From www.bloodjournal.org by guest on October 1, 2016. For personal use only.
A Secreted Proform of Neutrophil Proteinase 3 Regulates the Proliferation
of Granulopoietic Progenitor Cells
By Stefan Sköld, Bodil Rosberg, Urban Gullberg, and Tor Olofsson
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 a1-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 granulocytemacrophage colony-stimulating factor (GM-CSF). Experiments with highly purified CD341 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.
r 1999 by The American Society of Hematology.
L
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.
EUKEMIA-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 granulocytemacrophage progenitor cells (colony-forming unit granulocytemacrophage [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 a1-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
Blood, Vol 93, No 3 (February 1), 1999: pp 849-856
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 103 using a Pellicon Cassette System
From the Department of Hematology, Research Department 2,
University Hospital, Lund, Sweden.
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.
Address reprint requests to Tor Olofsson, PhD, Department of
Hematology, Research Department 2, E-blocket, University Hospital,
S-221 85 Lund, Sweden.
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.
r 1999 by The American Society of Hematology.
0006-4971/99/9303-0011$3.00/0
849
From www.bloodjournal.org by guest on October 1, 2016. For personal use only.
850
(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 3 106 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 3 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 3 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 (SDSPAGE). 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
SKÖLD ET AL
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 58-TTC GGA
AAG CTT GCC ACC ATG GCT CAC CGG CCC CCC AGC-38 (no.
1), plus downstream 58-GGG GCC ACC GCC GTC TCC GAA-38 (no.
2), and upstream 58-TTC GGA GAC GGC GGT GGC CCC-38 (no. 3),
plus downstream 58-T TCA GAA TTC CGC TGT GGG AGG GGC
GGT TCA-38 (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
From www.bloodjournal.org by guest on October 1, 2016. For personal use only.
PROTEINASE 3 AND REGULATION OF GRANULOPOIESIS
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 3 106 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 3 106 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% 6 2.4% (mean 6 standard deviation [SD];
range, 30.8 to 41.5; n 5 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.
CD341 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 CD341 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 CD341/
CD381 and CD341/CD382 cells, respectively. The CD341/CD381
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 3 106 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.
RESULTS
Purification of LAI. Figure 1A shows chromatography on
Phenyl-Sepharose, demonstrating the hydrophobic properties of
851
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).
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 5 3, P , .01).
The peptide fragments produced by CNBr cleavage were not
sufficiently separated by HPLC and instead we chose SDSPAGE 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
a1-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 a1-antitrypsin. CNBr fragments
From www.bloodjournal.org by guest on October 1, 2016. For personal use only.
852
SKÖLD ET AL
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).
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.
III-V were also isolated by electroelution and tested for LAI
activity; the a1-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 a1-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).
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).
Secreted PR3 in normal marrow CM. Nonphagocytic low
density marrow cells also produce LAI4 and CM collected after
Table 1. Immunoprecipitation of LAI From HL-60 CM
CFU-GM in
S-Phase (%)
Medium control
HL-60 CM untreated
HL-60 CM 1 anti-MPO
HL-60 CM 1 antielastase
HL-60 CM 1 anticathepsin G
HL-60 CM 1 anti-PR3 (MoAb)
HL-60 CM 1 anti-PR3
34.1
24.7*
21.7*
23.0*
22.1*
33.6
41.3
The number of CFU-GM in the medium control was 225 6 7/dish and
149 6 6/dish in the cytosine arabinoside-treated control. Anti-MPO
was used as a negative control antibody. All antibodies were rabbit
polyclonal except one, ie, monoclonal anti-PR3.
Abbreviations: MPO, myeloperoxidase; MoAb, monoclonal antibody. *Denotes significant reduction of CFU-GM in S-phase (P , .05).
Fig 4. Effect of CM from transfected cells on the S-phase of normal
CFU-GM. Control shows CFU-GM in S-phase (mean 6 SD) with
medium alone (n 5 4); HL-60 CM (n 5 4); RBL/Wild CM from
untransfected cells (n 5 2); RBL/PR3 proteinase 3 transfectant (n 5 4);
RBL/Cath G cathepsin G transfectant (n 5 2); RBL/Elastase transfectant (n 5 2); RBL/PR3/cat.del catalytically inactive PR3 transfectant
(n 5 3); 32D/PR3 (n 5 3); 32D/Cath G (n 5 2); 32D/Elastase (n 5 2). The
asterisk (*) denotes significant reduction of the number of CFU-GM in
S-phase (P F .01).
From www.bloodjournal.org by guest on October 1, 2016. For personal use only.
PROTEINASE 3 AND REGULATION OF GRANULOPOIESIS
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 6 32 (mean 6 SD) without and 175 6 21 with cytosine
arabinoside, respectively.
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% 6 4.3% (control,
n 5 4) to 20.3% 6 5.1% (n 5 6, P , .001), whereas the same
CM after immunoprecipitation (no measurable PR3 by ELISA)
was without effect; S-phase fraction was 37.8% 6 4.8% (n 5 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
853
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.
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 5 3, P 5 .43).
Table 2. Effect of Mature Neutrophil PR3 on CFU-GM
in S-Phase in Two Experiments
S-phase (%)
PR3 (ng/mL)
Exp
No. 1
Exp
No. 2
Medium control
15
30
60
125
250
RBL/PR3 20 ng/mL
34.8
29.8
28.9
41.6
26.5*
27.5*
18.6*
34.7
ND
36.0
36.3
41.5
31.5
19.9*
There were 204 6 5 and 268 6 32 colonies per dish in the controls of
exp no. 1 and exp no. 2, respectively. CM of PR3 transfectant RBL cells
(RBL/PR3) containing 20 ng/mL PR3 was included as positive control.
Abbreviation: ND, not done.
*Denotes significant reduction of CFU-GM in S-phase (P , .05).
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.
From www.bloodjournal.org by guest on October 1, 2016. For personal use only.
854
SKÖLD ET AL
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.
DISCUSSION
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 6 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 6 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 6 SD of five (G-CSF) and three (GM-CSF) experiments. The asterisk denotes statistically significant reduction of
S-phase in the positive controls (P F .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).
PR3 acts directly on the progenitor cells. To elucidate the
question whether PR3 interacts directly with CFU-GM progenitor cells, highly purified CD341 progenitor cells (.97% pure)
isolated by fluorescence-activated cell sorting were incubated
Fig 8. G-CSF abrogates the effect of PR3. (s) Shows the dosedependent 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 (d) 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 F .01). One representative experiment is shown. The number of
colonies per dish in the control without cytosine arabinoside was
244 6 26 (SD) and 159 6 12 with cytosine arabinoside, respectively.
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 a1-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 a1-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 a1-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 a1-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
Fig 9. Effect of RBL/PR3 CM on the S-phase fraction of CFU-GM
within a CD341 cell population isolated by cell sorting (mean values 6
SD, n 5 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 F .01).
From www.bloodjournal.org by guest on October 1, 2016. For personal use only.
PROTEINASE 3 AND REGULATION OF GRANULOPOIESIS
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 a1-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 steadystate granulopoiesis are not well understood and although
855
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 CD341 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 CD341 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.
REFERENCES
1. Olofsson T, Olsson I: Suppression of normal granulopoiesis in
vitro by a leukemia associated inhibitor (LAI) of acute and chronic
leukemia. Blood 55:975, 1980
2. Olofsson T, Olsson I: Biochemical characterization of a leukemia
associated inhibitor (LAI) suppressing normal granulopoiesis in vitro.
Blood 55:983, 1980
3. Olofsson T, Olsson I: Suppression of normal granulopoiesis in
vitro by a leukemia associated inhibitor (LAI) derived from a human
promyelocytic cell line (HL-60). Leuk Res 4:437, 1980
4. Olofsson T, Nilsson E, Olsson I: Characterization of the cells in
myeloid leukemia that produce leukemia associated inhibitor (LAI) and
demonstration of LAI-producing cells in normal bone marrow. Leuk
Res 8:387, 1984
5. Olofsson T: Leukemia associated inhibitor (LAI): Biological
characterization and purification of the active subunit, in Najman A,
Guigon M, Gorin NC, Mary JY (eds): The Inhibitors of Hematopoiesis.
Paris, France/London, UK, Colloque INSERM/John Libbey Eurotext,
1987, p 177
6. Gabay JE: Antimicrobial proteins with homology to serine
proteases, in Boman HG, Marsh J, Goode JA (eds): Antimicrobial
Peptides, vol 186. Ciba Foundation Symposium, Chichester, UK, Wiley,
1994, p 237
7. Gullberg U, Andersson E, Garwicz D, Lindmark A, Olsson I:
Biosynthesis, processing, and sorting of neutrophil proteins — Insight
into neutrophil granule development. Eur J Haematol 58:137, 1997
8. Borregaard N, Cowland JB: Granules of the human neutrophilic
polymorphonuclear leukocyte. Blood 89:3503, 1997
9. Weiss J: Leukocyte-derived antimicrobial proteins. Curr Opin
Hematol 1:78, 1994
From www.bloodjournal.org by guest on October 1, 2016. For personal use only.
856
10. Janoff A: Elastase and emphysema. Current assessment of the
protease-antiprotease hypothesis. Am Rev Respir Dis 132:417, 1985
11. Campanelli D, Melchior M, Fu Y, Nakata M, Shuman H, Nathan
C, Gabay JE: Cloning of cDNA for proteinase 3: A serine protease,
antibiotic, and autoantigen from human neutrophils. J Exp Med
172:1709, 1990
12. Labbaye C, Musette P, Cayre YE: Wegener autoantigen and
myeloblastin are encoded by a single mRNA. Proc Natl Acad Sci USA
88:9253, 1991
13. Rao NV, Wehner NG, Marshall BC, Gray WR, Gray BH, Hoidal
JR: Characterization of proteinase-3 (PR-3), a neutrophil serine proteinase—structural and functional properties. J Biol Chem 266:9540, 1991
14. Sturrock AB, Franklin KF, Rao GV, Marshall BC, Rebentisch
MB, Lemons RS, Hoidal JR: Structure, chromosomal assignment, and
expression of the gene for proteinase-3 — The Wegener’s granulomatosis autoantigen. J Biol Chem 267:21193, 1992
15. Bainton DF: Developmental biology of neutrophils and eosinophils, in Gallin JI, Goldstein IM, Snyderman R (eds): Inflammation:
Basic Principles and Clinical Correlates. New York, NY, Raven, 1992, p
303
16. Owen CA, Campbell MA, Sannes PL, Boukedes SS, Campbell
EJ: Cell surface-bound elastase and cathepsin G on human neutrophils:
A novel, non-oxidative mechanism by which neutrophils focus and
preserve catalytic activity of serine proteinases. J Cell Biol 131:775,
1995
17. Csernok E, Ernst M, Schmitt W, Bainton DF, Gross WL:
Activated neutrophils express proteinase 3 on their plasma membrane in
vitro and in vivo. Clin Exp Immunol 95:244, 1994
18. Halbwachs-Mecarelli L, Bessou G, Lesarve P, Lopez S, WitkoSarsat V: Bimodal distribution of proteinase 3 (PR3) surface expression
reflects a constitutive heterogeneity in the polymorphonuclear neutrophil pool. FEBS Lett 374:29, 1995
19. Cai TQ, Wright SD: Human leukocyte elastase is an endogenous
ligand for the integrin CD3 (CD11b/CD18, Mac-1, aMb2) and modulates polymorphonuclear leukocyte adhesion. J Exp Med 184:1213,
1996
20. Hasilik A: The early and late processing of lysosomal enzymes:
Proteolysis and compartmentation. Experientia 48:130, 1992
21. Lindmark A, Persson A-M, Olsson I: Biosynthesis and processing of cathepsin G and neutrophil elastase in the leukemic myeloid cell
line U-937. Blood 76:2374, 1990
22. McGuire MJ, Lipsky PE, Thiele DL: Generation of active
myeloid and lymphoid granule serine proteases requires processing by
the granule thiol protease dipeptidyl peptidase I. J Biol Chem 268:2458,
1993
23. Gullberg U, Lindmark A, Nilsson E, Persson A-M, Olsson I:
Processing of human cathepsin G after transfection to the rat basophil/
mast cell tumor line RBL. J Biol Chem 269:25219, 1994
24. Rao NV, Rao GV, Marshall BC, Hoidal JR: Biosynthesis and
SKÖLD ET AL
processing of proteinase 3 in U937 cells. Processing pathways are
distinct from those of cathepsin G. J Biol Chem 271:2972, 1996
25. Garwicz D, Lindmark A, Hellmark T, Gladh M, Jögi J, Gullberg
U: Characterization of the processing and granular targeting of human
proteinase 3 after transfection to the rat RBL and the murine 32D
leukemic cell lines. J Leukoc Biol 61:113, 1997
26. Tarr GE: Manual Edman sequencing system, in Shively JE (ed):
Methods of Protein Microcharacterization. Clifton, NJ, Humana, 1986,
p 155
27. Garwicz D, Lindmark A, Gullberg U: Human cathepsin G
lacking functional glycosylation site is proteolytically processed and
targeted for storage in granules after transfection to the rat basophilic/
mast cell line RBL or the murine myeloid cell line 32D. J Biol Chem
270:28413, 1995
28. Gullberg U, Lindmark A, Lindgren G, Persson A-M, Nilsson E,
Olsson I: Carboxyl terminal prodomain-deleted human leukocyte
elastase and cathepsin G are effectively targeted to granules and
enzymatically activated in the rat basophilic/mast cell line RBL. J Biol
Chem 270:12912, 1995
29. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR: Sitedirected mutagenesis by overlap extension using the polymerase chain
reaction. Gene 77:51, 1989
30. Baslund B, Petersen J, Permin H, Wiik A, Wieslander J:
Measurements of proteinase 3 and its complexes with a1-antiproteinase
inhibitor and anti-neutrophil cytoplasm antibodies (ANCA) in plasma. J
Immunol Methods 175:215, 1994
31. Olofsson T, Sallerfors B: Modulation of the production of
leukemia associated inhibitor (LAI) and its interaction with granulocytemacrophage colony-forming cells. Exp Hematol 15:1163, 1987
32. Sinha D, Bakhshi MR, Kirby EP: Complete cDNA sequence of
bovine a1-antitrypsin. Biochim Biophys Acta 1130:209, 1992
33. Sun J, Bird CH, Sutton V, McDonald L, Coughlin PB, De Jong
TA, Trapani JA, Bird PI: A cytosolic granzyme B inhibitor related to the
viral apoptotic regulator cytokine response modifier A is present in
cytotoxic lymphocytes. J Biol Chem 271:27802, 1996
34. Salvesen G, Enghild JJ: An unusual specificity in the activation
of neutrophil serine proteinase zymogens. Biochemistry 29:5304, 1990
35. Garwicz D, Lindmark A, Persson A-M, Gullberg U: On the role
of the proform-conformation for processing and intracellular sorting of
human cathepsin G. Blood 92:1425, 1998
36. Fujinaga M, Chernaia MM, Halenbeck R, Koths K, James MNG:
The crystal structure of PR3, a neutrophil serine proteinase antigen of
Wegener’s granulomatosis antibodies. J Mol Biol 261:267, 1996
37. Metcalf D: Hematopoietic regulators: Redundancy or subtlety?
Blood 82:3515, 1993
38. Bensinger WI, Price TH, Dale DC, Appelbaum FR, Clift R,
Lilleby K, Williams B, Storb R, Thomas ED, Buckner CD: The effects
of daily recombinant human granulocyte colony-stimulating factor
administration on normal granulocyte donors undergoing leukapheresis.
Blood 81:1883, 1993
From www.bloodjournal.org by guest on October 1, 2016. For personal use only.
1999 93: 849-856
A Secreted Proform of Neutrophil Proteinase 3 Regulates the Proliferation
of Granulopoietic Progenitor Cells
Stefan Sköld, Bodil Rosberg, Urban Gullberg and Tor Olofsson
Updated information and services can be found at:
http://www.bloodjournal.org/content/93/3/849.full.html
Articles on similar topics can be found in the following Blood collections
Hematopoiesis and Stem Cells (3364 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.