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Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 1044-1058
By
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.
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.
ENZYME 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 peroxidase-negative, form later
in maturation: specific or secondary granules,13 which
contain lactoferrin and many other substances, 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 post-translational 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.
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.
Antibodies and Marker Proteins
Subcellular Fractionation
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 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 × 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.37,38Detection 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 -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 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-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).
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.
Localization of the Man 6-P GP and LAMP in Developing BM
Neutrophils
Light Microscopy
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,
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 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.
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.
The goal of these studies was to acquire additional information on the
composition of lysosomes of human neutrophil 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
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 molecules, including CD43, CD34, P-selectin glycoprotein ligand-1, and Gly CAM-1. CD68 can also be found in liver and kidney.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 glycoproteins 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
Submitted May 12, 1997;
accepted September 23, 1997.
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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Abstract
Introduction
Abstract
-bis2
[ethane-sulfonic 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
(
-band), containing azurophilic granules; the intermediate band
(
-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.
), specific granules (lactoferrin, 
), gelatinase granules (gelatinase, 
), 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 (
) and LAMP 2 (
). 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, 
) 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.
