Published online before print June 8, 2005
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* Departments of Hematology, The Granulocyte Research Laboratory, and
Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark;
Department of Cell Biology, The Netherlands Cancer Institute, Amsterdam; and
Dako-Cytomation, Glostrup, Denmark
1Correspondence: The Granulocyte Research Laboratory, Department of Hematology, Rigshospitalet 4042, DK-2100 Copenhagen, Denmark. E-mail: borregaard{at}rh.dk
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Key Words: proHNP • HNP • secondary granules • polymorphonuclear leukocytes
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-defensins [human neutrophil peptide (HNP)-1–4], which possess a broad range of microbicidal, cytotoxic, and proinflammatory properties [1
2
3
4
5
6
7
8
]. HNP-1–3 constitute >97% of the HNP content in neutrophils and are almost identical in amino acid (aa) sequence [2
]. Thus, HNP-1 and HNP-3 are 30 aa long and differ from each other only in the first aa, and HNP-2 is 29 aa in length and arises through amino-terminal trimming of HNP-1 and/or HNP-3. Production of neutrophil HNPs is initiated at the promyelocytic stage of myelopoiesis and continues at a high level in myelocytes, metamyelocytes, and band cells [9
, 10
]. HNP-1 and -3 are synthesized as 94 aa preproHNPs with identical pre- and prosequences [11
, 12
]. The preproHNPs are converted to proHNPs by removal of a 19 aa endoplasmic reticulum (ER) signal peptide. In promyelocytes, the anionic 45 aa pro-piece is subsequently cleaved off, and the mature HNPs accumulate in azurophil granules [13
14
15
]. At this stage of myeloid cell development, sorting of HNPs to the granule compartment proceeds with great efficiency, and only minimal amounts of newly synthesized, unprocessed proHNPs escape along the constitutive, exocytic pathway [9
]. In contrast, proHNPs formed at later stages of myelopoiesis are not subjected to proteolytic processing and undergo a high degree of constitutive exocytosis in their full-length, 75 aa form [9
, 13
]. Studies of HNP biosynthesis have, however, indicated that not all proHNPs produced by myelocytes and metamyelocytes are routed out of the cells [9
]. Furthermore, small amounts of 75 aa proHNPs have been detected in crude homogenates of peripheral blood neutrophils [16
]. Together, these findings suggest that mature neutrophils not only store HNPs but also contain a reservoir of proHNPs. The present study was undertaken to explore this possibility. |
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For bacterial expression of proHNP-1, the cDNA sequence encoding proHNP-1 was amplified from a human bone marrow cDNA library using the primers 5'-CGAGGCCTCTCATCATCATCATCATCATGAGCCACTCCAGGCAAGAG-3', encoding a polyhistidine tag (underlined) and a StuI site at the 5' end of the proHNP gene, and 5'-GCAAGCTTGAGACCTCAGCAGCAGAATGCCCAG-3', containing a stop codon (bold), a HindIII site, and a BsaI site. The PCR product was restricted with StuI and BsaI and cloned into pRBI-DsbA (a kind gift from Dr. Rudi Glockshuber, Institute for Molecular Biology and Biophysics Zürich, Switzerland), restricted with HindIII and StuI. The pRBI-DsbA vector is designed for periplasmic coexpression of recombinant proteins and DsbA, the main thiol/disulfide isomerase of E. coli [17 ]. The plasmid was transformed into XL1-Blue and purified for sequencing as above.
The correctness of the cloned PCR fragments was assured by dideoxy dye terminator sequencing with AmpliTaq® DNA polymerase, FS (Perkin-Elmer, Applied Biosystems Division, Foster City, CA).
Purification of rproHNP-1 and neutrophil HNPs
Transfected High FiveTM cells were grown as a monolayer at 27°C in Express Five® serum-free medium (Invitrogen) with 10 µg/mL gentamycin and passaged at confluence. The supernatant containing secreted proHNP-1 was harvested every 2–3 days, and rproHNP-1 was purified using TalonTM metal affinity resin (Clontech), according to the protocol supplied by the manufacturer.
The recombinant E. coli strain expressing proHNP-1 was inoculated into 100 mL Luria-Bertani (LB) medium (1% NaCl, 1% tryptone, 0.5% yeast extract, pH 7.5; Bie & Berntsen, Rødovre, Denmark) with 0.1 mg/mL ampicillin and shaken overnight at 37°C. The suspension was diluted into 900 mL LB medium and shaken for 2 h at 25°C. After addition of isopropyl ß-D-thiogalactopyranoside to a final concentration of 1 mM, the cells were grown for an additional 6 h at 25°C and harvested by centrifugation. rproHNP-1 was extracted from the bacterial periplasm under nondenaturing conditions, essentially as described [18 ]. In brief, the bacteria were resuspended in 20 mL extraction buffer [50 mM Tris/HCl, 150 mM NaCl, 5 mM EDTA (Sigma Chemical Co., St. Louis, MO), 1 mg/mL polymyxin B (Sigma Chemical Co.), pH 7.5] and stirred for 2 h at 4°C. Following centrifugation, the supernatant was dialyzed into EDTA-free buffer (50 mM NaH2PO4, 10 mM Tris-HCl, 100 mM NaCl, pH 8.0), and rproHNP-1 was purified using TalonTM metal affinity resin.
Mature HNPs were isolated from peripheral blood neutrophils as described [15 ].
Synthesis of the 45-aa pro-piece of proHNP-1-3
The pro-piece of proHNP-1–3 (=residues 1–45 of proHNP-1–3) was purchased from Schafer-N (Copenhagen, Denmark). The peptide was synthesized using the Fmoc [N-(9-fluorenyl)methoxycarbonyl] strategy, purified by reverse-phase chromatography, and analyzed by high-pressure liquid chromatography coupled to a Shimadzu LCMS-QP8000 spectrometer. Furthermore, the identity of the peptide was verified by mass spectrometry (MS) and N-terminal aa sequence analysis as described below. The purity of the peptide was greater than 95%.
Peptide identification by MS and N-terminal sequence analysis
For verification of the identity of the insect cell-derived rproHNP-1, the synthetic proHNP pro-piece, and the endogenous proHNPs, gel pieces containing the purified proteins were reduced, alkylated using iodoacetamide, and digested by trypsin, and the resulting fragments were extracted as described [19
]. The extracts were loaded onto a C18 ZipTip (Millipore, Bedford, MA), washed with 0.5% formic acid, and eluted with 10 µL 0.5% formic acid in 50% acetonitrile. Aliquots of 0.5 µL of the purified peptide mixtures were analyzed in a Biflex matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) mass spectrometer (Bruker-Franzen, Bremen, Germany) using -cyano-4-hydroxycinnamic acid as matrix. For N-terminal aa sequence analysis, the peptide of interest was blotted onto a ProBlott polyvinylidene difluoride membrane (Applied Biosystems) and analyzed for 10 residues in a 494A Procise protein sequencer (Perkin-Elmer, Palo Alto, CA). Perkin-Elmer supplied all reagents and solvents.
Isolation of human neutrophils
Human neutrophils were isolated from buffy coats supplied by the hospital blood transfusion service. Erythrocytes were sedimented for 45 min by addition of an equal volume of 2% Dextran T-500 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) in 0.9% saline. The leukocyte-rich supernatant was aspirated, and the cells were pelleted by centrifugation at 200 g for 10 min. After resuspension in 0.9% saline, the cells were centrifuged through Lymphoprep (Nycomed Pharma AS, Oslo, Norway) at 400 g for 30 min to remove mononuclear cells. Remaining erythrocytes were lysed by hypotonic shock in ice-cold water for 30 s followed by restoration of tonicity with 1.8% saline. The cells were washed in saline and resuspended in the desired buffer. All steps except Dextran sedimentation were performed at 4°C.
Subcellular fractionation
Isolated neutrophils were resuspended at 3 x 107 cells/mL in 0.9% saline, incubated for 5 min with 5 mM diisopropylfluorophosphate (Aldrich Chemical Company, Milwaukee, WI), and centrifuged at 200 g for 10 min. The pelleted cells were resuspended at 3 x 107 cells/mL in disruption buffer {100 mM KCl, 3 mM NaCl, 1 mM Na2adenosine 5'-triphosphate, 3.5 mM MgCl2, 10 mM piperazine-N,N'-bis[2-ethanesulfonic acid] (PIPES), pH 7.2}, containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF; Sigma Chemical Co.), and disrupted by nitrogen cavitation (pressurized for 5 min) as described [20
]. Nuclei and intact cells were pelleted by centrifugation at 400 g for 15 min. Postnuclear supernatant (S1; 10 mL) was applied on top of a 2 x 14-mL two-layer Percoll (Amersham Pharmacia Biotech AB) gradient (1.05/1.12 g/mL) with 0.5 mM PMSF and centrifuged for 30 min at 37,000 g. This resulted in a gradient with three visible bands: the bottom band (-band), containing the azurophil granules; the intermediate band (ß-band), containing the peroxidase-negative granules, and the top band (
-band), containing the plasma membrane, secretory vesicles, and light membranes of the Golgi apparatus and ER [21
]. In some experiments, the S1 was applied on top of a three-layer Percoll gradient (1.05/1.09/1.12 g/mL) [22
] resulting in a gradient with four distinct bands: the -band, containing azurophil granules; the ß1-band, containing specific granules; the ß2-band, containing gelatinase granules; and the -band, containing the same organelles as in the two-layer gradient. The bands were harvested manually or aspirated from the bottom of the tube in fractions of 1 mL. Prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), gel filtration, and affinity chromatography, the Percoll was removed from the samples by ultracentrifugation, and the granules were disrupted in lysis buffer [phosphate-buffered saline (PBS) containing 1% Triton X-100 (Roche Diagnostics, Nutley, NJ), 3 mM EDTA, 200 U/mL aprotinin (Bayer, Leverkusen, Germany), 1.0 mM PMSF, and 100 µg/mL leupeptin (Sigma Chemical Co.)]. The granule membranes were subsequently pelleted by centrifugation, and the supernatants, containing the granule matrix proteins, were stored at –20°C until use.
Exocytosis
Isolated neutrophils were resuspended in Krebs-Ringer phosphate (KRP; 130 mM NaCl, 5 mM KCl, 0.95 mM CaCl2, 1.27 mM MgSO4, 5 mM glucose, 10 mM NaH2PO4/Na2HPO4, pH 7.4) at 5 x 107 cells/mL. Following a 5-min preincubation period at 37°C, 1 mL cells were stimulated with 1.0 µM ionomycin (Calbiochem, La Jolla, CA) or 2.5 µg/mL phorbol myristate acetate (PMA; Sigma Chemical Co.) and incubated at 37°C for 15 min. Stimulation was terminated by dilution with 1 vol ice-cold KRP and centrifugation at 200 g for 6 min. Control cells were kept on ice until dilution in KRP. The supernatant [exocytosed material from control cells (CSo), exocytosed material from cells stimulated with 1.0 µM ionomycin (ISo), exocytosed material from cells stimulated with 2.5 µg/mL PMA (PMASo)] was aspirated, and the pellet was resuspended in 1 mL KRP. Release of granule marker proteins was determined by enzyme-linked immunosorbent assay (ELISA) and calculated as amount in supernatant/(amount in supernatant+pellet). The release of proHNPs and HNPs was analyzed by immunoblotting as described below. Following stimulation with ionomycin, a portion of the supernatant was incubated at 37°C for 240 min before dilution in SDS-PAGE sample buffer (IS240).
Generation of antibodies
Antibodies against the proHNP-1 holo peptide were generated by DNA immunization of rabbits. First, the cDNA sequence of preproHNP-1 was amplified from a human bone marrow cDNA library using the primers 5'-GCGGGATCCGCCACCATGAGGACCCTCGCCATCC-3' containing a BamHI site and 5'-GCGTCTAGATCAGCAGCAGAATGCCCAG-3' containing an XbaI site. The PCR product was digested with BamHI and XbaI and cloned into the mammalian expression vector pcDNA3.1/Zeo (Invitrogen) under the control of the cytomegalovirus immediate-early promoter. The plasmid was transformed into E. coli XL1-Blue cells and purified through large-scale plasmid preparation using the Qiagen plasmid giga kit. Plasmid DNA was dissolved in sterile PBS at 2.5 mg/mL and stored at – 20°C until use.
Immunization of rabbits was carried out at The Animal Facilities, Department of Experimental Medicine, The Panum Institute, University of Copenhagen, Denmark. Preimmune serum was collected on the day of the first injection. Rabbits received intramuscular injection of 1.0 mg DNA every 4 weeks for 28 weeks, and serum was collected 3 weeks after each injection. The immunoglobulin G (IgG) fraction of the anti-proHNP-1 antiserum was isolated on a HiTrapTM Protein A column (Amersham Pharmacia Biotech AB). The antibodies were eluted with 3 M potassium thiocyanate and immediately dialyzed into PBS. A portion of the IgG was biotinylated as described [23 ] followed by dialysis into PBS. All antibodies were stored at 4°C with 0.1% azide protected from light.
Polyclonal anti-pro-piece antibodies were generated at Dako-Cytomation (Glostrup, Denmark). Rabbits received repetitive subcutaneous injections (four times at 2-week intervals and thereafter monthly) of a 200 µg synthetic proHNP pro-piece dissolved in sterile PBS (pH 7.0). The IgG fraction of the obtained anti-pro-piece antiserum was isolated and stored as described above.
Polyclonal anti-HNP-1–3 antibodies were raised against purified HNP-1–3 as described previously [15 ]. All needle injections and serum collections were performed according to institutional guidelines for animal care and handling.
ELISA procedures
The generated anti-proHNP-1 antibodies were used to establish a semiquantitative proHNP/HNP ELISA using 96-well flat-bottom immunoplates (Nunc, Roskilde, Denmark). The procedure was as follows: Plates were coated overnight with anti-proHNP-1 IgG, diluted 1/200 in carbonate buffer (50 mM Na2CO3/NaHCO3, pH 9.6); additional binding sites were blocked by incubation for 1 h with 200 µL/well buffer A [0.5 M NaCl, 3 mM KCl, 8 mM Na2HPO4/KH2PO4, 1% bovine serum albumin (BSA), 1% Triton X-100, pH 7.2]; samples and standards (purified neutrophil HNPs ranging from 12.5 to 800 ng/mL) were applied; biotinylated antibodies, diluted 1/100, were applied; avidin-peroxidase (Dako P347, Dako A/S, Glostrup, Denmark), diluted 1/1000, was added; color was developed by a 30-min incubation period in buffer B (0.1 M sodium phosphate/0.1 M citric acid buffer, pH 5.0) containing 0.04% o-phenylenediamine (Kem-En-Tech, Copenhagen, Denmark) and 0.03% H2O2 and stopped by addition of 100 µL/well 1 M H2SO4. The plates were washed three times in buffer C (0.5 M NaCl, 3 mM KCl, 8 mM Na2HPO4/KH2PO4, 1% Triton X-100, pH 7.2) between each step. Before development of color, an additional wash in buffer B was included. All incubations were performed at room temperature for 1 h after addition of 100 µL sample/well. Samples and antibodies were diluted in buffer A. Absorbance was read at 492 nm in a multiscan ascent ELISA reader (Labsystems, Helsinki, Finland). ELISAs for myeloperoxidase (MPO), lactoferrin, neutrophil gelatinase-associated lipocalin (NGAL), gelatinase, and albumin were performed as reported previously [24
25
26
27
28
].
SDS-PAGE and immunoblotting
SDS-PAGE and immunoblotting were performed according to standard procedures [29
, 30
]. Samples were reduced with mercaptoethanol. For immunoblotting, ImmobilonTM-PSQ transfer membranes (Millipore) were blocked for 1 h with 5% skimmed milk in PBS after transfer of the proteins from 14% polyacrylamide gels. The membranes were incubated overnight with anti-proHNP-1 antiserum (diluted 1/200), anti-pro-piece antiserum (diluted 1/200), or a mixture of anti-pro-piece IgG (diluted 1/200) and anti-HNP-1-3 IgG (diluted 1/500) in PBS containing 0.5% BSA. Subsequently, the membranes were incubated for 2 h with horseradish peroxidase (HRP)-conjugated porcine anti-rabbit Ig (Dako P217), diluted 1/1000, and color was developed using diaminobenzidine tetrachloride metal concentrate and stable peroxide substrate buffer (Pierce, Rockford, IL). Anti-His-tag immunoblotting was performed using a murine 6 x His monoclonal antibody (mAb; BD Biosciences, Palo Alto, CA), diluted 1/500, as primary antibody and HRP-conjugated rabbit-anti-mouse Ig (Dako P260), diluted 1/1000, as secondary antibody.
Affinity chromatography
Purified anti-pro-piece antibodies were immobilized on cyanogen bromide-activated Sepharose (Amersham Pharmacia Biotech AB) and packed into an anti-pro-piece column according to the instructions supplied by the manufacturer. A neutrophil ß-band (from 4x109 cells) was disrupted in lysis buffer with 0.5 M NaCl, and the material was applied to the column at 4°C. After extensive washing in the same buffer, bound proteins were eluted with glycin-HCl (0.2 M, pH 3.0) and neutralized by addition of 2.0 M Tris-HCl (pH 8.0). The eluted proteins were subjected to SDS-PAGE, as described, and their identity was determined by MS and N-terminal aa sequence analysis.
Immunoelectron microscopy
Isolated neutrophils were fixed for 24 h in 4% paraformaldehyde in 0.1 M PHEM buffer (120 mM PIPES, 50 mM HEPES, 8 mM MgCl2, 40 mM EGTA, pH 6.9) and subsequently processed for ultrathin cryosections as described [31
]. Cryosections of 45 nm were cut at –125°C using 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 [32
]. The grids were placed on 35 nm petri dishes containing 2% gelatine. For simple immunolabeling, the sections were incubated with anti-pro-piece IgG for 45 min, followed by 30 min incubation with protein A-conjugated colloidal gold (EM Lab, Utrecht University, The Netherlands). For double-immunolabeling, the procedure described by Slot et al. [33
] was followed with 10 and 15 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). Rabbit anti-human lactoferrin antibodies (Cappel Laboratories, Cochranville, PA) or mouse monoclonal anti-human MPO antibodies (CLB, Amsterdam, The Netherlands) were used in the double-immunolabeling studies. In control experiments, the primary antibody was substituted with a nonrelevant rabbit antibody.
Size-exclusion chromatography
Gel filtrations were performed using a Superose 12 column HR 10/30 (Amersham Pharmacia Biotech AB) equilibrated in PBS, pH 7.4, and ÄKTA-FPLC (Pharmacia, Uppsala, Sweden). Samples of 200 µL were applied to the column, and 60 fractions of 0.5 mL were collected and analyzed for their content of selected proteins/peptides.
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14 kDa in 14% SDS polyacrylamide gels under reducing conditions. |
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Table 1. Tryptic Fragments of Purified Insect Cell-Derived, His-Tagged rproHNP-1 and Endogenous proHNPs
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Figure 1. Detection of rproHNP-1 with anti-His-tag antibodies and anti-proHNP-1 antibodies in Western blotting. (A) Insect cell-derived rproHNP-1 visualized with 6 x His mAb. (B) E. coli-derived rproHNP-1 visualized with 6 x His mAb. (C) Recognition of purified neutrophil HNP-1–3 by the generated anti-proHNP-1 antibodies. (D) Recognition of insect cell-derived rproHNP-1 by the anti-proHNP-1 antibodies. The anti-proHNP-1 antibodies did not react with culture medium from nontransfected insect cells or lysates of nontransformed E. coli (data not shown).
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Figure 2. Size-exclusion chromatography. Samples of 200 µL were loaded onto a Superose 12 column equilibrated in PBS, and the fractions obtained were analyzed for marker proteins and proHNP-1 in ELISA. (A) Albumin (Sigma A-1887;  ), recombinant NGAL [27
] ( ), purified, endogenous HNP-1–3 ( ), and endogenous HNP-1–3 in a detergent-solubilized -band (from 1x109 neutrophils;  ) all resuspended in PBS. The proteins were subjected to gel filtration separately. (B) Purified insect cell-derived rproHNP-1 in PBS. (C) ProHNP-1 in a solubilized ß-band (from 1x109 neutrophils). (D) ProHNP-1 in exocytosed material from PMA-stimulated neutrophils.
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3.5 kDa HNPs. In neutrophils fractionated on a two-layer Percoll density gradient, the mature HNPs were found in the -band, as expected, and the putative proHNPs were found predominantly in the ß-band (Fig. 3C 3D
3E)
. The matrix proteins of a ß-band were subsequently subjected to size-exclusion chromatography using a Superose 12 column equilibrated in PBS. Under such nondenaturing conditions, the presumed neutrophil proHNPs in their "native" form displayed the same retention time as rproHNP-1 (Fig. 2B
and 2C)
. To further validate the identity of the neutrophil proHNPs, immunoblotting experiments were repeated using anti-pro-piece antibodies (Fig. 4
). These antibodies, like the anti-proHNP-1 antibodies, recognized a peptide migrating as rproHNP-1, when a homogenate of neutrophils was subjected to SDS-PAGE and immunoblotting (Fig. 4A
and 4B)
. This peptide was found in the ß-band of neutrophils fractionated on a two-layer Percoll density gradient (Fig. 4C
4D
4E)
. These results confirmed and strengthened the findings obtained with the anti-proHNP-1 antibodies. The proHNPs were finally affinity-purified from a solubilized ß-band on an anti-pro-piece antibody column. The identity of the purified peptides was confirmed by MALDI-TOF MS/Q-TOF tandem MS (Table 1) and N-terminal sequence analysis, which showed the previously reported N-terminal aa sequence of proHNP-1/3: EPLQARADEV [11
, 12
].
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Figure 3. Visualization of HNPs and proHNPs in neutrophils using anti-proHNP-1 antibodies. (A) Immunoblotting with preimmune serum of a neutrophil homogenate (1x108 cells/mL). (B) Immunoblotting with anti-proHNP-1 antibodies of the same sample. (C) Immunoblotting with anti-proHNP-1 antibodies of a neutrophil -band (from 1x109 cells). (D and E) Immunoblotting with anti-proHNP-1 antibodies of the corresponding ß- and -bands, respectively.
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Figure 4. Detection of rproHNP-1 and neutrophil proHNPs with anti-pro-piece antibodies. (A) Immunoblotting of insect cell-derived rproHNP-1. (B) Immunoblotting of a neutrophil homogenate (1x108 cells/mL). (C) Immunoblotting of a neutrophil -band (from 1x109 cells). (D and E) Immunoblotting of the corresponding ß- and -bands, respectively.
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Figure 5. Distribution profile of HNPs and proHNPs in neutrophils. Cells (3x108) were subjected to subcellular fractionation on a three-layer Percoll gradient. Fractions of 1 mL were aspirated from the bottom of the tube and analyzed for the following marker proteins in ELISA: MPO (azurophil granules, µg/mL), lactoferrin (LF; specific granules, µg/mL), gelatinase (gelatinase granules, µg/mLx10), albumin (secretory vesicles, ng/mLx1/10). The lower panel shows immunoblotting of the subcellular fractions with anti-proHNP-1 antibodies. No reactivity was observed in fractions 17–34.
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Figure 6. ProHNPs and HNPs in exocytosed material from neutrophils stimulated with ionomycin or PMA. After stimulation, the exocytosed material was analyzed by SDS-PAGE and immunoblotting with a mixture of anti-pro-piece antibodies and anti-HNP1–3 antibodies. For comparison, the percentage exocytosis of MPO (solid bars), lactoferrin (open bars), and gelatinase (shaded bars) in response to the same stimuli is presented in the lower panel (ELISA results).
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Figure 7. Specific detection of the 45-aa proHNP pro-piece in neutrophils. (A) Cryosections of intact neutrophils were labeled with purified anti-pro-piece IgG followed by protein A gold (10 nm). An area of a neutrophil shows anti-pro-piece labeling on the matrix of granules (arrows). (B) Double-labeling with anti-pro-piece antibodies and anti-lactoferrin antibodies. Cryosections of intact neutrophils were labeled with anti-pro-piece antibodies followed by protein A gold [pd, 10 nm (pd10)]. Subsequently, the sections were labeled with anti-lactoferrin antibodies followed by protein A gold [lf, 15 nm (If15)]. Anti-pro-piece labeling is observed in several lactoferrin-positive granules (arrows), i.e. specific granules. (C) Double-labeling with anti-MPO antibodies [MPO, 10 nm (MPO10)] and anti-pro-piece antibodies (pd15). No anti-pro-piece labeling is observed in MPO-positive (azurophil) granules.
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To estimate the content of HNPs and proHNPs in neutrophils, we performed repeated ELISA measurements on neutrophil homogenates; on -bands, containing the mature HNPs of azurophil granules (Figs. 2
and 3)
; on ß-bands, containing the unprocessed proHNPs of specific granules (Figs. 2
3
4)
; and on -bands, containing the plasma membrane, secretory vesicles, and light membranes of the ER and Golgi apparatus [21
]. By this approach, we found a mean total HNP/proHNP concentration in neutrophils of 4.9 µg HNP/proHNP ±1.05 (SD) per 106 neurophils (n=3), a mean concentration of HNPs in the -band of 4.5 µg HNP ± 1.50 (SD) per 106 neurophils (n=3), and a mean concentration of proHNPs in the ß-band of 0.26 µg proHNPs ± 0.07 (SD) per 106 neutrophils (n=3). No HNP/proHNP was detected in the -band. Thus, our results indicate that mature neutrophils harbor 4.5 µg mature HNPs, stored within azurophil granules, and 0.26 µg unprocessed proHNPs, contained mainly within specific granules. The estimated total HNP/proHNP content of 4.9 µg per 106 cells is in agreement with earlier calculations [37
]. Furthermore, in accordance with previous reports [16
], our findings indicate a high intracellular HNP/proHNP ratio.
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-defensins were among the first antimicrobial peptides of innate immunity to be discovered and characterized. Six -defensins are currently known in man: HNP-1–4 of the neutrophilic granulocyte and human defensin (HD)-5 and -6 of intestinal Paneth cells [38
]. The -defensins are polycationic as a result of a high content of arginine residues and share a conserved cysteine framework, which stabilizes a characteristic triple-stranded ß-sheet motif through three disulfide bridges. They probably exert their antimicrobial activities by formation of multimeric, transmembrane pores in targeted bacteria, enveloped viruses, and fungi [39
].
In contrast to mature HNPs, the much larger proHNPs do not interact with membrane bilayers and are devoid of antimicrobial and cytotoxic potential [40
, 41
]. Structural analyses of various mammalian prodefensins have indicated that the anionic pro-pieces generally charge-neutralize the cationicity of the mature peptides [42
]. Furthermore, a recent study of murine -defensins (termed cryptdins) has provided evidence that the ß-sheet structure of native cryptdin4 is destabilized in pro-cryptdin4 or after in vitro incubation of mature cryptdin4 with its pro-piece [43
]. Together, these findings suggest that the pro segments interfere with the ability of mature -defensins to permeabilize membranes by balancing their charge and attenuating their ß-sheet topology. In human promyelocytes, this interference is believed to prevent the mature HNPs from interacting with the membranes of the ER and Golgi apparatus as they travel toward azurophil granules [44
]. However, the biological significance of the neutrophil proHNPs after secretion from myelocytes, metamyelocytes, and band cells is unknown.
As earlier studies had indicated the presence of proHNPs in peripheral blood neutrophils [16 ], we decided to determine the subcellular localization of the neutrophil proHNPs. Antibodies against the entire proHNP-1 sequence were generated by DNA vaccination of rabbits, and antibodies against the 45 aa proHNP pro-piece were raised by conventional immunization procedures. In combination, these antibodies allowed us to confirm the presence of unprocessed proHNPs in human neutrophils. The neutrophil proHNPs were found to colocalize and comobilize with the specific granule protein lactoferrin in subcellular fractionation experiments and release studies, respectively, and their localization in specific granules was validated through immunoelectron microscopy. Thus, human neutrophils not only store large amounts of processed 29–39 aa HNPs in their azurophil granules but also contain a more easily mobilized reservoir of unprocessed proHNPs.
We have previously demonstrated that the biosynthetic window of several granule proteins corresponds to the timing of their mRNA expression. Furthermore, we have shown that granule proteins synthesized at a given stage of myelopoiesis are sorted to granules formed concomitantly [10 , 45 46 47 ]. These and other observations have given rise to the targeting-by-timing hypothesis of granulogenesis, which states that granule proteins simply fill into granules as they are produced [45 , 48 ]. In agreement with this hypothesis, we here show that neutrophil defensins, produced primarily by promyelocytes and myelocytes [10 , 47 ], are packaged in granules formed at these stages of myelopoiesis, i.e., azurophil granules and specific granules [49 ]. Clearly, the present study does not in any way prove the correctness of the targeting-by-timing hypothesis, and we cannot formally rule out the possibility that proHNPs synthesized by band cells or mature neutrophils contribute to the pool of proHNPs contained within specific granules. We have, however, previously demonstrated that large amounts of proHNPs are synthesized by promyelocytes, myelocytes, and metamyelocytes, while band cells and mature neutrophils produce small amounts of proHNPs [9 , 10 ]. Furthermore, myelocytes and metamyelocytes retain a substantial proportion of newly synthesized proHNPs [9 ]. We therefore find it most probable that the proHNPs of specific granules were sorted for storage in these granules at the myelocytic/metamyelocytic stage of myelopoiesis.
As shown in Figure 5 , mature HNPs are found exclusively in high-density azurophil granules. Moreover, our N-terminal sequence analysis revealed that the proHNPs of specific granules are completely unprocessed. These findings indicate that the proHNP-processing enzyme is not active beyond the promyelocyte/myelocyte transition point. Thus, whereas the genes encoding the proHNPs are expressed by promyelocytes, myelocytes, metamyelocytes, band cells, and mature neutrophils [10 , 11 , 16 , 47 ], transcription of the gene encoding this as-yet unidentified protease probably stops as myeloid cells progress from the promyelocytic to the myelocytic stage [50 ]. Furthermore, the high intracellular HNP/proHNP ratio of mature neutrophils and the incomplete retention of proHNPs in myelocytes and metamyelocytes suggest that the cationic HNPs are targeted to granules with greater efficiency than the slightly anionic proHNPs [9 , 16 ]. Whether this is a consequence of interactions between cationic regions of the HNPs and anionic residues in the granule lining (e.g., proteoglycans) remains to be determined.
The localization of proHNPs in specific granules implies that these peptides, in contrast to the mature HNPs, are subjected to a high degree of regulated secretion during the neutrophil-mediated inflammatory response [51
]. Moreover, our release experiments indicate that the proHNPs are not rapidly converted to mature HNPs by neutrophil proteases following exocytosis (Figs. 2D
and 6) . This observation raises the possibility that the neutrophil proHNPs may be involved in yet-uncharacterized biological processes in their full-length, 75-aa form. Recently, the pro form of the intestinal -defensin HD-5 was shown to possess significant antimicrobial activity against Listeria monocytogenes and Salmonella typhimurium [41
]. This finding clearly demonstrates that at least some human prodefensins should not be regarded simply as inert defensin precursors. However, further studies will be needed to determine whether the proHNPs, like their intestinal counterparts, are capable of executing independent, biological functions following secretion from myeloid cells.
Received November 25, 2004; revised May 2, 2005; accepted May 9, 2005.
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in a subpopulation of cytoplasmic granules Blood 90,1255-1266-defensins and -defensin precursors with membranes. Prosegment inhibition of peptide association with biomimetic membranes J. Biol. Chem. 278,13838-13846This article has been cited by other articles:
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  C. L. Wilson, A. P. Schmidt, E. Pirila, E. V. Valore, N. Ferri, T. Sorsa, T. Ganz, and W. C. Parks Differential Processing of {alpha}- and {beta}-Defensin Precursors by Matrix Metalloproteinase-7 (MMP-7) J. Biol. Chem., March 27, 2009; 284(13): 8301 - 8311. [Abstract] [Full Text] [PDF]
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