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PHAGOCYTES
From the Department of Biochemistry, Center for the
Study of Host Resistance, McGill Cancer Center, McGill University,
Montreal, QC, Canada; the Division of Cell Biology, The Netherlands
Cancer Institute, Amsterdam, Netherlands; INRS-Institut
Armand-Frappier, Laval, QC, Canada; the Division of Cell Biology, The
Hospital for Sick Children, Toronto, Canada; and the Department of
Hematology, University of Copenhagen, Copenhagen, Denmark.
Mutations at the Nramp1 gene cause susceptibility to
infections with intracellular pathogens. In human blood,
polymorphonuclear (PMN) leukocytes are the most abundant site of
NRAMP1 messenger RNA (mRNA) expression, suggesting that
NRAMP1 plays an important role in the activity of these cells. By
Northern blot analysis, NRAMP1 mRNA was only detected in
most mature neutrophils from bone marrow (band and segmented cells). A
high-affinity polyclonal rabbit antihuman NRAMP1 antibody directed
against the amino terminus of the protein was produced and used to
study cellular and subcellular localization of the protein in primary
human neutrophils. Subcellular fractionation of granule populations
together with immunoblotting studies with granule-specific markers
indicate that NRAMP1 expression is primarily in tertiary granules.
These granules are positive for the matrix enzyme gelatinase and the
membrane subunit of the vacuolar H+/ATPase and can be
recruited for exocytosis by treatment of neutrophils with phorbol
myristate acetate. Immunogold studies by cryoelectron microscopy with
primary neutrophils confirm that a majority (75%) of NRAMP1-positive
granules are also positive for gelatinase, but they also suggest
further heterogeneity in this granule population. Presence of NRAMP1 in
tertiary granules is in agreement with the late-stage appearance of
NRAMP1 mRNA during neutrophil maturation in bone marrow.
Finally, immunofluorescence studies of Candida albicans-containing phagosomes formed in neutrophils
indicate that NRAMP1 is recruited from tertiary granules to the
phagosomal membrane on phagocytosis, supporting a role for NRAMP1 in
the antimicrobial defenses of human neutrophils.
(Blood. 2002;100:268-275) In the mouse, naturally occurring1 or
experimentally introduced mutations2 at the
Nramp1 gene cause susceptibility to infection with several
unrelated intracellular parasites such as Salmonella,
Leishmania, and Mycobacterium (reviewed by
Skamene et al3). These mutations impair the ability of
macrophages to restrict intracellular replication of these microbes,
suggesting an important role of the Nramp1 protein in bacteriostatic or
bactericidal defense mechanisms of these cells.4
Nramp1 messenger RNA (mRNA) is expressed in primary mouse
macrophages and in J774 and RAW297 macrophage cell lines, where its
expression can be strongly induced by exposure to interferon- In humans, population-based association studies and linkage analyses in
multigeneration pedigrees have demonstrated an association between
polymorphic variants at NRAMP1 and susceptibility to
tuberculosis and leprosy in endemic areas of disease and in the
outbreak situation.14-17 Additional reports have also
suggested association of NRAMP1 polymorphisms with
inflammatory diseases in humans such as rheumatoid
arthritis18-20 and Crohn disease.21
Interestingly, functional polymorphisms have been identified in the
promoter of the NRAMP1 gene in the form of sequence
variations in a Z-DNA-forming dinucleotide repeat.22 These polymorphisms define a 4-allele system that shows significant functional properties in transcriptional activity with reporter genes.22 Searle and Blackwell22
proposed a hypothesis in which chronic activation of the
NRAMP1 promoter in allele 3 of this polymorphism is
associated with autoimmune disease susceptibility, while showing a
protective effect against infectious diseases. The reverse would be
true for the low-expressing allele 2 at NRAMP1.
Human NRAMP1 mRNA is expressed in lungs, spleen, and liver
but is highest in peripheral blood leukocytes.23,24 There,
polymorphonuclear leukocytes represent by far the major site of
NRAMP1 mRNA expression followed to a lesser degree by
monocytes. Migration of monocytes to tissues (alveolar macrophages) or
maturation in vitro is associated with increased NRAMP1
expression compared with blood monocytes.24 Studies in the
promyelocytic leukemia cell line HL-60 indicate that differentiation
toward either the monocytic pathway (with vitamin D3 and
phorbol ester) or the granulocyte pathway (dimethylformamide, dimethyl
sulfoxide [DMSO]) is concomitant to strong induction of
NRAMP1 mRNA expression.24 Together, these
results have suggested that NRAMP1 may play an important
role in the antimicrobial response or inflammatory process mediated by
polymorphonuclear (PMN) leukocytes.
Both PMN leukocytes and macrophages are capable of engulfing
and destroying microorganisms by the action of toxic radicals, ions,
and proteolytic enzymes produced by these cells. Neutrophils contain
different types of granules that can be recruited for release by
exocytosis or for fusion to phagosomes containing ingested particles.25 These granules are divided into 3 or 4 subtypes, depending on the purification method used to isolate them.
The biochemical properties and content of bactericidal species of these
granules have been extensively characterized in human neutrophils (reviewed in Borregaard et al26). Simple separation by
sedimentation on density gradients yields 4 major fractions: a
cytosolic soluble fraction, a PM/secretory vesicle fraction (positive
for alkaline phosphatase and nicotinamide adenine dinucleotide
phosphate [NADPH] oxidase), a specific granule fraction (secondary
and tertiary granules, positive for lactoferrin and vitamin
B12 binding protein), and the azurophil granules (positive
for myeloperoxidase and The role of the NRAMP1 protein in the antimicrobial defenses of the
neutrophil remains unknown. However, the previous localization of the
murine Nramp1 protein to the lysosomal compartment of
macrophages9 suggests that it may also be present in one or
more granule populations in neutrophils. To address this issue, we have
generated a polyclonal antibody against the human NRAMP1 protein and
used this reagent to study cellular and subcellular localization of
NRAMP1 protein in human neutrophils.
Cell isolation procedures
Cell culture
RNA isolation and Northern blotting Total cellular RNA was extracted from the different BM cell populations by a single-step procedure.32 The NRAMP1 hybridization probe was a 1.2-kilobase (kb) cDNA fragment corresponding to positions 120 to +1120 of the published
sequence.23 Control hybridization probe for the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was produced and used
exactly as described.29 The membrane was washed for
2 × 15 minutes at 60°C in 2 × sodium chloride/sodium citrate
(SSC; 1 × SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0) 0.5%
sodium dodecyl sulfate (SDS) followed by 2 × 15 minutes in 0.2 × SSC, 0.5% SDS. The membrane was stripped by boiling in 0.1% SDS
before rehybridization.
Subcellular fractionation Neutrophils were resuspended in Krebs-Ringer phosphate (130 mM NaCl, 5 mM KCl, 1.27 mM MgSO4, 0.95 mM CaCl2, 5 mM glucose, 10 mM NaH2PO4/Na2HPO4, pH 7.4) at 3 × 107 cells/mL and divided into 2 equal portions. One portion was kept on ice as control, the other was stimulated at 37°C for 15 minutes with 2 µg/mL phorbol myristate acetate (PMA). The stimulation was stopped by adding 2 volumes of ice-cold buffer. Control and stimulated cells were sedimented at 200g for 10 minutes and resuspended in ice-cold buffer before subcellular fractionation. Individual granule fractions were purified from resting and PMA-stimulated neutrophils exactly as previously described.33 Briefly, neutrophils were subjected to nitrogen cavitation followed by centrifugation to remove nuclei and unbroken cells. The resulting postnuclear supernatant was applied on top of a discontinuous, 3-layer Percoll gradient (1.050/1.09/1.12 g/mL) and centrifuged at 37 000g for 30 minutes at 4°C. Gradients were aspirated from the bottom through a peristaltic pump attached to a fraction collector set to deliver 1 mL in each fraction. Fractions 1 to 6, 7 to 12, 13 to 18, and 19 to 24 were pooled in 4 distinct populations. Percoll was removed by centrifugation, and the biologic material was resuspended in 1 mL PBS. Aliquots of each sample were assayed for the presence of marker proteins corresponding to individual compartments (indicated in parenthesis): myeloperoxidase ( band/azurophils or primary granules), lactoferrin ( 1 band/specific or secondary granules), gelatinase (2 band/gelatinase or tertiary granules), and albumin ( band; secretory vesicles).28
Production and purification of NRAMP1 antibodies A glutathione-S-transferase (GST) fusion protein was constructed by subcloning in plasmid vector pGEX (Pharmacia, Montreal, QC, Canada) a NRAMP1 N-terminal cDNA sequence (nucleotides 1 to 177, corresponding to amino acids 1 to 59; GST-59NT) obtained by PCR amplification from full-length NRAMP1 cDNA template using oligonucleotides 5HF (5'TCG GAT CCT CAA TGA CAG GTG AC-3') and 5HR (5'-TAG AAT TCG CAG GCT GAA GGT G-3'). The PCR product was digested with BglII and EcoRI (restriction enzymes sites are underlined in the oligonucleotide sequence), prior to subcloning into pGEX-2 digested with BamHI and EcoRI to create the in-frame GST fusion. The fusion proteins were produced in Escherichia coli as previously described.31 Approximately 0.4 to 0.8 mg GST-59NT protein was injected in New Zealand white male rabbits as previously described.8 The polyclonal antiserum was then purified by an affinity chromatography protocol against the same NRAMP1 peptide segment (1-59 aas) fused to dihydrofolate reductase (DHFR), as previously described.31Immunofluorescence and immunocytochemistry Cells were fixed with Bouin solution for 20 minutes and permeabilized (or not) with Triton-X100 (0.1% in PBS) for 10 minutes. Cells were incubated at 20°C in blocking solution (bovine serum albumin [BSA] 1%, 10% heat-inactivated goat serum for CHO cells or BSA 1%, 20% heat-inactivated donkey serum for HL-60) for 1 hour, prior to incubation with primary antibodies for 2 hours at 20°C. Dilution of primary antibodies in blocking solution was as follows: anti-NRAMP1 antibody (rabbit polyclonal), 1/50 to 1/75 or nonimmune serum, 1/50 to 1/75. After washing with PBS-0.5% Tween 20, secondary antibodies were incubated for 1 hour at room temperature. Dilution of secondary antibodies was as follows: goat Texas red-antirabbit 1/400 and donkey Alexa Fluor488-antirabbit, 1/400 (Molecular Probes). Cells were examined by using a Nikon optical fluorescence microscope with a ×60 to ×100 objective (×600 to ×1000 magnification) or by using a confocal microscope. For immunocytochemistry, after permeabilization with Triton X-100, CHO cells were incubated in (1%) H2O2 in PBS for 10 minutes to inhibit endogenous peroxidase activity. Primary antibody incubation was followed by incubation with a swine secondary antirabbit immunoglobulin G (IgG; 1/100; DAKO) for 1 hour at room temperature and subsequently with a rabbit peroxidase antiperoxidase (1/100; DAKO). Specific staining was revealed by using 3'-diaminobenzidine tetrahydrochloride in solution. Finally, cells were counterstained with 0.1% methylene blue in PBS, dehydrated, and mounted with Permount.Phagocytosis DMSO-differentiated HL-60 cells were seeded in Poly-D-Lysine-coated glass slides (Biocoat; BD-Falcon) and further incubated for 1 day to promote adherence. Heat-inactivated Candida albicans was opsonized for 15 minutes at 25°C with rabbit complement (Biotest). Cells were washed twice with PBS, and the culture medium was replaced by phagocytosis buffer (Hanks balanced salt solution with Ca++ and Mg2+ [Sigma], 0.2% BSA [Fisher]) containing opsonized C albicans A at a cell-to-C albicans ratio of 1:10. Incubation was at 37°C for 30 minutes. Slides were washed and processed for light microscopy or epi-immunofluorescence.Immunoblotting Crude membrane preparations from control CHO/U937 cells (5 µg) and aliquots (25 µg) of enriched granule fractions were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on 10% acrylamide gels followed by transfer to a polyvinylidene diflouride (PVDF) membrane (16 hours, 4°C).31 Primary antibodies were used at the following dilution: rabbit antihuman NRAMP1, 1/250; mouse anti-c-Myc (9E10) 1/100; rabbit antigelatinase, 1/2500; rabbit antimyeloperoxidase, 1/2500, rabbit antihuman serum albumin, 1/2500; rabbit antilactoferrin, 1/2500; and rabbit anti- subunit (39 kd) of
the mammalian V-type H+-ATPase, 1/400.
Electron microscopy Neutrophils from peripheral blood were fixed for 24 hours in 4% paraformaldehyde in 0.1 M PHEM (240 mM PIPES, 100 mM HEPES, 8 mM MgCl2, 40 mM EGTA, pH 6.9) and then processed for ultrathin cryosectioning, as previously described.34 Cryosections (45 nm) were cut at125°C with the use of diamond
knives (Drukker, Cuijk, The Netherlands) in an ultracryomicrotome
(Leica Aktiengesellschaft, Vienna, Austria) and transferred with a
mixture of sucrose and methylcellulose onto formvar-coated copper
grids.35 The grids were placed on 35-mm Petri dishes
containing 2% gelatin. For immunolabeling, the sections were incubated
with rabbit antihuman NRAMP-1 (1/25) for 45 minutes, followed by a
30-minute incubation with 10-nm protein A-conjugated colloidal gold
(EM Lab, Utrecht University). For double immunolabeling with rabbit
anti-NRAMP-1 and rabbit antigelatinase (1/300), the procedure
described by Slot et al36 was followed with 15- and 10-nm
protein A-conjugated colloidal gold probes. After immunolabeling, the
cryosections were embedded in a mixture of methylcellulose and uranyl
acetate and examined with a Philips CM 10 electron microscope
(Eindhoven, The Netherlands).
NRAMP1 mRNA expression in developing polymorphonuclear leukocytes The development of neutrophils from stem cell precursors involves different maturational steps, including the acquisition of specific granules.25 The mRNAs encoding proteins present in different types of granules display a unique and sometimes diagnostic pattern of expression during the maturation process of neutrophils. For example, mRNA for myeloperoxidase, a protein present in primary/azurophyl granules, appears very early in neutrophil precursors and is largely absent from mature cells, whereas the mRNAs for gelatinase (tertiary granules) and for CD35 and fMLP-R (secretory vesicles) appear late during differentiation and remain present in mature cells.29 Concordance of temporal expression profiles between the mRNA for a novel granule protein and that previously established for mRNAs of matrix or membrane proteins of unique granule populations can provide initial clues on the site of expression of this novel protein. With the use of a discontinuous Percoll gradient protocol, BM cells of the neutrophil lineage can be fractionated into 3 cell populations consisting of early immature (primarily myeloblasts and promyelocytes found in band 3), late immature (primarily myelocytes and metamyelocytes found in band 2), and mature neutrophils (primarily band and segmented neutrophils found in band 1).29 Total RNA was prepared from these BM populations as well as from circulating PMN leukocytes, separated by gel electrophoresis and probed with a human NRAMP1 cDNA fragment (Figure 1A). NRAMP1 mRNA expression was detected in circulating PMN leukocytes and also in the most mature neutrophil precursors corresponding to band 1 (Figure 1A). The apparent faster electrophoretic mobility of NRAMP1 mRNA in the sample of band 1 cells compared with mature PMN leukocytes is due to a gel artifact that also affects migration of GAPDH control mRNA in the same samples. However, no NRAMP1 expression was detectable in RNA preparations from band 2 and band 3 cells, suggesting that NRAMP1 is not expressed in less mature cells (myeloblasts, promyelocytes, myelocytes, and metamyelocytes). These results suggest that NRAMP1 mRNA expression occurs late in neutrophil maturation, with expression persisting in circulating PMN leukocytes. Previously, band 1 cells were shown to express mRNAs corresponding to markers of tertiary granules and secretory vesicles (gelatinase, fMLP-R, CD35), suggesting that the NRAMP1 protein may be expressed in these granule populations.
Production and characterization of rabbit antihuman NRAMP1 antisera To further study expression and subcellular localization of the NRAMP1 protein in human neutrophils, a specific antiserum raised against the first 59 amino acid residues of NRAMP1 was developed and purified by affinity.31 The specificity of the resulting antiserum was assessed by immunoblotting (Figure 2) by using membrane fractions from CHO cells or human U937 cells transfected or not with a human NRAMP1 cDNA epitope tagged with a c-Myc antigenic peptide inserted at the C-terminus of the protein. The affinity-purified rabbit anti-NRAMP1 polyclonal antiserum specifically detected a protein species of apparent molecular mass 80 to 100 kd in the NRAMP1-transfected cells that was absent from the membrane fractions of control cells (Figure 2i-ii). NRAMP1 migrated as a broad immunoreactive species of size considerably larger than that predicted from the cDNA sequence (61 kd), suggesting that the protein is extensively modified by posttranslational modification in CHO and U937 cells, possibly by N-linked glycosylation, as previously reported for mouse Nramp1.8 The NRAMP1 protein detected by this polyclonal antiserum displayed electrophoretic mobility characteristics identical to that observed if a mouse anti-c-Myc monoclonal antibody was used to probe the immunoblot (Figure 2Aiii), confirming the specificity of the anti-NRAMP1 antiserum. Finally, the antihuman NRAMP1 antibody produced could also recognize the NRAMP1 protein by immunohistochemistry (Figure 2Bi-ii) and by immunofluorescence (Figure 2Biii-iv) in transfected CHO cells.
Detection of NRAMP1 in human neutrophil granules by immunoblotting Granule populations were prepared from human neutrophils by separation on a 3-step discontinuous Percoll gradient.33 After centrifugation, 4 bands are formed at the interface of the gradient steps; these correspond (from the bottom) to the-band,
which contains primarily azurophil granules, the 1-band enriched for the specific or secondary granules, the 2-band enriched for the gelatinase-positive tertiary granules, and the -band that contains secretory vesicles and plasma membranes.33 One-milliliter
fractions were collected from the bottom of the tube and the fractions
1 to 6, 7 to 12, 13 to 18, and 19 to 24 were pooled in 4 populations designated fractions: 1, 2, 3, and 4 (Figure
3). Biologic material from these pools
(1-4) were recovered by centrifugation and were assayed for the
presence of granule-specific markers by either enzyme-linked
immunoassay (ELISA; Figure 3A-D) and by immunoblotting after
SDS-PAGE (Figure 3E-H), using specific antibodies. The soluble matrix
marker protein myeloperoxidase was used as a marker for the
-band,37 lactoferrin for the 1-band,37
gelatinase for the 2-band, and human serum albumin (HSA) for the
-band.38 Results from both analyses were consistent
and, despite some degree of overlap, showed clear enrichment of primary
(-band), secondary (1-band), tertiary (2-band), and secretory
vesicles (-band) in the 1-, 2-, 3-, and 4-pooled fractions,
respectively (Figure 3).
The presence of NRAMP1 protein was then investigated in these fractions
by immunoblotting (Figure 4).
Interestingly, NRAMP1 was found expressed mostly in the
Effect of PMA treatment on NRAMP1-positive neutrophil granules Recruitment of different granule populations for exocytosis can be induced by stimulation with compounds such as PMA or fMLP and follows established kinetics. This recruitment can be used to confirm the identity of an NRAMP1-positive granule population and to determine if NRAMP1 can be delivered to the PM after fusion of this granule population. In these experiments, neutrophils were treated with 10 6 M PMA, a treatment that has been previously
established to induce recruitment of secretory vesicles and tertiary
and secondary but not primary granules.28 After nitrogen
cavitation and granule separation on Percoll gradient, fractions were
collected as 4 pools, as described in Figures 3 and 4. Such fractions
were then analyzed by ELISA and immunoblotting for the presence of
gelatinase (Figure 5A-B, respectively) and for other granule-specific
matrix markers (data not shown). Immunoblotting data indicate that PMA treatment of neutrophils resulted in disappearance of approximately 80% of gelatinase reactivity, demonstrating tertiary granule
recruitment to the plasma membrane for exocytosis (Figure 5A-B).
Analysis of the same fractions with the anti-NRAMP1 antiserum (Figure
5C) indicates that PMA treatment of neutrophils results in drastic reduction of level of NRAMP1 protein in the 2 granule fraction in a
manner and proportion very similar to that observed for the matrix
marker gelatinase. These results support the contention that NRAMP1 is
expressed in the membrane of PMA-recruitable, gelatinase-containing tertiary granules. Interestingly, disappearance of NRAMP1 from the
tertiary granules was not accompanied by a concomitant appearance of
NRAMP1 in the plasma membrane/secretary vesicles-containing fraction (Figure 5A), as has been previously observed for other membrane proteins.39 These results suggest the possibility
that NRAMP1 may not be stable at the plasma membrane.
Electron microscopy detection of NRAMP1 in human neutrophils The subcellular localization of NRAMP1 was also examined by immunoelectron microscopy. For this examination, human neutrophils were harvested and 45-nm thin cryosections were incubated with the rabbit anti-NRAMP1 polyclonal antiserum (Figure 6A), and specific immune complexes were revealed with protein A-conjugated colloidal gold (10-nm particles). Although few gold particles were detected, immunoreactive NRAMP1 was found associated with granules in neutrophils (arrows in Figure 6A) but was absent from the cytosol or from the nucleus and mitochondria. Interestingly, gold particles were found at the membrane but not in the matrix, in agreement with the fact that NRAMP1 is an integral membrane protein, and thus possibly expressed in the membrane of neutrophil granules. As results in Figures 1, 4, and 5 strongly suggested that NRAMP1 may localize to tertiary granules, additional double-labeling experiments were carried out. In this case, NRAMP1 was labeled with 15-nm gold particles, and gelatinase was revealed with a specific antibody coupled to 10-nm gold particles. In these experiments, several granules could be seen heavily labeled by the antigelatinase antibody (Figure 6B; small arrows). Labeling appeared to be in the lumen of the granule in agreement with the known matrix location of this protein in tertiary granules. Although a large proportion of granules were positive for gelatinase only (96 of 115, ~83%; see "Discussion"), the majority (14 of 19, ~75%) of NRAMP1-positive granules were also positive for gelatinase (Figure 6B; large arrows), in agreement with the proposal that NRAMP1 is present in gelatinase granules. Interestingly, a subpopulation of NRAMP1-positive granules (5 of 19; ~25%) were negative for gelatinase (Figure 6B), suggesting that NRAMP1 can also be present in gelatinase-negative granules.
Recruitment of NRAMP1 to phagosomes in HL-60-derived granulocytes Localization of NRAMP1 protein to tertiary granules in primary human neutrophils suggests that NRAMP1 may be recruited to the membrane of phagosomes, after phagocytosis of microbial particles by these cells. This possibility was tested by using HL-60 cells differentiated in vitro into neutrophils following treatment with DMSO. We have previously shown that this treatment results in robust induction of NRAMP1 mRNA24 as well as other markers of the granulocytic lineage. HL-60-derived neutrophils were allowed to ingest heat-killed, opsonized C albicans cells for 30 minutes at 37°C. Cells were then permeabilized and analyzed by immunofluorescence for reactivity with anti-NRAMP1 antiserum (Figure 7). In intact, particle-free, differentiated HL-60 cells, NRAMP1 staining appeared intracellular and punctate, with no obvious labeling of the plasma membrane (Figure 7Ai; arrowheads). Staining was specific and absent when cells were incubated with either normal preimmune serum (Figure 7Aii) or secondary antibody alone (Figure 7Aiii). This staining is similar to that previously observed for the mouse Nramp1 protein expressed in primary macrophages9 or for the human NRAMP1 protein overexpressed in transfected CHO cells (Figure 2). In individual cells having engulfed C albicans, NRAMP1 staining appeared concentrated at the periphery of the internalized particles (Figure 7Ai; arrows). This staining could result from active recruitment of NRAMP1 to the phagosomal membrane or could be an optical artifact caused by displacement of unfused NRAMP1-positive vesicles by the internalized particle. To distinguish between the 2 possibilities, C albicans phagosomes formed in HL-60-derived neutrophils were analyzed for NRAMP1 reactivity by confocal microscopy (Figure 7Bi-ii). This analysis revealed NRAMP1 staining concentrated at the periphery of individual phagosomes, with 80% (67 of 83 scored) of phagosomes scoring positive. This finding strongly suggests active NRAMP1 recruitment at that site following fusion with tertiary granules.
Neutrophils are polymorphonuclear leukocytes that play a primordial role in the first line of defense against bacterial infections. Neutrophils possess a large number of bacteriostatic and bactericidal enzymes, one of the most important being the production of superoxide anions via NADPH oxidase. However, continuous production of such oxygen radicals by neutrophils can cause inflammatory reactions and is seen in autoimmune disorders.40 We have previously reported very high levels of NRAMP1 mRNA expression in human neutrophils, either in primary cells or in HL-60 cells induced to differentiate along the granulocytic pathway.24 The fact that neutrophils (1) play a key role in antimicrobial defenses and in inflammatory reaction and (2) express high-level NRAMP1 mRNA and that NRAMP1 polymorphisms are associated with susceptibility to infections and autoimmune diseases in humans suggest that the NRAMP1 may play an important role in neutrophil function. In the present study, we have used a rabbit antihuman NRAMP1 antibody
to demonstrate robust NRAMP1 protein expression in human neutrophils.
Subcellular fractionation studies indicate that NRAMP1 protein
copurifies with a granule population but shows very little if any
expression in the plasma membrane fraction of these cells. The
NRAMP1-positive granule population is positive for the matrix marker
gelatinase and is also enriched for the 39-kd Tertiary granules can be recruited to fuse with phagosomes into which they deliver enzymes that are important for the destruction of ingested bacteria.25 Their matrix is characterized by the presence of the protease gelatinase, which is related to collagenase and heparanase present in other granules of these cells. They also contain cytochrome b, which consists of the heme-containing, membrane-bound gp91-phox and p22-phox subunits that ultimately form the superoxide-producing complex NADPH oxidase. Therefore, recruitment of tertiary granules to phagosomes formed in neutrophils after ingestion of microbial pathogens would be expected to deliver the NRAMP1 protein to the membrane of such phagosomes. This proposition was verified by immunofluorescence and confocal microscopy on HL-60 cells differentiated into granulocytes and that clearly show association of NRAMP1 to C albicans-containing phagosome. This situation is reminiscent to that seen in mononuclear phagocytes such as monocytes and macrophages in which the mouse Nramp1 protein localizes to Lamp-1-positive lysosomes in resting cells and can be quickly recruited to the membrane of phagosomes containing either inert particles or live bacteria.4,9,10 These findings also suggest that NRAMP1 may have the same biochemical activity and play the same functional role in neutrophils and macrophages. In macrophages, Nramp1 functions as a divalent cation transporter.11-13 Our group has recently shown that Nramp1 functions as an efflux pump at the phagosomal membrane,11 possibly removing from the phagosomal lumen divalent cations that are either essential to bacterial growth or essential to the activity of bacterial detoxifying enzymes.7 The activity of microbially encoded superoxide dismutases may be essential for survival of bacteria ingested by neutrophils, and it is tempting to speculate that removal of the Mn2+/Fe2+ cofactors that are essential for catalytic activity of such enzymes may be the mechanism by which NRAMP1 contributes to the antimicrobial defenses of the neutrophil. In macrophages, divalent cation transport by Nramp1 is strictly pH
dependent and can be abrogated by folimycin and bafilomycin, potent
inhibitors of the vacuolar H+/ATPase, the enzyme
responsible for phagosomal acidification.11 This strict
requirement of acidic pH for Nramp protein transport has also been
established for the mammalian Nramp2 homolog,42 but it is
also the hallmark of the eukaryotic and prokaryotic Nramp superfamily
(reviewed by Cellier et al6 and Forbes and Gros7). The acidic pH may be required for either
protonation of key amino acid residues in the formation of a putative
transport site, or the Tertiary granules can also be recruited to fuse with the plasma membrane during degranulation. They contain a pool of membrane glycoproteins such as the fMLP receptor, the FcRIII receptor, and the C3bi receptor that are important for proper adhesion and migration of neutrophils in tissues. Therefore, it may be expected that degranulation of neutrophils may also recruit NRAMP1 to the plasma membrane. Results in Figure 5 indeed verify that NRAMP1 disappears from the tertiary granule fraction on degranulation experimentally induced by PMA treatment. However, this result is not accompanied by appearance of NRAMP1 in the plasma membrane of these cells, which when analyzed in the same experiment remains largely negative for NRAMP1 expression. This situation is similar to the membrane-bound metalloproteinase MT6/MMP-25 that is localized primarily to the membrane of gelatinase granules.39 This situation suggests that NRAMP1 may be unstable at the plasma membrane and is possibly targeted for degradation. Therefore, the activity of NRAMP1 in neutrophils may be limited to possible transport function at the phagosomal membrane on recruitment from tertiary granules. These findings suggest a possible role of NRAMP1 in neutrophil function and provide a link with the association of NRAMP1 with susceptibility to infectious diseases and inflammatory diseases noted in genetic studies.
We thank Hans Janssen for technical assistance and Nico Ong for assistance with electron microscopy.
Submitted November 19, 2001; accepted March 4, 2002.
Supported by National Institutes of Health grant AI355237 (P.G.) and the Danish Medical Research Council (N.B.). F.C.-H. is supported by a postdoctoral fellowship from Milestone Medica Corporation. P.G. and S.G. are International Research Scholars of the Howard Hughes Medical Institute (HHMI) and Distinguished Scientists of the Canadian Institutes for Health Research.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Philippe Gros, Department of Biochemistry, McGill University, 3655 Sir William Osler Promenade, Rm 907, Montreal, Quebec, Canada, H3G-1Y6; e-mail: gros{at}med.mcgill.ca.
1. Malo D, Vogan K, Vidal S, et al. Haplotype mapping and sequence analysis of the mouse Nramp gene predict susceptibility to infection with intracellular parasites. Genomics. 1994;23:51-61[CrossRef][Medline] [Order article via Infotrieve].
2.
Vidal S, Tremblay ML, Govoni G, et al.
The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene.
J Exp Med.
1995;182:655-666 3. Skamene E, Schurr E, Gros P. Infection genomics: Nramp1 as a major determinant of natural resistance to intracellular infections. Annu Rev Med. 1998;49:275-287[CrossRef][Medline] [Order article via Infotrieve].
4.
Govoni G, Canonne-Hergaux F, Pfeifer CG, et al.
Functional expression of Nramp1 in vitro in the murine macrophage line RAW264.7.
Infect Immun.
1999;67:2225-2232 5. Govoni G, Gauthier S, Billia F, Iscove NN, Gros P. Cell-specific and inducible Nramp1 gene expression in mouse macrophages in vitro and in vivo. J Leukoc Biol. 1997;62:277-286[Abstract]. 6. Cellier MF, Bergevin I, Boyer E, Richer E. Polyphyletic origins of bacterial Nramp transporters. Trends Genet. 2001;17:365-370[CrossRef][Medline] [Order article via Infotrieve]. 7. Forbes JR, Gros P. Divalent-metal transport by NRAMP proteins at the interface of host-pathogen interactions. Trends Microbiol. 2001;9:397-403[CrossRef][Medline] [Order article via Infotrieve]. 8. Vidal SM, Pinner E, Lepage P, Gauthier S, Gros P. Natural resistance to intracellular infections: Nramp1 encodes a membrane phosphoglycoprotein absent in macrophages from susceptible (Nramp1 D169) mouse strains. J Immunol. 1996;157:3559-3568[Abstract].
9.
Gruenheid S, Pinner E, Desjardins M, Gros P.
Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome.
J Exp Med.
1997;185:717-730 10. Searle S, Bright NA, Roach TI, et al. Localisation of Nramp1 in macrophages: modulation with activation and infection. J Cell Sci. 1998;111:2855-2866[Abstract].
11.
Jabado N, Jankowski A, Dougaparsad S, Picard V, Grinstein S, Gros P.
Natural resistance to intracellular infections: natural resistance-associated macrophage protein 1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane.
J Exp Med.
2000;192:1237-1248 12. Goswami T, Bhattacharjee A, Babal P, et al. Natural-resistance-associated macrophage protein 1 is an H+/bivalent cation antiporter. Biochem J. 2001;354:511-519[CrossRef][Medline] [Order article via Infotrieve].
13.
Kuhn DE, Lafuse WP, Zwilling BS.
Iron transport into mycobacterium avium-containing phagosomes from an Nramp1(Gly169)-transfected RAW264.7 macrophage cell line.
J Leukoc Biol.
2001;69:43-49 14. Abel L, Sanchez FO, Oberti J, et al. Susceptibility to leprosy is linked to the human NRAMP1 gene. J Infect Dis. 1998;177:133-145[Medline] [Order article via Infotrieve]. 15. Alcais A, Sanchez FO, Thuc NV, et al. Granulomatous reaction to intradermal injection of lepromin (Mitsuda reaction) is linked to the human NRAMP1 gene in Vietnamese leprosy sibships. J Infect Dis. 2000;181:302-308[CrossRef][Medline] [Order article via Infotrieve].
16.
Bellamy R, Ruwende C, Corrah T, McAdam KP, Whittle HC, Hill AV.
Variations in the NRAMP1 gene and susceptibility to tuberculosis in West Africans.
N Engl J Med.
1998;338:640-644 17. Greenwood CM, Fujiwara TM, Boothroyd LJ, et al. Linkage of tuberculosis to chromosome 2q35 loci, including NRAMP1, in a large aboriginal Canadian family. Am J Hum Genet. 2000;67:405-416[CrossRef][Medline] [Order article via Infotrieve]. 18. Esposito L, Hill NJ, Pritchard LE, et al. Genetic analysis of chromosome 2 in type 1 diabetes: analysis of putative loci IDDM7, IDDM12, and IDDM13 and candidate genes NRAMP1 and IA-2 and the interleukin-1 gene cluster. IMDIAB Group. Diabetes. 1998;47:1797-1799[Medline] [Order article via Infotrieve]. 19. Sanjeevi CB, Miller EN, Dabadghao P, et al. Polymorphism at NRAMP1 and D2S1471 loci associated with juvenile rheumatoid arthritis. Arthritis Rheum. 2000;43:1397-1404[CrossRef][Medline] [Order article via Infotrieve].
20.
Shaw MA, Clayton D, Atkinson SE, et al.
Linkage of rheumatoid arthritis to the candidate gene NRAMP1 on 2q35.
J Med Genet.
1996;33:672-677 21. Hofmeister A, Neibergs HL, Pokorny RM, Galandiuk S. The natural resistance-associated macrophage protein gene is associated with Crohn's disease. Surgery. 1997;122:173-179[CrossRef][Medline] [Order article via Infotrieve].
22.
Searle S, Blackwell JM.
Evidence for a functional repeat polymorphism in the promoter of the human NRAMP1 gene that correlates with autoimmune versus infectious disease susceptibility.
J Med Genet.
1999;36:295-299
23.
Cellier M, Govoni G, Vidal S, et al.
Human natural resistance-associated macrophage protein: cDNA cloning, chromosomal mapping, genomic organization, and tissue-specific expression.
J Exp Med.
1994;180:1741-1752 24. Cellier M, Shustik C, Dalton W, et al. Expression of the human NRAMP1 gene in professional primary phagocytes: studies in blood cells and in HL-60 promyelocytic leukemia. J Leukoc Biol. 1997;61:96-105[Abstract]. 25. Smolen JE, Boxer LA. Functions of neutrophils. In: Beutler E,Lichtman MA,Collier BS,Kipps T,Seligsohn U, eds. Williams Hematology. New York, NY: McGraw Hill; 2001:761-784. 26. Borregaard N, Kjeldsen L, Lollike K, Sengelov H. Granules and secretory vesicles of the human neutrophil. Clin Exp Immunol. 1995;101(suppl 1):6-9[Medline] [Order article via Infotrieve].
27.
Maridonneau-Parini I, de Gunzburg J.
Association of rap1 and rap2 proteins with the specific granules of human neutrophils. Translocation to the plasma membrane during cell activation.
J Biol Chem.
1992;267:6396-6402 28. Sengelov H, Follin P, Kjeldsen L, Lollike K, Dahlgren C, Borregaard N. Mobilization of granules and secretory vesicles during in vivo exudation of human neutrophils. J Immunol. 1995;154:4157-4165[Abstract]. 29. Cowland JB, Borregaard N. The individual regulation of granule protein mRNA levels during neutrophil maturation explains the heterogeneity of neutrophil granules. J Leukoc Biol. 1999;66:989-995[Abstract]. 30. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77-89[Medline] [Order article via Infotrieve].
31.
Canonne-Hergaux F, Gruenheid S, Ponka P, Gros P.
Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron.
Blood.
1999;93:4406-4417 32. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159[Medline] [Order article via Infotrieve]. 33. Kjeldsen L, Sengelov H, Borregaard N. Subcellular fractionation of human neutrophils on Percoll density gradients. J Immunol Methods. 1999;232:131-143[CrossRef][Medline] [Order article via Infotrieve].
34.
Calafat J, Janssen H, Stahle-Backdahl M, Zuurbier AE, Knol EF, Egesten A.
Human monocytes and neutrophils store transforming growth factor-alpha in a subpopulation of cytoplasmic granules.
Blood.
1997;90:1255-1266 35. Liou W, Geuze HJ, Slot JW. Improving structural integrity of cryosections for immunogold labeling. Histochem Cell Biol. 1996;106:41-58[CrossRef][Medline] [Order article via Infotrieve].
36.
Slot JW, Geuze HJ, Gigengack S, Lienhard GE, James DE.
Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat.
J Cell Biol.
1991;113:123-135
37.
Cramer E, Pryzwansky KB, Villeval JL, Testa U, Breton-Gorius J.
Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold.
Blood.
1985;65:423-432 38. Borregaard N, Kjeldsen L, Lollike K, Sengelov H. Ca(2+)-dependent translocation of cytosolic proteins to isolated granule subpopulations and plasma membrane from human neutrophils. FEBS Lett. 1992;304:195-197[CrossRef][Medline] [Order article via Infotrieve].
39.
Kang T, Yi J, Guo A, et al.
Subcellular distribution and cytokine- and chemokine-regulated secretion of leukolysin/MT6-MMP/MMP-25 in neutrophils.
J Biol Chem.
2001;276:21960-21968 40. Fantone JC, Ward PA. Polymorphonuclear leukocyte-mediated cell and tissue injury: oxygen metabolites and their relations to human disease. Hum Pathol. 1985;16:973-978[Medline] [Order article via Infotrieve]. 41. Cellier M, Belouchi A, Gros P. Resistance to intracellular infections: comparative genomic analysis of Nramp. Trends Genet. 1996;12:201-204[CrossRef][Medline] [Order article via Infotrieve]. 42. Gunshin H, Mackenzie B, Berger UV, et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature. 1997;388:482-488[CrossRef][Medline] [Order article via Infotrieve].
43.
Forgac M.
Structure and properties of the vacuolar (H+)-ATPases.
J Biol Chem.
1999;274:12951-12954
© 2002 by The American Society of Hematology.
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  H.-R. Jiang, D. S. Gilchrist, J.-F. Popoff, S. E. Jamieson, M. Truscott, J. K. White, and J. M. Blackwell Influence of Slc11a1 (formerly Nramp1) on DSS-induced colitis in mice J. Leukoc. Biol., April 1, 2009; 85(4): 703 - 710. [Abstract] [Full Text] [PDF]
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 H. K. Bayele, C. Peyssonnaux, A. Giatromanolaki, W. W. Arrais-Silva, H. S. Mohamed, H. Collins, S. Giorgio, M. Koukourakis, R. S. Johnson, J. M. Blackwell, et al. HIF-1 regulates heritable variation and allele expression phenotypes of the macrophage immune response gene SLC11A1 from a Z-DNA forming microsatellite Blood, October 15, 2007; 110(8): 3039 - 3048. [Abstract] [Full Text] [PDF]
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 K. Turcotte, S. Gauthier, D. Malo, M. Tam, M. M. Stevenson, and P. Gros Icsbp1/IRF-8 Is Required for Innate and Adaptive Immune Responses against Intracellular Pathogens J. Immunol., August 15, 2007; 179(4): 2467 - 2476. [Abstract] [Full Text] [PDF]
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 H. J. Knowles, D. R. Mole, P. J. Ratcliffe, and A. L. Harris Normoxic Stabilization of Hypoxia-Inducible Factor-1{alpha} by Modulation of the Labile Iron Pool in Differentiating U937 Macrophages: Effect of Natural Resistance-Associated Macrophage Protein 1. Cancer Res., March 1, 2006; 66(5): 2600 - 2607. [Abstract] [Full Text] [PDF]
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A. Fortier, G. Min-Oo, J. Forbes, S. Lam-Yuk-Tseung, and P. Gros Single gene effects in mouse models of host: pathogen interactions J. Leukoc. Biol., June 1, 2005; 77(6): 868 - 877. [Abstract] [Full Text] [PDF]
|
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P. Massullo, L. J. Druhan, B. A. Bunnell, M. G. Hunter, J. M. Robinson, C. B. Marsh, and B. R. Avalos Aberrant subcellular targeting of the G185R neutrophil elastase mutant associated with severe congenital neutropenia induces premature apoptosis of differentiating promyelocytes Blood, May 1, 2005; 105(9): 3397 - 3404. [Abstract] [Full Text] [PDF]
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N. Borregaard, K. Theilgaard-Monch, J. B. Cowland, M. Stahle, and O. E. Sorensen Neutrophils and keratinocytes in innate immunity--cooperative actions to provide antimicrobial defense at the right time and place J. Leukoc. Biol., April 1, 2005; 77(4): 439 - 443. [Abstract] [Full Text] [PDF]
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K. Turcotte, S. Gauthier, L.-M. Mitsos, C. Shustik, N. G. Copeland, N. A. Jenkins, J.-C. Fournet, P. Jolicoeur, and P. Gros Genetic control of myeloproliferation in BXH-2 mice Blood, March 15, 2004; 103(6): 2343 - 2350. [Abstract] [Full Text] [PDF]
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J. R. Forbes and P. Gros Iron, manganese, and cobalt transport by Nramp1 (Slc11a1) and Nramp2 (Slc11a2) expressed at the plasma membrane Blood, September 1, 2003; 102(5): 1884 - 1892. [Abstract] [Full Text] [PDF]
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L. A. Guilloteau, J. Dornand, A. Gross, M. Olivier, F. Cortade, Y. L. Vern, and D. Kerboeuf Nramp1 Is Not a Major Determinant in the Control of Brucella melitensis Infection in Mice Infect. Immun., February 1, 2003; 71(2): 621 - 628. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
pH may provide a portion or the entire
electrochemical gradient required to energize transport. In the present
study, immunoblotting of granule populations purified from primary
neutrophils indicates that the NRAMP1-positive granule population is
also positive for the