytic activity. Amino acid sequencing of
biosynthetically radiolabeled PR3 showed that PR3 from transfected
cells is secreted after synthesis as proforms retaining amino terminal
propeptides. In contrast, mature PR3 extracted from mature neutrophils
has only minor activity. The inhibitory effect of secreted PR3 is
reversible and abrogated by granulocyte (G)- or granulocyte-macrophage
colony-stimulating factor (GM-CSF). Experiments with highly purified
CD34+ bone marrow cells suggested that PR3 acts directly
on the granulopoietic progenitor cells. These observations suggest a
role for PR3 in regulation of granulopoiesis, and possibly in
suppression of normal granulopoiesis in leukemia.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
LEUKEMIA-ASSOCIATED inhibitor (LAI) was
described as a large glycoprotein found in the conditioned media of
leukemia cells1,2 and the human promyelocytic cell line
HL-60.3 LAI reversibly reduces the fraction of normal
granulocyte-macrophage progenitor cells (colony-forming unit
granulocyte-macrophage [CFU-GM]) in S-phase, whereas leukemic
clonogenic cells seem to be unresponsive. It was postulated that LAI
could have a pathophysiologic role in the suppression of normal
hematopoiesis characteristic for acute leukemia and provide a growth
advantage for the leukemia cells due to overproduction. The observation
that LAI is produced by a subpopulation of normal bone marrow
cells4 suggested that it also might have a growth
regulatory role in normal hematopoiesis. LAI was purified from HL-60
conditioned medium (CM) and the biological activity shown to reside in
a 125-kD component.5 Now, we have determined the identity
of LAI and present evidence that the 125-kD component is a complex of
bovine  1-antitrypsin (added to the culture medium by fetal calf
serum [FCS]) and PR3. Proteinase 3 (PR3) belongs to a family of
neutrophil serine proteases, where the other members are leukocyte
elastase and cathepsin G, and the catalytically inactive
azurocidin.6-8 Their microbicidal properties, which are
independent of their proteolytic activity, make them important for
microbial killing during phagocytosis.9 In inflammation,
their proteolytic activity is responsible for digestion of matrix
components such as elastin, fibronectin, and collagen. In emphysema,
leukocyte elastase is regarded as the major enzyme responsible for
destruction of connective tissue in the lung,10 and PR3 is
known as the autoantigen in Wegener's granulomatosis.11-14
The serine proteases are synthesized almost exclusively in
promyelocytes and stored in the primary granules.7,8,15 Activation of mature neutrophils may lead to translocation of elastase,
cathepsin G, and PR3 to the cell surface,16-18 possibly as
part of a mechanism to facilitate egress of the neutrophils from blood
vessels.19
Recent studies have shown that during synthesis minor portions of the
catalytically inactive proforms of serine proteases escape granule
targeting and are secreted.20-25 This phenomenon has been
regarded as an imperfection of the granule targeting process and the
secreted proforms have so far not been ascribed any function.
Therefore, the findings described in this report that a secreted
proform of PR3 can downmodulate DNA synthesis in normal hematopoietic
progenitor cells adds another property to neutrophil proteases and
implies a novel function of PR3 as a putative negative feedback
regulator of granulopoiesis.
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MATERIALS AND METHODS |
Large-scale production of HL-60 cell CM.
This has been described in detail previously5; briefly,
HL-60 cells were expanded in RPMI with 10% FCS in a 10-L glass bottle
with intermittent stirring and CM was harvested daily by use of a
peristaltic pump connected to a plasmapheresis filter, which allows
recirculation of the cells back to the bottle. CM was immediately
concentrated 10× using a Pellicon Cassette System (Millipore
Corp, Milford, MA) equipped with a PTHK Cassette filter with cutoff at
100,000 molecular weight (MW), and stored frozen.
For production of HL-60 CM to be used as a positive control in the
assay of CFU-GM in S-phase and for immunoprecipitation (see below),
fresh HL-60 cells were harvested, resuspended in RPMI 5% FCS, and
incubated at 2 × 106 cells/mL at 37°C for 3 to 4 hours. The cell-free supernatant was filter sterilized and stored frozen.
Chromatography on Phenyl-Sepharose.
A total of 1 mol/L ammonium sulfate, 0.02% Tween 20, and 0.02% sodium
azide was added to the concentrated HL-60 CM and 5 to 600 mL at a time
applied to a Phenyl-Sepharose column (2.5 × 45 cm) (Pharmacia
Fine Chemicals, Uppsala, Sweden) equilibrated in 1 mol/L ammonium
sulfate, 0.1 mol/L sodium phosphate buffer pH 6.0, 0.02% Tween 20, and
0.02% sodium azide (starting buffer). The CM was applied at a rate of
20 mL/h, and the column was washed with starting buffer (200 mL) before
elution with gradient 1 (200 mL starting buffer and 200 mL
H2O) immediately followed by gradient 2 (150 mL
H2O and 150 mL 70% ethylene glycol); 10-mL fractions were
collected and the absorbance at 280 nm measured. Gradient 1 was
registered by measuring conductivity and gradient 2 by refraction index. To assay the content of LAI 0.5-mL aliquots of every second fraction were mixed with 5 mL McCoy's medium 1% FCS and then washed on XM100 filters (Amicon Corp, Lexington, MA) with 15 mL
McCoy's medium 1% FCS and concentrated to 2 mL before filter sterilization.
Ion exchange chromatography.
Fractions from Phenyl-Sepharose chromatography with LAI-activity (10 to
12 fractions from each chromatogram, two chromatograms at a time) were
pooled and washed on XM100 filters with 20 mmol/L Tris pH 7.5, 0.05%
Tween 20 and concentrated to 10 mL. This material was then applied to a
MonoQ column (1 × 10 cm attached to a Pharmacia FPLC System;
Pharmacia Fine Chemicals) and eluted with 1 mol/L NaCl in 20 mmol/L
Tris pH 7.5, 0.05% Tween 20, at 1 mL/min increasing to 0.5 mol/L NaCl
over a period of 40 minutes; 1-mL fractions were collected. Aliquots of
0.1 to 0.5 mL were mixed with McCoy's medium 1% FCS and washed and
concentrated to 2 mL on XM100 filters before filter sterilization and
assay of LAI activity.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE).
Pooled fractions from the MonoQ separation were taken to preparative
SDS-PAGE.5 Samples were run under reducing conditions, but
not boiled before electrophoresis to avoid destruction of biological
activity. One lane with sample was silver-stained before being used as
a guide to cut out the 125-kD band from the unstained part of the gel
previously shown to contain the LAI-activity.5 Protein was
electroeluted from the gel pieces (Bio-Rad electro-eluter model 422, Bio-Rad Lab, Richmond, CA) and precipitated in 90% ethanol and 50 µg/mL dextran T500 (Pharmacia Fine Chemicals) in the
cold overnight. The precipitate was collected by centrifugation, taken
to dryness, and used for cyanogen bromide (CNBr) cleavage.
CNBr cleavage.
Electroeluted material containing the 125-kD component was dissolved in
70% formic acid with 0.5% 2-mercaptoethanol, and a 50-fold to
100-fold molar excess of CNBr was added; the reaction was allowed to
continue for 24 hours under nitrogen in the dark at room
temperature.26 Afterward the reaction mixture was diluted 1:3 with water and 0.1% trifluoroacetic acid (TFA) and 10%
acetonitrile was added and the sample run on high-performance liquid
chromatography (HPLC) (Vydac C4 column, The Separations Group,
Hesperia, CA) to remove salt and dextran. Protein containing fractions
were added with 80 mmol/L urea and taken to dryness and then dissolved in sample buffer for SDS-PAGE and Western blotting.
Western blot and amino acid sequencing.
After CNBr cleavage of the 125-kD component, the peptide fragments were
electrophoresed on SDS-PAGE and blotted onto polyvinylidene fluoride
(PVDF) membranes by semidry blot. The membranes were stained in Coomassie blue (0.1% in 50% methanol) and stained bands cut out of the membrane and subjected to automated amino acid sequencing (BioMolecular Resource Facility, University of Lund). CNBr
cleavage fragments isolated on preparative SDS-PAGE were also
electroeluted and tested for LAI activity.
Transfectant cell lines.
Rat RBL and murine 32D cells transfected with human neutrophil
PR3,25 cathepsin G,23,27 or
elastase28 and known to secrete proforms of the transfected
proteins, were cultured for 1 to 3 days to produce conditioned media,
which then were tested for LAI activity.
cDNA and site-directed mutagenesis.
Full-length cDNA for human PR3 was cloned into expression vector as
described.25 To create an enzymatically inactive mutant of
PR3 (PR3/cat.del), Ser 203 (numeration from the ATG translational initiation site) in the catalytical amino acid triad of the enzyme was
substituted with glycine by use of site-directed mutagenesis as
described.29 The polymerase chain reaction (PCR) primers in
the two amplifications were upstream 5'-TTC GGA AAG CTT
GCC ACC ATG GCT CAC CGG CCC CCC AGC-3' (no. 1), plus
downstream 5'-GGG GCC ACC GCC GTC TCC GAA-3' (no.
2), and upstream 5'-TTC GGA GAC GGC GGT GGC CCC-3'
(no. 3), plus downstream 5'-T TCA GAA TTC CGC TGT GGG
AGG GGC GGT TCA-3' (no. 4), respectively (start and
stop codons in bold, restriction enzyme sites underlined, codons for
Gly 203 in italics). The PCR product was cloned into pcDNA3-plasmid and
individual clones were isolated and sequenced to verify the mutation
and the integrity of the reading frame.
Transfection procedure.
The rat basophilic/mast cell line RBL and murine myeloblast-like 32D
cells were grown as described.27 Exponentially growing cells were transfected by electroporation as previously
described.23,27 Individual clones growing in the presence
of geneticin were isolated, expanded in mass cultures, and screened for
expression of PR3 by biosynthetic labeling.23,27 Clones
with the most pronounced expression were selected for further experiments.
Immunoprecipitation.
For immunoprecipitation, 2.5 mL HL-60 CM was incubated 18 hours with 10 µL of the following antibodies: anti-PR3 monoclonal antibody 4A3,
rabbit anti-PR3 antibody30 (both a generous gift from Dr
Jörgen Wieslander, Wieslab, Lund, Sweden), rabbit anticathepsin G, rabbit antielastase, and rabbit antimyeloperoxidase.21 A total of 10 mg protein A-Sepharose was added to each tube, and the
incubation was continued under rotation for another 4 hours before
centrifugation to pellet the Sepharose particles. The supernatant was
withdrawn, filter sterilized, and tested for remaining LAI activity.
Immunoblot analysis.
Purified PR3 from mature neutrophils (same as used as standard in
enzyme-linked immunosorbent assay [ELISA]) and electroeluted 125-kD
component from preparative SDS-PAGE was dot blotted onto nitrocellulose
paper and incubated with monoclonal antibodies against PR3 (1:500
dilution) for 2 hours. Nonspecific binding sites were blocked by
incubation with 2% bovine serum albumin (BSA). Alkaline
phosphatase-conjugated goat antimouse antibodies were then applied
(1:1,000 dilution) (DAKO A/S, Copenhagen, Denmark) for 60 minutes and
bound alkaline phosphatase activity visualized using
bromochloroindolyl/nitroblue tetrazolium substrate according to the
manufacturer's description.
ELISA for human PR3.
The concentration of free PR3 in HL-60 CM and CM of PR3-transfected RBL
and 32D cell lines, respectively, was measured by ELISA as
described30; the monoclonal anti-PR3 antibodies used as
capture antibodies, the secondary rabbit anti-PR3 antibody, and the
purified human neutrophil PR3 used as standard,30 were all
generous gifts from Dr Jörgen Wieslander. The standard curve ranged from 1 to 200 ng/mL and the detection limit was 3 ng/mL.
Radiosequence analysis of secreted PR3.
This was performed as described previously.25 To determine
the amino terminal sequence of secreted PR3, RBL cells transfected with
PR3/cat.del were grown for 6 hours in isoleucine-free RPMI medium with
3% dialyzed FCS and supplemented with [3H]-isoleucine
(100 µCi/mL) to achieve metabolic labeling of synthesized proteins.
After pulse labeling, the cell-free supernatant was collected and
subjected to immunoprecipitation using the rabbit anti-PR3 antibody.
The immunoprecipitate was taken to SDS-PAGE, electroblotted to a PVDF
membrane, and after localization of the radiolabeled PR3 by
autoradiography as a single band at approximately 35 kD, the band was
excised from the PVDF membrane and subjected to amino acid sequencing.
The initial 10 degradation cycles were assayed for radioactivity by
scintillation counting.
Normal marrow cell CM.
To obtain LAI from normal bone marrow, low density marrow cells were
isolated on Lymphoprep (Nycomed, Oslo, Norway) and phagocytic cells
removed by carbonyl iron4 before incubation at 5 × 106 cells/mL in McCoy's medium 10% FCS at 37°C for 5 hours. The cell-free CM was harvested and tested for S-phase reducing
activity before and after immunoprecipitation with rabbit anti-PR3
antibodies as described above.
Assay of CFU-GM in S-phase.
This was performed as previously described3,5 with minor
modifications. Briefly, human bone marrow mononuclear cells obtained by
separation on Lymphoprep were incubated in duplicates at 1.5 × 106 cells/mL with an equal volume of McCoy's medium 1%
FCS (control), and the different CM from HL-60 cells, wild-type and
transfected RBL and 32D cells, respectively, as well as the purified
PR3 used in the ELISA, for 60 minutes (or as indicated in text) before addition of 2 µg/mL of cytosine arabinoside (Cytosar, Upjohn, Kalamazoo, MI) to one of the tubes for another 45 minutes
to kill cells in S-phase. The tube without cytosine arabinoside serves as control within each pair of tubes and to verify that the added CM
does not have unspecific cytotoxic effects on the colony-forming cells.
Cells were washed three times and cultured in four replicates in 0.3%
agar in McCoy's medium with 15% FCS and 10% CM from the bladder
carcinoma cell line 5637 as colony-stimulating factor or a combination
of recombinant human (rh) G-CSF (Neupogen; Roche, Basel,
Switzerland) and rhGM-CSF (Leucomax; Schering-Plough,
Kenilworth, NJ), 20 ng/mL of each. CFU-GM colonies of more
than 50 cells were counted in an inverted microscope after a 10-day
incubation at 37°C in 5% CO2 in humidified air. The
difference in number of colonies between the control tube without
cytosine arabinoside and the tube incubated with cytosine arabinoside
is a measure of the number of CFU-GM in S-phase. Normally, 35.5% ± 2.4% (mean ± standard deviation [SD]; range, 30.8 to 41.5; n = 25) of CFU-GM are in S-phase, which means that all CFU-GM are in cell
cycle. LAI activity results in a reduced S-phase fraction without
reduction of the number of colonies in the control tubes, ie, it has no cytotoxic effects. Instead of cytosine arabinoside tritiated thymidine or hydroxyurea can be used to kill cells in S-phase with similar results.31 In three experiments, the marrow cells were
cultured in methylcellulose with erythropoietin (GIBCO-BRL, Life
Technologies, Gaithersburg, MD) for assay of burst-forming
unit-erythroid (BFU-E) in S-phase.
CD34+ progenitor cells as target cells.
Mononuclear cells of human marrow were labeled with monoclonal
anti-CD34-fluorescein isothiocyanate (FITC) and
anti-CD38-phycoerythrin (PE) (Becton Dickinson, San Jose,
CA) at 4°C for 30 minutes and washed twice in Iscove's modified
Dulbecco's medium (IMDM) with 20% FCS before
fluorescence-activated cell sorting on a FACS Vantage flow cytometer
equipped with the Turbo Sort Option (TSO) (Becton Dickinson) in a
two-step procedure; first CD34+ cells within an extended
lymphocyte gate with low side scatter were enriched by high speed
sorting (20,000 cells/s) and then resorted at lower speed (1,500 cells/s) into CD34+/CD38+ and
CD34+/CD38 cells, respectively. The
CD34+/CD38+ cells (purity >97%) were
incubated at 20 to 30,000 cells/mL with 10% FCS in McCoy's medium at
37°C for 60 minutes before addition of 50% CM of PR3 transfected
RBL cells (or medium alone to the control) for another 60 minutes,
followed by cytosine arabinoside for 45 minutes as described above. To
minimize cell losses, 1.5 × 106 autologous blood
mononuclear cells were added to each tube during washing before culture
in agar as described above; this addition of blood mononuclear cells
does not affect colony formation.
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RESULTS |
Purification of LAI.
Figure 1A shows chromatography on
Phenyl-Sepharose, demonstrating the hydrophobic properties of LAI;
essentially all LAI activity bound to the column and eluted with 15%
to 50% ethylene glycol. Figure 1B shows ion exchange chromatography on
MonoQ showing a charge heterogeneity of LAI in accordance with previous
observations.2 The LAI activity eluted between 0.10 to 0.15 mol/L NaCl (pool 1) was quantitatively insufficient for further
attempts of purification. Pool 2 eluted between 0.24 to 0.27 mol/L
NaCl, and pool 3 between 0.37 to 0.41 mol/L NaCl and were used for
further purification on preparative SDS-PAGE as shown in
Fig 2; lane A is silver-stained and used as
a guide for excision of the LAI containing 125-kD bands and lane B
shows the resulting purified component after electroelution and ethanol
precipitation. Aliquots of this material reduced the fraction of CFU-GM
in S-phase from 37.2% to 22.5% (n = 3, P < .01).
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| Fig 1.
Purification of LAI. (A) Shows chromatography of HL-60 CM
on Phenyl-Sepharose (1/40 similar chromatograms). Protein concentration
is shown as absorbance at 280 nm; gradient 1 is shown as the left part
of the dotted line and was measured as conductivity (C), and gradient 2 is the right part of the dotted line registered as percentage ethylene
glycol (EG%). The insert shows percentage of CFU-GM in S-phase. (B)
Shows ion exchange chromatography on MonoQ FPLC (1/22 similar). The
gradient of increasing NaCl is shown as a dotted line and three regions
of material that was pooled are shown (p1-3). Other symbols as in
(A).
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| Fig 2.
Isolation of the 125-kD component of LAI. Lanes A and B
show preparative SDS-PAGE for isolation of the 125-kD component marked
by an arrow in lane A and the resulting electroeluted material in lane
B. Lane C shows the peptide fragments after CNBr cleavage blotted onto
a PVDF membrane and stained with Coomassie blue. Bands I through V were
excised for amino acid sequencing. The position of MW markers is
indicated.
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The peptide fragments produced by CNBr cleavage were not sufficiently
separated by HPLC and instead we chose SDS-PAGE and blotting onto PVDF
membranes for amino acid sequencing (Fig 2, lane C). There were three
major bands at 31 kD, 27 kD, and 23 kD (III-V); sequencing of
the 31-kD component (band III) failed at three different occasions,
whereas the amino terminal sequence of band IV and V were identical
(LSLGAKGNT) and identified as amino acids 64-72 of bovine
1-antitrypsin.32 This sequence was confirmed in two
additional experiments. The faint bands at approximately 40 kD and 67 kD varied in intensity between different preparations, but were also
derived from bovine 1-antitrypsin. CNBr fragments III-V were also
isolated by electroelution and tested for LAI activity; the
1-antitrypsin-derived fragments had no effect on DNA synthesis,
whereas the 31-kD fragment had LAI activity suggesting that it is
identical to LAI; CFU-GM in S-phase was 36% in the control and 11%
with the 31-kD fragment in one experiment.
Immunoprecipitation of LAI and immunoblot analysis.
Because the 31-kD fragment could not be identified by amino acid
sequencing and the association with 1-antitrypsin suggested an
identity with the neutrophil serine proteases, we investigated this
possibility by subjecting HL-60 CM to immunoprecipitation. Antibodies
against myeloperoxidase (control), elastase, and cathepsin G were
without effect, whereas antibodies against PR3, both the monoclonal and
the polyclonal antibodies, neutralized the LAI activity, suggesting
that LAI is identical to PR3 (Table 1). This was further substantiated by results from immunoblot analysis showing that the electroeluted 125-kD component contained PR3 (Fig 3). However, the 31-kD CNBr fragment
had lost its immunoreactivity (not shown).
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| Fig 3.
Immunoblot analysis of the 125-kD component isolated by
electroelution from preparative SDS-PAGE. A total of 100, 50, and 25 ng
purified PR3 from mature neutrophils was applied to a nitrocellulose
membrane in dots A, B, and C, respectively; E shows the reaction of the
125-kD component from one SDS-PAGE gel, and D the equivalent volume of
electroelution buffer (negative control).
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Secreted PR3 has LAI activity.
To show that PR3 can downregulate the S-phase fraction of normal
CFU-GM, we next tested CM from transfected cell lines with stable
expression of human PR3. As controls, we used untransfected RBL
wild-type cells and RBL or 32D cells transfected with human neutrophil
cathepsin G and elastase. Figure 4 shows
that CM from PR3 transfected cells did reduce the fraction of CFU-GM in
S-phase, comparable to what is seen with HL-60 CM, whereas CM from RBL and 32D cells transfected with cathepsin G or elastase had no such
effect. The concentration of PR3 in CM, as measured by ELISA, ranged
from 29 to 35 ng/mL in different preparations of HL-60 CM, and 21 to 48 ng/mL in CM of RBL and 32D cells transfected with PR3 used in these
experiments. Interestingly, CM from RBL cells transfected with a
catalytically inactive form of PR3 (PR3/cat.del; Ser203-Gly) (28 to 34 ng/mL) was equally effective as CM from cells transfected with
wild-type PR3 (Fig 4). Figure 5 shows that there is a dose-response relationship between concentration of secreted
PR3 in CM and reduction of CFU-GM in S-phase. However, human PR3
purified from the granule fraction of normal mature neutrophils had
insignificant effects within the range 15 to 60 ng/mL PR3 and only
minor effects at concentrations above 60 ng/mL (Table 2).
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| Fig 4.
Effect of CM from transfected cells on the S-phase of
normal CFU-GM. Control shows CFU-GM in S-phase (mean ± SD) with
medium alone (n = 4); HL-60 CM (n = 4); RBL/Wild CM from
untransfected cells (n = 2); RBL/PR3 proteinase 3 transfectant (n = 4); RBL/Cath G cathepsin G transfectant (n = 2); RBL/Elastase
transfectant (n = 2); RBL/PR3/cat.del catalytically inactive PR3
transfectant (n = 3); 32D/PR3 (n = 3); 32D/Cath G (n = 2); 32D/Elastase (n = 2). The asterisk (*) denotes significant
reduction of the number of CFU-GM in S-phase (P < .01).
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| Fig 5.
Dose-response relationship between secreted PR3 and
reduction of S-phase of CFU-GM. RBL/PR3 transfectant cells were grown
for 3, 6, 24, and 72 hours and the resulting CM tested for
S-phase-reducing activity. The concentration of PR3 in ng/mL was
measured by ELISA. The number of colonies per dish in the control was
268 ± 32 (mean ± SD) without and 175 ± 21 with cytosine
arabinoside, respectively.
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Secreted PR3 in normal marrow CM.
Nonphagocytic low density marrow cells also produce LAI4
and CM collected after a 5-hour incubation of such cells was tested for
S-phase reduction before and after immunoprecipitation of PR3.
Untreated CM (14 to 17 ng/mL PR3) significantly reduced the fraction of
CFU-GM in S-phase from 36.8% ± 4.3% (control, n = 4) to
20.3% ± 5.1% (n = 6, P < .001), whereas the same CM
after immunoprecipitation (no measurable PR3 by ELISA) was without
effect; S-phase fraction was 37.8% ± 4.8% (n = 6, not
significant). Normal plasma from five donors was also tested
repeatedly, but no S-phase-reducing activity was found in any case
(data not shown).
PR3 is secreted as a proform.
Previous studies have shown that early during synthesis PR3 exists in
proforms retaining an amino terminal propeptide25 not
present in purified mature PR3.13 The molecular size of the
secreted proform of PR3 is approximately 35 kD, whereas the processed
form targeted to granules is about 32 kD.25 Mature PR3 as
found in extracts of azurophil granules is 29 kD.30 The amino acid sequence of mature PR3 starts with an isoleucine, which makes it possible to study the amino terminal sequence of secreted PR3
after biosynthetic radiolabeling with [3H]-isoleucine.
Radiolabeled PR3 was isolated by immunoprecipitation, SDS-PAGE, and
Western blot, and subjected to amino acid sequencing. The first 10 amino acids of mature PR3 are IVGGHEAQPH and the seven preceding amino
acids of the propeptide are GAARAAE. As shown in
Fig 6, the two first amino acids lacked
radioactivity, indicating the presence of a propeptide of two amino
acids in the secreted form of PR3. The major peak of radioactivity is
in the third amino acid residue corresponding to isoleucine in position one of the mature PR3. In this case, the amino terminal sequence of
secreted PR3 would be AEIVGGHEAQPH. However, as demonstrated previously
for intracellular proforms of PR3,25 a minor peak of
radioactivity in amino acid number eight suggests that an alternative proform containing a seven amino acid propeptide also is secreted, corresponding to the amino terminal sequence GAARAAEIVGGHEAQPH.
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| Fig 6.
Radiosequencing of PR3 secreted from RBL/PR3 transfectant
cells during a 6-hour incubation in the presence of
[3H]-isoleucine. PR3 in the CM was immunoprecipitated and
isolated on SDS-PAGE and transferred by Western blot to a PVDF membrane
from which the radioactive band at 35 kD was excised and subjected to
amino acid sequencing. The columns show the radioactivity of the first
10 cycles of sequencing, corresponding to the first 10 amino acids.
Blank (bl) shows the background activity.
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PR3 activity is reversible and counteracted by CSF.
When bone marrow cells incubated with RBL/PR3 CM for 2 hours are washed
and left in fresh medium for 20 hours, the downmodulation of DNA
synthesis is reversed as shown in Fig 7A.
When G-CSF or GM-CSF at 20 ng/mL is added together with PR3 CM (2 to 8 ng/mL PR3) for 2 or 20 hours, the DNA synthesis inhibitory activity of
PR3 is abrogated (Fig 7B and C). Figure 8
shows that the effect of G-CSF decreases with increasing concentration
of PR3; at 0.3 to 1.5 ng/mL PR3 G-CSF significantly abrogated the
effect of PR3, but was without effect at 15 ng/mL PR3. The extended
incubations of marrow cells with PR3 CM for up to 20 hours did not
reduce the number of colony-forming cells, although the fraction of
CFU-GM in S-phase remained at a low level as long as PR3 was present, thus showing that PR3 CM did not have any cytotoxic effect toward the
progenitor cells (data not shown). The inhibitory effect of PR3 may be
restricted to granulopoietic progenitors, as PR3 CM did not reduce the
S-phase fraction of BFU-E (control mean value, 40.4%; PR3 CM, 41.3%;
n = 3, P = .43).
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| Fig 7.
Reversibility and modulation by CSF of PR3 activity. (A)
Shows CFU-GM in S-phase after a 2-hour incubation with PR3 CM followed
by washing of the cells and continued incubation in fresh medium for
another 20 hours before addition of cytosine arabinoside. The middle
bar shows CFU-GM in S-phase at 2 hours when incubation with PR3 was
interrupted. Mean ± SD of three experiments. (B) Shows 2 to 5 hours'
incubation with PR3 CM with and without G-CSF present at 20 ng/mL. Mean ± SD of five experiments. (C) Shows similar experiments with 20 hours' incubation with and without G-CSF or GM-CSF present at 20 ng/mL
before addition of cytosine arabinoside. Mean ± SD of five (G-CSF)
and three (GM-CSF) experiments. The asterisk denotes statistically
significant reduction of S-phase in the positive controls (P < .01). The concentration of PR3 in these experiments was 1.5 to 7.5 ng/mL. The number of colonies per dish in the controls in these
experiments ranged from 140 to 343, median, 244. G-CSF or GM-CSF in
itself did not change the fraction of CFU-GM in S-phase (data not
shown).
|
|
View larger version (18K):
[in this window]
[in a new window]
| Fig 8.
G-CSF abrogates the effect of PR3. ( ) Shows the
dose-dependent reduction of CFU-GM in S-phase at 0.3, 1.5, and 7.5 ng/mL PR3 (2%, 10%, and 50% RBL/PR3 CM, respectively) and ( )
shows S-phase fraction with the addition of G-CSF 20 ng/mL during a
2-hour incubation before addition of cytosine arabinoside. At 0.3 and
1.5 ng/mL PR3, G-CSF resulted in a statistically reduced effect of PR3
(P < .01). One representative experiment is shown.
The number of colonies per dish in the control without cytosine
arabinoside was 244 ± 26 (SD) and 159 ± 12 with cytosine
arabinoside, respectively.
|
|
PR3 acts directly on the progenitor cells.
To elucidate the question whether PR3 interacts directly with CFU-GM
progenitor cells, highly purified CD34+ progenitor cells
(>97% pure) isolated by fluorescence-activated cell sorting were
incubated with CM of PR3 transfected RBL cells. As shown in
Fig 9, PR3 reduced the fraction of CFU-GM
in S-phase to the same extent as when bone marrow mononuclear cells
were used as target cells, which suggests that PR3 acts directly on the
progenitor cells.
View larger version (25K):
[in this window]
[in a new window]
| Fig 9.
Effect of RBL/PR3 CM on the S-phase fraction of CFU-GM
within a CD34+ cell population isolated by cell sorting
(mean values ± SD, n = 8). The number of colonies in the controls
varied from 52 to 176 (mean, 98) per dish in these experiments. The
asterisk denotes significant reduction of CFU-GM in S-phase (P < .01).
|
|
| |
DISCUSSION |
We show in this report that the S-phase-reducing activity towards
normal granulopoietic progenitor cells purified from HL-60 CM is a
complex of 1-antitrypsin and a secreted proform of PR3. Although the
31-kD CNBr fragment holding the S-phase inhibitory activity was not
identifiable by amino acid sequencing, there are three lines of
evidence for identity between the 31-kD fragment and PR3. First, the
125-kD complex in fact contains PR3 as shown by immunoblot staining.
Second, antibodies against PR3 precipitated the S-phase-reducing
capacity of HL-60 CM, whereas antibodies against the other serine
proteases, cathepsin G and elastase, did not. Third, transfected human
PR3 secreted by RBL or 32D cells reduced the fraction of CFU-GM in
S-phase in a manner indistinguishable from that of HL-60 CM.
Furthermore, CM from HL-60 cells and from the PR3 transfectant cell
lines had approximately the same concentration of PR3. The
radiosequencing data showing two different amino terminals of the
secreted form of PR3 could explain the difficulties in obtaining
interpretable amino acid sequences from the 31-kD CNBr fragment.
It is noteworthy that the majority of PR3 in CM is in a free form not
complexed with 1-antitrypsin,25 and it is probably the
free form that primarily is responsible for the activity towards the
granulopoietic progenitor cells. However, PR3 form stable and
SDS-resistant complexes with 1-antitrypsin as described for other
serpin-serine protease complexes.33 Due to the strong hydrophobic properties of PR3, the free form was largely lost during
purification through unspecific adsorbance to column materials, thus
explaining the recovery of PR3 only in complex with 1-antitrypsin after extensive purification.
The S-phase reduction is dose-dependent and reaches full effect at 15 to 30 ng/mL PR3, which corresponds to 0.5 to 1 nmol/L concentration; it
should be emphasized that a reduction of the S-phase from 35% to 20%
corresponds to more than 40% reduction of the number of progenitor
cells in DNA synthesis, which means that even a seemingly modest
reduction of the percentage of cells in S-phase extended over time will
result in profound inhibition of cell production.
Why then is PR3 purified from mature neutrophil granules much less
active, only marginally affecting the S-phase fraction of CFU-GM at 10 times the concentration of secreted PR3 in CM? This discrepancy is
probably best explained by the structural differences between PR3
stored in granules and the secreted form of PR3. Amino acid sequencing
of PR3 extracted and purified from polymorphonuclear neutrophil
granules has shown that the overwhelming majority of PR3 stored in
primary granules is of the mature form without an amino terminal
propeptide.13 However, during synthesis, the serine
proteases retain amino terminal propeptides, which keeps the proform
catalytically inactive,7,34 presumably to protect the cell
interior from proteolysis.35 The protease does not become
catalytically active until the amino terminal propeptide is removed by
dipeptidyl peptidase, a process that takes place after targeting for
storage in the primary granules.20,22,33 There is evidence
that removal of the propeptide results in a conformational change where
the amino terminal of the mature protein becomes hidden.36
As shown in this report, it is mainly proforms of PR3 retaining an
amino terminal dipeptide, and to a lesser extent, a septapeptide that
is secrected during synthesis. This would suggest that the
S-phase-reducing activity of PR3 is dependent on the amino terminal
propeptide or preservation of the tertiary structure of the proform
rather than preservation of the propeptide itself. At present, this is
an unsolved problem currently under study. Nevertheless, the activity
towards CFU-GM is independent of proteolytic activity, as demonstrated
by a catalytically silent mutant of PR3 transfected to RBL cells. In
addition, previous studies showed that inhibition of protein synthesis
by cycloheximide abrogated the secretion of the S-phase-reducing
factor, demonstrating that it is synthesized immediately before
secretion and not released from a preformed intracellular storage
compartment, and no S-phase-reducing activity was found in the granule
fraction of leukemia cells, but rather in the microsomal
fraction.2,3
The secretion of PR3 during synthesis is not a phenomenon restricted to
myeloid leukemia cells, HL-60 cells, or the transfected cell lines
described in this report, but also do occur with normal marrow cells,
as shown here and previously demonstrated for LAI.4 There
is reason to believe that the secretion of PR3 is localized to the bone
marrow compartment because synthesis of PR3 is restricted to the
promyelocytes7,8 normally not present in peripheral blood.
Normal plasma contains low levels of PR3 all complexed with
1-antitrypsin,30 and although it is not known in detail, it is generally believed that it derives from mature neutrophils and
therefore the majority of it, if not all, is in the mature form. In any
case, normal plasma does not reduce the fraction of CFU-GM in S-phase.
The hematopoietic system has the capacity to rapidly respond with
accelerated cell production when needed during infection or after
bleeding, but it is also strictly regulated to maintain the numbers of
peripheral blood cells within narrow ranges during steady state. A
number of hematopoietic growth factors necessary for survival and
proliferation of hematopoietic stem cells have been identified, among
which G-CSF is the most important for production of
neutrophils37 and has the capacity to rapidly increase the production of neutrophils after administration in vivo.38
However, the mechanisms for maintenance of steady- state granulopoiesis are not well understood and although G-CSF probably is involved in a
continuous stimulation of neutrophil production, little is known
whether a negative regulator is involved in steady-state granulopoiesis. The secreted proform of PR3 could possibly fulfill such
a role, and a finding of special interest is that G-CSF, and GM-CSF,
both are able to abrogate the effect of PR3 on DNA synthesis in
granulopoietic progenitors. The observation that PR3 probably acts
directly on CD34+ progenitor cells shows that PR3 and G-CSF
may have the same target cells. The lack of effect of PR3 on erythroid
progenitors BFU-E is compatible with the assumption that the
downmodulation of DNA synthesis by PR3 is restricted to granulopoiesis.
These observations suggest that PR3 and G/GM-CSF could function as
counteracting regulators of proliferation within the granulopoietic compartment.
We hypothesize that the secretion of a proform of PR3 reflects the
number of promyelocytes and serves as a normal feedback regulator of
the proliferation of granulopoietic progenitor cells within the
CD34+ population. Because the promyelocyte is at an
intermediate stage of development from progenitor cell to mature
neutrophil, this mechanism would provide a sensitive instrument for
fine tuning of steady-state granulopoiesis. With regard to myeloid
leukemia and the initial observations of PR3 as a leukemia-associated
inhibitor, the disturbances in maturation and granule formation that
characterize myeloid leukemia could lead to increased secretion of PR3
and thereby contribute to the suppression of normal granulopoiesis. The
relevance of this model is now further investigated.
| |
ACKNOWLEDGMENT |
We thank Ann-Maj Persson and Eva Nilsson for expert technical assistance.
| |
FOOTNOTES |
Submitted June 9, 1998; accepted October 2, 1998.
Supported by grants from the Swedish Cancer Foundation, the Swedish
Medical Research Council (Project No. 11546), the Swedish Pediatric
Cancer Foundation, Alfred Österlund Foundation, Greta and
Johan Kock Foundation, the Crafoord Foundation, and the Medical Faculty
of Lund.
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 Tor Olofsson, PhD, Department of
Hematology, Research Department 2, E-blocket, University Hospital,
S-221 85 Lund, Sweden.
| |
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Top
Abstract
Introduction