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Azurophilic Granules of Human Neutrophilic Leukocytes Are Deficient
in Lysosome-Associated Membrane Proteins but Retain the Mannose
6-Phosphate Recognition Marker
By A.-M. Cieutat, P. Lobel, J.T. August, L. Kjeldsen, H. Sengeløv, N. Borregaard, and D.F. Bainton
During granulocyte differentiation in the bone marrow (BM),
neutrophilic leukocyte precursors synthesize large amounts
of lysosomal enzymes. These enzymes are sequestered into
azurophilic storage granules until used days later for digestion of phagocytized microorganisms after leukocyte emigration to inflamed tissues. This azurophil granule population
has previously been defined as a primary lysosome, ie, a
membrane-bound organelle containing acid hydrolases that
have not entered into a digestive event. In this study,
azurophil granules were purified and shown to contain large
amounts of mannose 6-phosphate-containing glycoproteins
(Man 6-P GP) but little lysosome-associated membrane
proteins (LAMP). In addition, the fine structural localization
of Man 6-P GP and LAMP was investigated at various stages
of maturation in human BM and blood. Man 6-P GP were
present within the azurophilic granules at all stages of
maturation and in typical multivesicular bodies (MVB) as
well as in multilaminar compartments (MLC), identified by
their content of concentric arrays of internal membranes.
LAMP was absent in all identified granule populations, but
was consistently found in the membranes of vesicles, MVB,
and MLC. The latter compartment has not been previously
described in this cell type. In conclusion, the azurophilic
granules, which contain an abundance of lysosomal enzymes and Man 6-P GP, lack the LAMP glycoproteins. By
current criteria, they therefore cannot be classified as lysosomes, but rather may have the functional characteristics of
a regulated secretory granule. Rather, the true lysosomes of
the resting neutrophil are probably the MVB and MLC.
Finally, the typical ‘‘dense bodies’’ or mature lysosomes
described in other cells are not present in resting neutrophils.
r 1998 by The American Society of Hematology.
E
stances, and the gelatinase or tertiary granules, which form
mainly at the band cell stage.14 Also, highly mobilizable
secretory vesicles that contain endocytosed plasma proteins
such as albumin have recently been characterized.15,16
Since de Duve’s original discovery and description of
lysosomes, considerable progress has been made in defining the
components of lysosomes in many cell types.17 In most other
previously investigated cell types, the synthesis and posttranslational processing of the lysosomal enzymes occur over
the relatively short time of a few hours.18 During their transport
from the endoplasmic reticulum to the Golgi complex, the
newly synthesized lysosomal hydrolases are modified to contain
mannose 6-phosphate (Man 6-P), which binds to Man 6-P
receptors in the Golgi complex effecting the targeting of the
hydrolases to an acidic endosome where the complexes dissociate.17,19 The receptors recycle back to the Golgi complex or to
the plasma membrane, while the lysosomal enzymes reach the
mature lysosome where they are rapidly dephosphorylated.
Thus, at steady state, the lysosome contains the bulk of the
dephosphorylated hydrolases while the phosphorylated forms
are in endosomes.20 Another defining feature of lysosomes is
the presence of unique transmembrane glycoproteins, identified
as closely related lysosome-associated membrane proteins
(LAMP) LAMP-1 and LAMP-221,22 reviewed by Kornfeld and
Mellman.17 LAMPs are among the most densely glycosylated
glycoproteins known, with carbohydrate comprising 55% to
65% of the total mass.23,24 While the function of these proteins
is unknown, it has been speculated that one of their roles is to
protect the lysosomal membrane from degradation by lysosomal acid hydrolyses, since their luminal domains are very
resistant to proteolysis.25 However, it is unlikely that this simple
explanation defines the complete role of the glycoproteins,
because, for one thing, it does not explain the different levels of
tissue expression of LAMP 1 and 2. Moreover, Cuervo and
Dice26 have recently reported that LAMP-2 is a receptor for the
selective uptake of proteins into lysosomes for subsequent
degradation.
NZYME CYTOCHEMISTRY and subcellular fractionation have shown that lysosomal enzymes are synthesized
early in the maturation of neutrophilic leukocytes in bone
marrow (BM).1-5 These enzymes are stored within azurophilic
(or primary) granules for 10 to 14 days before being used during
phagocytosis. The azurophilic granules were considered by de
Duve6 to be classic examples of primary lysosomes since they
are membrane-bound organelles that contain acid hydrolases
that have not yet entered into a digestive event. These granules,
which are formed at the promyelocytic stage (see Table 1),7-9 are
the major source of acid hydrolases and many other proteins
such as myeloperoxidase (MPO), granulocyte elastases, and
nonglycosylated defensins. However, little is known of their
membranes. Only CD6310,11 and CD6812 have been demonstrated in the membrane of azurophilic granules. Two other
granule populations, which are nonlysosomal and peroxidasenegative, form later in maturation: specific or secondary
granules,13 which contain lactoferrin and many other sub-
From the Department of Pathology, University of California San
Francisco School of Medicine, CA; Center for Advanced Biotechnology
and Medicine and Department of Pharmacology, UMDNJ-Robert Wood
Johnson Medical School, Piscataway, NJ; Granulocyte Research Laboratory, University Hospital, Copenhagen, Denmark; and the Department of Pharmacology and Molecular Sciences, The Johns Hopkins
University School of Medicine, Baltimore, MD.
Submitted May 12, 1997; accepted September 23, 1997.
Supported by Grants No. HLB-31610 and DK-10486 from the
National Institutes of Health (Bethesda, MD); The Danish Medical
Research Council; and The Danish Cancer Society, Copenhagen,
Denmark.
Address reprint requests to D.F. Bainton, MD, University of California, San Francisco School of Medicine, San Francisco, CA 94143-0400.
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 1998 by The American Society of Hematology.
0006-4971/98/9103-0019$3.00/0
1044
Blood, Vol 91, No 3 (February 1), 1998: pp 1044-1058
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LAMPs ARE ABSENT FROM NEUTROPHIL GRANULES
Table 1. Granules of the Human Neutrophil
Characteristic Protein(s)
Azurophilic/primary
granules
Specific/secondary
granules
Gelatinase/tertiary
granule
Myeloperoxidase
elastase, defensins
Lactoferrin, NGAL,
B12-binding protein
Gelatinase
Time of Formation
Promyelocyte
Myelocyte, metamyelocyte
Band cell
Historically, two major types of storage granules were definitively
identified by the presence or absence of MPO. The MPO-positive
azurophil (primary) granules are formed only during the promyelocyte stage and are reduced in number by mitosis. It has recently been
shown that the peroxidase negative granules constitute a continuum
from early appearing granules (myelocyte stage) that contain lactoferrin but no gelatinase to granules that contain both lactoferrin and
gelatinase (metamyelocyte stage) to granules that contain gelatinase
but no lactoferrin (band cell stage). The aforementioned granules that
contain lactoferrin are referred to as specific (secondary) granules
while the granules that contain gelatinase but not lactoferrin are
referred to as gelatinase (tertiary) granules. Thus, the mature circulating neutrophil contains the three major granule types. When appropriately stimulated, these cells move from blood to tissues and, within
seconds, the granules may release their contents in endocytic vacuoles
or, by fusion with the plasma membrane, to the exterior of the cell.
As a result of these advances, lysosomes are now defined as
vesicular compartments with (1) a high concentration of
LAMPs, (2) a full complement of mature dephosphorylated
lysosomal enzymes, (3) the absence of cation-independent Man
6-P receptor, and (4) an acid pH.17 This definition has largely
been observed from studies of rapidly dividing tissue culture
cell lines and leaves open a number of questions concerning
lysosomal biosynthesis, including the pathway of lysosomal
synthesis in neutrophilic leukocytes, an important professional
phagocyte. We decided to reexamine the concept in this
important cell type by asking two questions: (1) Where are the
LAMPs and Man 6-P GP located in developing and mature
neutrophils? (2) Does the azurophilic granule fit the more
modern definition of a lysosome?
We have addressed these questions by application of fractionation methods and immunolabeling using thawed cryosections
to study the distribution of the Man 6-P marker and distribution
of LAMP-1 and LAMP-2 in mature human blood neutrophils.
In addition, human BM neutrophils at different stages of
maturation were examined by light and electron microscopy.
These studies show the absence of LAMP-1 and LAMP-2 in
membranes of azurophil granules. The findings suggest that,
despite the high content of lysosomal acid hydrolases, the
azurophil granule has the characteristics of a regulated secretory
granule rather than being a true lysosome. LAMP proteins were
identified, for the first time in neutrophils, in a multilaminar
compartment (MLC) identified by its content of concentric
arrays of internal membranes, and in typical multivesicular
bodies (MVB). These multilaminar and multivesicular bodies
are possibly the true precursors to the ‘‘housekeeping’’ lysosomes of this cell type, during its relatively short life span of 2
to 3 weeks.
1045
MATERIALS AND METHODS
Antibodies and Marker Proteins
Monoclonal antibodies (MoAbs) H5G11 and H4A3 (antihuman
LAMP-1), H4B4 (anti-LAMP-2) (all IgG1,K) were derived from mice
immunized with human adherent peripheral blood (PB) cells.21,22
Polyclonal rabbit antibodies against human gelatinase and against
human albumin were raised as described by Kjeldsen et al27 and
Borregaard et al,16 respectively. Lactoferrin antibodies were obtained
from Accurate Chemical Co (Westbury, NY).
Azurophil granules present in subcellular fractions were identified by
myeloperoxidase and their contents were measured by enzyme linked
immunosorbent assay (ELISA).28 Specific and gelatinase granules were
identified by lactoferrin and gelatinase, respectively, and measured by
ELISA.27,29 Secretory vesicles were identified by albumin and measured
by ELISA.16 Plasma membranes were identified by HLA class I and
measured by ELISA.30
Subcellular Fractionation
Subcellular fractionation was performed as previously described.16,31
Briefly, isolated neutrophils were disrupted by nitrogen cavitation in 13
mL relaxation buffer (100 mmol/L KCl, 3 mmol/L NaCl, 1 mmol/L
ATPNa2, 3.5 mmol/L MgCl2, 10 mmol/L Piperazine N,N8-bis2 [ethanesulfonic acid] [Pipes] pH 7.2 containing 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF). Nuclei and intact cells were sedimented by
centrifugation at 400g for 15 minutes (P1) and 10 mL of the postnuclear
supernatant (S1) was applied on top of a 28 mL two-layer Percoll
density gradient (1.05/1.12 g/mL) containing 0.5 mmol/L PMSF and
centrifuged at 35,000g for 30 minutes. This resulted in the generation of
separate fractions that could be visually identified in the gradient: the
bottom band (a-band), containing azurophilic granules; the intermediate band (b-band), containing specific and gelatinase granules; the top
band (g-band), containing plasma membranes and secretory vesicles.
The gradient was aspirated from the bottom and dispersed into fractions
of 1.4 mL each through a fraction collector.
Detection of Man 6-P Containing Glycoproteins
(Man 6-P GP) in Subcellular Fractions
Samples of 100 µL from each fraction were diluted with 150 µL
saline. This mixture was added to 250 µL sodium dodecyl sulfate
(SDS)-reducing sample buffer. After boiling for 5 minutes, the proteins
of 125-µL samples were resolved on a 5% to 20% acrylamide SDS
gradient gel and transferred to 0.2-µm nitrocellulose filters.32 The filters
were probed with radioiodinated sCI-MPR as described by Valenzano et
al.33 Briefly, membranes were treated at 4°C with blocking buffer
(phosphate-buffered saline [PBS] containing 1 mg/mL bovine serum
albumin [BSA] and 0.2% Tween-20) for two hours to quench nonspecific binding sites, incubated with 3 nmol/L 125I-labeled sCI-MPR (,1
Ci/µmol) in blocking buffer for 16 hours, and then rinsed 10 times for 30
seconds in PBS containing 0.2% Tween-20. The binding of sCI-MPR
was detected and quantified with a phosphorimager (Molecular Dynamics, Sunnyvale, CA). The probe detects the phosphorylated glycoproteins which retain the Man 6-P recognition marker and are subsequently
called Man 6-P GP.
Detection of LAMP in Subcellular Fractions
LAMP were detected as previously described by Mane et al.22
Briefly, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed as described by Laemmli and proteins were transferred to
0.2-µm nitrocellulose filters (Bio-Rad Laboratories, Richmond, CA)
essentially as described by Towbin et al.34 Mouse-MoAbs H4A3 and
H4B4 directed against LAMP-1 and LAMP-2, respectively, were then
applied at a dilution of 1:3,000 (from stock of 1 mg/mL) in PBS
containing 0.1% Tween-20 and incubated overnight. The nitrocellulose
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1046
filters were then washed in PBS, 0.1%Tween-20, and developed by the
Amersham Chemiluminescence Method as described by the manufacturer. The films were scanned using the CREAM system, version 4.0
(Kem-En-Tec, Copenhagen, Denmark).
Immunoprecipitation
Immunoprecipitation was performed after lysis of 3 3 107 cells/mL
in lysis buffer (10 mmol/L HEPES, 100 mmol/L KCl, 25 mmol/L NOG
[N-octylglucoside]; 0.2% cetyl-trimethyl ammonium bromide (CTAB),
1 mmol/L PMSF, 200 KIU Aprotinin, 100 µg/mL leupeptin, 1 mmol/L
EGTA). Samples of 200 µL were incubated with anti-MPO coupled to
Sepharose particles. The precipitates were washed five times and resuspended to 200 µL of SDS sample buffer. Each sample was divided in two,
each of which was further run on SDS-PAGE, one of which was further
processed for transfer to nitrocellulose and quantitation of Man 6-P content.
Preparation of Human Leukocytes
for Morphologic Examination
Normal human leukocytes from PB, anticoagulated in heparin and
freed of erythrocytes by sedimentation in Dextran, were washed in
Hanks’ balanced salt solution. The cells were fixed in 4% paraformaldehyde-lysine, using the buffers of McLean and Nakane35 or in 2%
paraformaldehyde, 0.05% glutaraldehyde, in 0.1 mol/L phosphate
buffer (pH 7.4), for 30 minutes, at 4°C. The cells were then washed
thoroughly in the same buffer containing 3% (wt/vol) sucrose.
Immunolabeling Using Thawed Cryosections
The cell pellet was embedded in a 20% Polyvinyl pyrrolidone
(PVP-10; Sigma, St Louis, MO)/2.1 mol/L sucrose solution, frozen and
stored in liquid nitrogen. Sections were cut on a Reichert-Jung Ultracut
FC-4 (Buffalo, NY). The techniques used for preparing ultrathin
cryosection and the immunocytochemistry have been previously described.36 The primary antibody was used at the following dilutions: all
LAMP MoAbs were used as undiluted ascites fluid; antigelatinase,
1/100; antialbumin, 1/50; antilactoferrin, 1/1,000 in .5 mol/L NaCl/
PBS. After several washes, the immunogold probes were used at a
dilution of 1/50 in .1% BSA/PBS, pH 8.2. The gold probes were goat
antimouse IgG and IgM-gold, 5 nm or 10 nm (GAM-5 or GAM-10), and
goat antirabbit IgG-gold, 5 nm or 10 nm (GAR-5 or GAR-10) with
optical density (O.D.) at 520 nm of 2.5 (Amersham, Arlington Heights,
IL). A nonimmune purified rabbit IgG was used as a control for
polyclonal antibodies, and normal mouse serum or the control hybridoma, for MoAbs. Double-labeling experiments to localize H4B4 and
albumin were performed by combining the monoclonal H4B4 and
polyclonal albumin in the primary antibody incubation, washing, and
subsequently combining GAM and GAR for the immunogold incubation. After several washes, the sections were then stained with a neutral
pH uranyl acetate oxalate and embedded in methyl cellulose/uranyl
acetate as described previously.37,38
Detection of Man 6-P GP on Ultrathin Cryosections of
Human BM and Blood by Electron Microscopy
Leukocytes from five normal donors were separated from blood by
Dextran sedimentation, or from BM from two normal donors and
enriched for different maturation stages, as described previously.14 The
ultrathin cryosections were incubated for 2 hours at 4°C, in 1% BSA,
0.2% Tween-20 and PBS: and then incubated overnight with the
biotinylated sC1-MRP (1 mg/100 mL) in PBS plus 1% BSA and 5
mmol/L b-glycerolphosphate. The phosphorylated enzymes were detected the next day by streptavidin-gold, 5 or 10 with O.D. at 520 nm of
approximately 5.0 (Sigma) diluted to 1:20. The sections were then
further processed as described above. For double-label, the antibody
was applied first, after washing; the biotinylated sC1-MPR was then
applied and allowed to incubate overnight. For controls, the sections
were preincubated with 10 mmol/L of Man 6-P for 1 hour before
CIEUTAT ET AL
applying the biotinylated sC1-MPR.The grids were then further processed as described above.
Detection of Man 6-P GP on Blood or BM Smears by
Light Microscopy
Samples of human blood or BM were spread on a cover slip, fixed in
acetone: methanol: 37% formaldehyde (19:19:2) at 4°C for 90 seconds,
then washed in PBS and further processed as previously described by
Sleat et al.39 Briefly, the slides were treated for 2 hours at 4°C in PBS,
1% BSA, 2% Tween 20, and then incubated overnight with the
biotinylated sCI-MPR (1 mg/100 mL) in PBS, 1% BSA, and 5 mmol/L
b-glycerophosphate. For controls, slides were incubated with 10
mmol/L of Man 6-P 1 hour before the biotinylated sCI-MPR was
applied. The phosphorylated enzymes were detected the next day by
avidin-linked alkaline phosphatase and the substrate Vector Red (Vectastain
ABC kit; Vector, Burlingame, CA). Levamisole was also present to inhibit
neutrophil endogenous alkaline phosphatase (Vectastain ABC kit; Vector).
RESULTS
Subcellular Localization of Man 6-P GP and LAMP
in Mature Neutrophils
Subfractionation Studies
The subcellular localization of Man 6-P GP and LAMP was
determined in the different neutrophilic fractions separated by
density on a Percoll gradient. The fractions were characterized
by use of the following markers (Table 2): myeloperoxidase for
azurophilic granules, lactoferrin for specific/secondary granules, gelatinase for gelatinase/tertiary granules, albumin for the
secretory vesicles, and HLA class I for the plasma membrane.
On blots of subcellular fractions, Man 6-P–containing proteins
were quantitatively analyzed by use of the sCI-MPR probe. This
probe is a biotinylated or radioiodinated derivative of a soluble form
of the cation-independent Man 6-P receptor isolated from fetal
bovine serum.33,39 The probe binds Man 6-P containing proteins with
nanomolar affinity, and is used in much the same manner as an
antibody is to detect a specific substrate. The biotinylated receptor
binds its ligand; subsequently the biotin moiety binds with either
avidin-conjugated alkaline phosphatase for detection at the light
microscopy level or streptavidin-gold for detection at the fine
structural level. This assay of Valenzano et al33 on fractions also
allows for the detection of phosphorylated lysosomal enzymes and
other glycoproteins.
The subcellular distribution of LAMP-1 and 2 (Fig 1A) and
Table 2. Specific Markers for Neutrophil Compartments
Granules
Azurophilic granules
Content Markers
Membrane Markers
Lysosomal enzymes, CD63, CD68
myeloperoxidase,
M6P-glycoproteins
Lactoferrin
CD11b, cytochrome b
Gelatinase
CD11b, cytochrome b
Specific granules
Gelatinase granules
Other Compartments
Secretory vesicles Albumin
Multivesicular
M6P-glycoproteins
bodies & multilaminar compartments
Plasma membrane
Alkaline phosphatase, CD35, CD11b,
cytochrome b
LAMP-2/LAMP-1
HLA-1
References are found in the text, except for CD35.65
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LAMPs ARE ABSENT FROM NEUTROPHIL GRANULES
A
1047
B
C
D
Fig 1. Subcellular distribution of LAMP 1 and 2 and Man 6-P GP in neutrophils. Neutrophils from PB were isolated, disrupted by nitrogen
cavitation, and fractionated on a two-layer Percoll Density gradient. (A) LAMP 1 and 2 in subcellular fractions. Other fractions of 1.4 mL were
collected and assayed for markers of azurophil granules (MPO, M), specific granules (lactoferrin, Q), gelatinase granules (gelatinase, R),
secretory vesicles (albumin, X), and plasma membranes (HLA, class I, *). Samples from each fraction were centrifuged to remove Percoll and
subjected to SDS-PAGE. The separated proteins were blotted to nitrocellulose membranes and probed with antibody against LAMP 1 (W) and
LAMP 2 (U). The immunoblots were developed by chemiluminescence and quantitated by scanning (bold lines). (B) Man 6-P GP in subcellular
fractions. Fractions of 1.4 mL were collected and assayed for markers of azurophil granules (MPO, M), specific granules (lactoferrin, W),
gelatinase granules (gelatinase, U), secretory vesicles (albumin, X ), and plasma membranes (HLA, class I, *). Samples from each fraction were
centrifuged to remove Percoll and subjected to SDS-PAGE. The separated proteins were blotted to nitrocellulose membranes and probed with
the sCI-MPR (,) and quantified by phosphorimager (bold line). (C) Man 6-P GP in relation to myeloperoxidase. Isolated neutrophils were disrupted
by nitrogen cavitation and the postnuclear supernatant was immunoprecipitated with anti-MPO coupled to sepharose particles, divided into
two, and subjected to SDS-PAGE and Coomassie blue staining for protein (left panel) or to SDS-PAGE followed by transfer to nitrocellulose and
probed with sCI-MPR (right lane). From left to right: Lane 1, protein profile in immunoprecipitate; lane 2, protein profile in supernatant after
immunoprecipitation; lane 3, Man 6-P GP in immunoprecipitate; lane 4, Man 6-P GP in supernatant after immunoprecipitation. (D) Profile of Man
6-P GP in fraction #3 and #10.
Man 6-P GP (Fig 1B) in fractions from mature neutrophils are
shown in Fig 1. It is striking that these do not colocalize to the
same fractions. While Man 6-P GP essentially colocalizes with
myeloperoxidase in the most dense fraction (Fig 1B), LAMP-1
and 2 are found in less dense fractions that also contain the
markers lactoferrin and gelatinase (Fig 1A), and in small
amounts in fractions 15 and 16.
To test whether or not myeloperoxidase was the major Man
6-P–containing protein, neutrophils were solubilized and precipitated with polyclonal antibodies against MPO. This precipita-
tion was effective as judged by reduction of myeloperoxidase
from 93 µg/mL in the solubilized cells to 0.31 µg/mL in the
supernatant after immunoprecipitation. The precipitation was
specific for MPO since the MPO bands at 62 kD and 12 kD, and
the IgG at 50 kD are the only major protein bands seen in the
SDS-PAGE profiles (Fig 1C). By comparing the content of Man
6-P in supernatant and pellet as revealed by binding of the
radioiodinated fragment of the CI-mannose 6-P receptor to the
blotted proteins we found that the majority of the receptor
binding is associated with myeloperoxidase. Other, smaller
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1048
CIEUTAT ET AL
Fig 2. Electron micrographs of frozen thin sections from fractions containing azurophil granules, section of fractions # 2 (a and c), and specific
granules # 9 (b and d). (a) Frozen thin section of fraction #2, which is labeled for Man 6-P GP with biotinylated probe, and detected with
streptavidin gold-10. Most of the organelles in fraction #2 appear to be azurophilic granules labeled for Man 6-P GP (Ag). Some of the granules
have extracted contents (Ag), while others retain their content (Ag8). This variation in content preservation has been previously observed in
intact cells. (b) Fraction #10 contained typical specific granules (Sg) negative for Man 6-P GP. Indeed, compared to (a), only a few gold particles
were present, and they were in MLC (arrow) and rare granular structures. Fractions 2 (c) and 10 (d) were labeled for LAMP-2 with 10 nm gold.
Fraction 2 consisted mostly of azurophilic granules (Ag and Ag8); these granules were devoid of immunoreactivity (c). Most of the organelles in
fraction 10 appear to be specific granules (Sg), which are not labeled, whereas the gold labeling is present in MLC (arrows). Tissue preparation for
figures of electron micrographs, except 8: Fractions or cells were fixed in 2% paraformaldehyde, 0.05% glutaraldehyde for 1 hour at 4°C
embedded in sucrose, frozen and processed for ultrathin-cryosectioning. For Fig 8b and c: leukocytes were fixed in 4% paraformaldehyde-lysine,
using the buffers of McLean and Nakane.35
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LAMPs ARE ABSENT FROM NEUTROPHIL GRANULES
1049
Fig 3. Electron micrographs of frozen thin sections from human BM labeled by a biotinylated sCI-MPR probe. The Man 6-P GP are visualized
with streptavidin 10 nm. (a) Early immature granulocytic cell of the BM. (b) The Golgi complex (GC) of an immature cell is labeled for Man 6-P GP.
(c and d) In this immature neutrophil from BM (portion of the promyelocyte or an early myelocyte from [a]), the highest levels of Man 6-P GP are
found within the newly formed azurophilic granules (Ag), some of which have retained their density (Ag8) as in (c), while others appear extracted
(d) but retain immunolabel. At intermediate stages of maturation (myelocytes), an MLC is first observed (Inset, [d]).
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1050
CIEUTAT ET AL
Fig 4. Electron micrograph of frozen thin section of mature blood neutrophils (a), Man 6-P GP is detected with the sCI-MPR–biotinylated
probe and visualized with streptavidin gold (5 nm). (b) High levels of Man 6-P GP are found within the Ag, and in vesicular structures (V) probably
an MVB, but not in Sg.
bands probably represent lysosomal enzymes. The Man 6-P GP
profiles differ in the different fractions of Fig 1B. This can be
observed in Fig 1D, which gives the Man 6-P GP profile of
fraction #3 and #10, the latter showing a much more diverse
profile than #3, which is dominated by MPO.
We next performed immunocytochemistry for Man 6-P GP
and LAMP on frozen thin sections of individual fractions
characterized in Fig 1. The azurophilic granules from fractions
2 and 3 (Fig 2a) were shown to be strongly labeled for Man 6-P
GP. However, the small amount of Man 6-P GP present in
fractions 9 and 10 was not in specific granules but was seen in
vacuoles containing concentric arrays of internal membranes,
called MLC (Fig 2b), in MVB, and in rare small granules (not
shown). Fractions 15 and 16 contained no label for Man 6-P GP
(not illustrated). When the fractions were examined for LAMP,
no label was found in the azurophil granule fraction (Fig 2c).
The most interesting finding was in fraction 10, the one highest
in LAMP; the marker was not found in the numerous specific
granules located there, but was detected in MLC (Fig 2d and
inset) and in rare MVB (not illustrated).
Fractions 15 and 16 contained numerous small vesicles, some
of which were undoubtedly vesiculated plasma membranes as
well as occasional large vacuoles. No Man 6-P GP was found in
these fractions, consistent with the quantitative data presented
in Fig 1B. Occasionally, label for LAMP-2 could be seen as
isolated gold particles on the membranes of small vesicles. On
the other hand, small vesicles heavily labeled for albumin could
be found, as one would have predicted (not illustrated).
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LAMPs ARE ABSENT FROM NEUTROPHIL GRANULES
1051
Fig 5. Electron micrograph of
a frozen thin section of an immature neutrophil labeled for
LAMP-2, using the MoAb H4B4.
At lower magnification the cell
can be identified as a promyelocyte (see b). The Golgi region of
this cell can be seen at higher
magnification in a. Most of the
gold grains are present in vesicular structures (arrows). The
vesicles (v) are concentrated near
the Golgi complex (Gc) and the
centriole (C). Note the absence
of label on the plasma membrane (PM). Nucleus (Nu).
Localization of the Man 6-P GP and LAMP
in Developing BM Neutrophils
Light Microscopy
Man 6-P GP in BM smears was labeled and visualized by
biotinylated sCI-MPR in combination with streptavidinalkaline phosphatase. A bright red reaction product was detected
in cells at all stages of neutrophil maturation (from promyelocytes to mature neutrophils) as well as on eosinophils. This
labeling was especially strong in promyelocytes and myelocytes. Other cells contained lesser amounts of reaction product,
such as in megakaryocytes, or none at all, eg, in nucleated
erythroblastic or mature red blood cells. The specificity of the
probe was shown in a control experiment by a marked decrease
in labeling when the sCI-MPR was preincubated with Man 6-P.
Staining was also absent when the probe was omitted. The
MoAb against LAMP-2 was applied to the same BM smears,
and was detected in the neutrophilic cells at all stages of
maturation.
Electron Microscopy
Man 6-P GP. At the ultrastructural level, in immature
promyelocytes and myelocytes (Fig 3a) Man 6-P GP was
present in the Golgi complex (Fig 3b) and, to a greater extent, in
the newly formed azurophilic granules presumably because
they are concentrated in that organelle. The labeling was
especially strong within the matrix of the azurophilic granules
(Fig 3c and d), as predicted by the data previously presented in
the fractions. In areas near azurophilic granules with high
labeling, we frequently found ‘‘spillage’’ of the antigen in the
nearby cytoplasm (Fig 3c and d). This artifact has been
previously observed with the low molecular weight defensins40
and proteinase 3.41 A possible explanation is that the membranous compartments are cut open by the cryostat method, and
some of the granule contents become soluble and may partially
relocate during the subsequent incubation procedures. Man 6-P
GP was also detected in structures containing concentric arrays
of internal membranes, the so-called MLC, which had not been
described in neutrophils, but was observed in a related cell line,
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1052
CIEUTAT ET AL
Fig 6. Electron micrograph of a frozen thin section of myelocytes immunolabeled for LAMP-2. In (a), large vesicles (V) and the MLC are labeled
with GAM-10 (arrowheads). Note the absence of labeling in large Ag as well as in the Sg and PM. In inset, higher magnification of another
multilaminar compartment positive for LAMP-2. (b) Two organelles contain label (arrowheads). The typical MVB are labeled for LAMP-2 as is the
MLC. The membranes of the small granules (Sg) are not labeled nor is the Gc. Nucleus (Nu).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
LAMPs ARE ABSENT FROM NEUTROPHIL GRANULES
1053
Fig 7. Electron micrograph of frozen thin section of blood neutrophils labeled with LAMP-2. (a) LAMP-2 (arrows) is found in small vesicles
around the Gc. (b) In larger vesicular structures (V). (c) LAMP-2 is present in this MLC, and in (d) the MVB. The Ag are stained for myeloperoxidase
and appear dense. Nucleus (Nu).
HL60.22 Other organelles, with the exception of rare multivesicular bodies, were not labeled. Intense labeling for Man 6-P GP
within the matrix of azurophilic granules of intact mature
neutrophils was also confirmed (Fig 4a and b). No staining was
observed when control preparations were preincubated with 10
mmol/L of Man 6-P, or when the probe or streptavidin-gold
label were omitted (not shown).
LAMP. At the early stages of maturation, in promyelocytes
(Fig 5a and b), LAMP labeling was found in vesicles near the
Golgi complex (Fig 5a). Later (at the myelocyte stage), LAMP
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1054
CIEUTAT ET AL
Fig 8.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
LAMPs ARE ABSENT FROM NEUTROPHIL GRANULES
was found mainly in vesicles of various sizes, and the multilaminar compartments (Fig 6a, inset), as well as in typical multivesicular bodies (Fig 6b). In contrast, as could be predicted from
the fractionation data, LAMP labeling was absent from azurophilic granules (Figs 6 and 7), but was found in vesicles near the
Golgi cisternae and in larger vesicles (Fig 7a and b) as well as in
the multilaminar compartment and multivesicular bodies (Fig
7c and d). LAMP was not seen in the azurophilic granules
stained in Fig 7d for peroxidase. No label was seen in other
organelles, plasma membrane, Golgi cisternae, or mitochondria.
Negative controls were seen when the antibody was omitted or a
normal mouse serum or X63 were used. In general, H4B4, the
antibody for LAMP-2, was more suitable for immunolabeling
than the other LAMP-1 antibodies. In the different maturational
stages of neutrophils, at no time was LAMP seen in azurophilic
granules or in the two other granule populations.
As mentioned above, the LAMP-positive organelles are
vesicular in nature and their numbers vary with the stage of
maturation as follows: (1) in promyelocytes and early myelocytes, small vesicles (50 to 150 nm) 16%; larger vesicles (150 to
500 nm) 83%; and rare MLC or typical MVB. (2) In late
myelocytes and metamyelocytes, small vesicles (17%), larger
vesicles (73%) and the appearance of MLC (9%), and typical
MVB (1%). (3) In mature neutrophils, small vesicles (16%),
larger vesicles (76%), MLC (3%), and typical MVB (4%). A
total of 520 positive compartments were counted.
Double labeling. The differential localization of the specific probes was further investigated by double-labeling experiments (Fig 8). Double labeling with M6-P GP probe and
LAMP-2 showed colocalization in the multilaminar compartment (Fig 8a, inset). Double labeling with a rabbit polyclonal
antibody against lactoferrin clearly showed that there was no
colocalization of lactoferrin (goat antirabbit-gold 5 nm) with the
Man 6-P GP (streptavidin gold-10nm) (Fig 8a). The reverse
labeling of lactoferrin with goat antirabbit-gold (10 nm) and
Man 6-P GP with streptavidin gold (5 nm) confirmed this
differential localization (not shown). Man 6-P GP did not
colocalize with albumin (not shown). Double labeling with
antibodies against lactoferrin and LAMP-2 (Fig 8b) and gelatinase and LAMP (Fig 8c) confirmed that the LAMP glycoproteins
were in different compartments from the granules. Furthermore,
albumin was found in vesicular structures other than those
labeled for LAMP (Fig 8d). We conclude that in mature intact
cells, as predicted by the fractionation data, the major repository
of Man 6-P GP is the azurophil granule, that of LAMP is the
multilaminar compartment, and that both are found in the
multilaminar compartment.
DISCUSSION
The goal of these studies was to acquire additional information on the composition of lysosomes of human neutrophil
1055
leukocytes. The significant new findings of this article are as
follows: (1) The azurophil granules contain phosphorylated
glycoproteins, which retain the Man 6-P recognition marker for
several days. (2) Azurophilic granules, despite their high
content of lysosomal enzymes, do not contain LAMPs. (3) A
multilaminar structure (MLC) that had not been previously
observed in neutrophilic leukocytes contains both Man 6-P GP
and LAMP and appears early in the maturation of neutrophils,
but its function is unknown.
Man 6-P GP
Detailed studies by other investigators42 conducted mainly in
fibroblasts, have shown the Man 6-P recognition marker as
necessary for the sorting of newly synthesized acid hydrolases.
Man 6-P–containing acid hydrolases bind to the Man 6-P
receptors and are targeted from the secretory pathway to the
lysosomal pathway. The Man 6-P is hydrolyzed to yield the
dephosphorylated hydrolases found in mature lysosomes. In
most cultured cells, Man 6-P recognition marker is a transient
modification that is rapidly removed from lysosomal enzymes
when they reach a compartment where Man 6-P is cleaved. For
instance, kinetic data from mouse lymphoma cells showed that
the Man 6-P recognition marker remained on lysosomal enzymes within the cells for only 1.4 hours.43 However, this is not
universally the case, as in mouse L(Rec-) cells that lack the
sCI-MPR, where the Man 6-P has a half-life of 19 hours.44
Supporting these in vitro results, a preliminary survey of mouse
tissues using the sCI-MPR probe at the light level indicates that
there are low steady state levels of proteins that retain the Man
6-P recognition marker in most cell types, but some cell types
(eg, neurons and Sertoli cells) also contain relatively high
levels.45
It has long been recognized that MPO is formed early in
neutrophil maturation, during the promyelocyte stage, and that
synthesis ceases when the cell enters the myelocyte stage of
maturation. Since MPO is one of the major glycoproteins with
Man 6-P, this is consistent with our observation that Man 6-P
GP is present within the azurophilic granules of neutrophils
from the early stages of maturation and is maintained even in
the mature circulating cell. This means that proteins with the
Man 6-P recognition markers are stored in the azurophilic
granules for at least 14 days. This is much longer than has been
noted in other cell types to date. The fact that the Man 6-P GP
content is very high in azurophilic granules indicates that the
conditions that allow for the removal of Man 6-P may not exist
in these granules.
The sorting mechanism of lysosomal enzymes was initially
demonstrated by the finding that fibroblasts from patients with
I-cell disease (mucolipidosis) were unable to sort acid hydrolases because the cells were deficient in a phosphotransferase,
an enzyme needed to add the Man 6-P recognition marker.42
;
Fig 8. Portions of human neutrophils with double labeling to identify various organelles. (a) Double labeling with sCI-MPR–biotinylated and
Lactoferrin 1/1,000. The specific granules are characterized by the presence of lactoferrin (lf), with the small gold-5 nm, (arrowhead), which does
not colocalize with the Man 6-P GP, as visualized with streptavidin 10 nm (*) inside the azurophilic granules. Inset: the multilaminar compartment
is double labeled for the Man 6-P GP, as observed with streptavidin 10 nm (*), and LAMP-2, gold-5 nm, (arrow). (b) Double labeling with LAMP-2
(arrows). Portions of human neutrophils, which demonstrate that LAMP-2 is localized in membranes of vesicular structures, and do not
colocalize with lf, the marker for specific granules. The large gold (GAM-10) labels the location of LAMP-2 in vesicles (arrows), whereas the small
gold (GAR-5), (arrowheads) detects lf. (c) LAMP-2 (GAM-5), (arrows) and gelatinase (gel), (GAR-10), (arrowheads) do not colocalize either. (d)
Albumin (alb) (GAR-5), (arrowheads) is found in different vesicular structures from LAMP-2 (GAM-10), (arrows).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1056
However, this mechanism is not exclusively used by all cell
types. For example, hepatocytes and leukocytes from patients
with this deficiency successfully target enzymes to the lysosome.42 There is accumulating evidence for alternative lysosomal enzyme targeting pathways.46,47 The sorting pathway of
lysosomal enzymes in neutrophils is controversial. Nauseef et
al,48 have shown that MPO in HL60 cells, a promyelocytic cell
line, contains the Man 6-P marker, but there is no evidence that
the Man 6-P receptor is involved in the MPO targeting to the
azurophilic granules. Moreover, the defensins, other azurophilic
granule proteins, lack the Man 6-P marker, hence, they cannot
use this mechanism for sorting to the azurophilic granules.40,49,50 It is likely that the content of different granules
reflects the profile of proteins synthesized and transported to the
trans-Golgi network at the time of formation of the individual
granules during different stages of maturation. This is supported
by the finding that the heterogeneity of granules reflects the
known order of biosynthesis of granule proteins,5,13 and by the
finding that the granule protein neutrophil gelatinase-associated
lipocalin, which is found exclusively in specific granules of
normal neutrophils51 will localize to azurophilic granules when
expressed in promyelocytic HL60 cells.52
LAMP and Lysosomes
Combining immunolabeling with cell fractionation studies of
mature blood neutrophils, we demonstrate that the typical
membrane markers of lysosomes (LAMP-1 and LAMP-2) are
confined to the MLC as well as to small vesicles and typical
multivesicular bodies of human neutrophils, and are not present
in the three major granule types. These data are in agreement
with those of Dahlgren et al53 who also recently studied the
distribution of LAMP in subcellular fractions of human neutrophils and concluded that LAMP-1 and LAMP-2 are present ‘‘in
the specific granule-enriched fraction and in the light membrane
fraction, but not in the azurophil granules.’’ Our direct analysis
of the specific granule fraction itself clarifies the situation and
shows that LAMPs are not present in specific granules. The only
known azurophil granule membrane proteins are CD6310 and
CD68 (see Table 1). CD63, also referred to as granulophysin,11
ME491,54 or LIMP-1C, has been identifed by Fukuda24 to be
another lysosomal membrane marker, LAMP-324 because it
shares a cytoplasmic Gly-Tyr motif essential for lysosomal
trafficking during receptor-mediated endocytosis with LAMP-1
and -2. It differs, however, in that it is predicted to traverse the
plasma membrane four times (the tetraspan family), unlike
LAMP-1 or 2 which are typical type 1 transmembrane proteins.
Vischer and Wagner55 recently showed the presence of CD63 in
the membranes of Weibel-Palade bodies, which are the regulated secretory granules in endothelial cells. In both endothelial
cells and neutrophils, granules containing CD63 can be relocated to the plasma membrane by activation,11 whereas in
neutrophils, LAMP-1 and LAMP-2 are not translocated after
activation (unpublished data). CD63 seems to be expressed
mainly in hematopoietic and endothelial cells. Additionally,
Saito et al12 have shown that CD68 is present on the membranes
of azurophil granules in neutrophils. Less is known about
CD68; it is a 110-kD transmembrane glycoprotein recently
cloned by Holness and Simmons.56 It seems that CD68 is a
member of a growing family of hematopoietic mucin-like
CIEUTAT ET AL
molecules, including CD43, CD34, P-selectin glycoprotein
ligand-1, and Gly CAM-1. CD68 can also be found in liver and
kidney.
Although the phagocytic pathway of neutrophils has been
clearly defined, it is less well-appreciated that neutrophils are
also active in pinocytosis of soluble proteins and recycling of
plasma membrane receptors. Multivesicular bodies have already been described as LAMP-positive in HL60.22 Multivesicular bodies are not easily seen in resting neutrophils that have not
been exposed to chemoattractants, but in activated cells they are
important and serve as sites of internalization of markers of
fluid phase pinocytosis.57 Berger et al58 showed that multivesicular bodies from resting neutrophils express few LAMP vesicles,
but they subsequently fuse with LAMP-positive structures,
rendering them positive as well, and they then mature as
prelysosomal compartments.58 In related studies, the uptake of
eosinophil peroxidase into vesicular structures in neutrophils
was observed by Zabucchi et al.59 Of more relevance, Borregaard et al16 showed that neutrophils contain endocytosed
plasma proteins, particularly albumin, although this occurs
mainly at the myelocyte stage of maturation.
It is likely that the special structures, the multilaminar
compartment and the multivesicular bodies, are the prelysosomal structures of the neutrophils, and may serve in the early
or late endosomal compartment. Mane et al22 showed a marked
accumulation of LAMP on the membrane of phagocytic vacuoles containing opsonized erythrocytes in U937 cells, apparently delivered to these sites in multivesicular vacuoles. We also
observed this in human neutrophils that have phagocytized
bacteria (unpublished observation, January 1997). This means
that MVB and MLC must fuse with the newly formed endosome because the vacuole containing degraded bacteria contains LAMPs. Finally, it should be mentioned that we did not
observe ‘‘dense bodies’’ that have been referred to as mature
lysosomes in most other cells.6,17
Azurophil Granules Correspond to Regulated
Secretory Granules
The absence of LAMP-1 and LAMP-2 in azurophilic granule
membranes suggests that, despite its high content of lysosomal
enzymes, the neutrophil azurophilic granule has the characteristics of a regulated secretory granule membrane rather than being
a true lysosome. This may also be true in other cell types such as
the acrosomes of sperm cells,2 but this awaits further investigation. The lack of LAMP in azurophil granules suggests that
these granules are not part of a dynamic endosomal lysosomal
compartment, but behave more as regulated storage granules
that are mobilized to the phagosome during ingestion of
microorganisms.
The Multilaminar Compartment of the Neutrophils
This compartment has not been previously recognized in this
cell type in Epon embedded material. However, this multilaminar compartment was previously observed in ultracryosections
of neutrophils60 and in the promyelocytic cell line HL60.22 Our
data show that this compartment is present in BM neutrophils
from intermediate to late stages of maturation, and in circulating
neutrophils, but is most easily sampled in myelocytes. The
compartment that contains the lysosomal membrane glycopro-
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LAMPs ARE ABSENT FROM NEUTROPHIL GRANULES
teins LAMP-1 and LAMP-2 and with Man 6-P recognition
markers is morphologically similar to the prelysosomal compartment first observed in normal rat kidney cells by ultracryosectioning and believed to be part of the endocytic pathway. This
compartment was found to be positive for the Man 6-P
receptor.20,61,62
This compartment was also similar to the multilaminar
compartment termed MII C63 characterized by the colocalization of the major histocompatibility class II (MHC II) protein,
lysosomal hydrolases, and the lysosomal membrane glycoproteins, but lacking the CI-MPR. Gosselin et al64 found that
stimulated neutrophils express MHC II molecules; however,
using immunocytochemical methods, we were unable to detect
sufficient labeling to draw any definite conclusions.
As mentioned previously, O’Brien et al45 using the same
sCI-MPR probe at the light microscopic level on mouse tissues
found that Man 6-P GP is abundant in vesicular structures of
mouse testis and brain but was absent from most of the other
tissues tested. Furthermore, the distribution of LAMP and Man
6-P GP was distinct. Also, malignant cells in a subset of human
breast carcinomas exhibited high levels of Man 6-P GPs.39 This
suggests that a special class of granules contains proteins which
retain the Man 6-P recognition marker and may serve unique
functions. Additional studies will be necessary to fully appreciate the meaning of this distribution in various cell types.
ACKNOWLEDGMENT
We thank Ivy Hsieh and Yvonne Jacques for the excellent technical
assistance and David Geller for his help editing, and Silvia Molina for
preparation of the manuscript.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1998 91: 1044-1058
Azurophilic Granules of Human Neutrophilic Leukocytes Are Deficient in
Lysosome-Associated Membrane Proteins but Retain the Mannose
6-Phosphate Recognition Marker
A.-M. Cieutat, P. Lobel, J.T. August, L. Kjeldsen, H. Sengeløv, N. Borregaard and D.F. Bainton
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