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Originally published online as doi:10.1189/jlb.0105042 on June 7, 2005

Published online before print June 7, 2005
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(Journal of Leukocyte Biology. 2005;78:462-470.)
© 2005 by Society for Leukocyte Biology

Highly glycosylated {alpha}1-acid glycoprotein is synthesized in myelocytes, stored in secondary granules, and released by activated neutrophils

Kim Theilgaard-Mönch*, Lars C. Jacobsen*, Thomas Rasmussen{dagger}, Carsten U. Niemann*, Lene Udby*, Rehannah Borup{ddagger}, Maged Gharib§, Peter D. Arkwright, Adrian F. Gombart||, Jero Calafat**, Bo T. Porse{ddagger} and Niels Borregaard*,1

* The Granulocyte Research Laboratory, Department of Hematology, and
{ddagger} Section of Gene Therapy Research, Department of Clinical Biochemistry, Rigshospitalet, and
{dagger} Department of Hematology, Herlev Hospital, University of Copenhagen, Denmark,
§ Department of Hematology, Royal Manchester Children’s Hospital, United Kingdom;
Booth Hall Children’s Hospital, University of Manchester, United Kingdom;
|| Division of Hematology/Oncology, Cedars-Sinai Medical Center, Burns and Allan Research Institute, University of California, Los Angeles, School of Medicine; and
** Department of Cell Biology, The Netherlands Cancer Institute, Amsterdam

1 Correspondence: Department of Hematology-4042, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, 2100 Copenhagen-Ø, Denmark. E-mail: borregaard{at}rh.dk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{alpha}-1-Acid glycoprotein (AGP) is an acute-phase protein produced by hepatocytes and secreted into plasma in response to infection/injury. We recently assessed the transcriptional program of terminal granulocytic differentiation by microarray analysis of bone marrow (BM) populations highly enriched in promyelocytes, myelocytes/metamyelocytes (MYs), and BM neutrophils. These analyses demonstrated a transient, high mRNA expression of genuine secondary/tertiary granule proteins and AGP in MYs. In agreement with this, immunocytochemistry revealed the presence of AGP protein and the secondary granule protein lactoferrin in cells from the MY stage and throughout granulocytic differentiation. Immunoelectron microscopy demonstrated the colocalization of AGP and lactoferrin in secondary granules of neutrophils. This finding was substantiated by the failure to detect AGP and lactoferrin in blood cells from a patient with secondary/tertiary (specific) granule deficiency. In addition, Western blot analysis of subcellular fractions isolated from neutrophils revealed that neutrophil-derived AGP, localized in secondary granules, was abundant and highly glycosylated compared with endocytosed, plasma-derived AGP localized in secretory vesicles. Exocytosis studies further demonstrated a marked release of AGP and lactoferrin by activated neutrophils. Finally, induction of CCAAT/enhancer-binding protein (C/EBP)-{varepsilon} in a myeloid cell line was shown to increase AGP transcript levels, indicating that AGP expression in myeloid cells, like in hepatocytes, is partially regulated by members of the C/EBP family. Overall, these findings define AGP as a genuine secondary granule protein of neutrophils. Hence, neutrophils, which constitute the first line of defense, are likely to serve as the primary local source of AGP at sites of infection or injury.

Key Words: granulocytes • granule proteins • specific granule deficiency • acute-phase proteins • C/EBP{varepsilon}


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are essential players in innate immune defense of mammalian hosts against microorganisms. Within minutes of injury, neutrophils migrate to sites of infection and initiate a first line of defense using a number of distinct mechanisms. These defense mechanisms include phagocytosis of microorganisms, release of reactive oxygen intermediates (ROIs), as well as release of proteases and antimicrobial proteins stored in granules [1 , 2 ]. De novo synthesis of chemokines and cytokines, which recruit additional effector cells (monocytes, T cells, more neutrophils) and regulate their inflammatory response, constitutes another defense mechanism of neutrophils [3 ].

Proinflammatory cytokines generated by leukocytes and stromal cells in response to infections or injury trigger a systemic acute-phase reaction, which is characterized by a marked increase of a variety of plasma proteins synthesized in the liver. These plasma proteins, which collectively are termed acute-phase proteins (APPs), are considered essential for the re-establishment of systemic homeostasis and can be subcategorized according to their key functions including (1) coagulation and fibrinolysis, (2) protease inhibition, (3) clearance of toxic substances, and (4) modulation of the immune response [4 ].

{alpha}-1-Acid glycoprotein (AGP), also known as orosomucoid, is an APP and a member of the lipocalin protein family. Being a lipocalin, AGP contains a conserved binding site for the transport of hydrophobic ligands [5 ]. Another characteristic of AGP is its high level of glycosylation, which along with the hydrophobic binding site, is thought to mediate various immunomodulatory effects in vitro and in vivo.

AGP has been shown to modulate the immune response of neutrophils by inhibiting their migration and generation of ROIs [6 , 7 ]. In addition, AGP has been demonstrated to inhibit lymphocyte-mediated lysis of allogeneic target cells and to antagonize the mitogenic response of lymphocytes to lipopolysaccharide (LPS), phytohemagglutinin, and alloantigens [8 , 9 ]. Other reported functions of AGP include the induction of pro- and anti-inflammatory cytokine production by mononuclear cells [10 , 11 ], the inhibition of platelet aggregation [12 ], and the formation of complexes with LPS, resulting in neutralization and enhanced clearance of LPS [13 ]. In vivo administration of AGP has further been demonstrated to protect animals against lethal challenge with Klebsiella pneumoniae. This protective effect of AGP was a result of a reduced bacterial spread or a higher bacterial clearance, as animals treated with AGP demonstrated a significantly lower bacterial load in blood and a variety of organs [14 ]. Two other animal models of septic shock have shown that AGP administration maintained perfusion of vital organs by reducing capillary leakage [15 , 16 ]. Hence, the net effect of AGP is the re-establishment of systemic homeostasis following infections by propagating various anti-inflammatory activities and nonspecific bacterial resistance.

At present, AGP is mostly thought of as an APP synthesized by the liver. However, recent data have demonstrated inducible AGP expression by type II alveolar epithelial cells and alveolar macrophages [17 , 18 ]. These findings indicate that AGP not only modulates the immune response systemically but also locally.

We provide evidence that AGP is a genuine secondary granule protein of neutrophils, which is exocytosed immediately in response to activation. As neutrophils constitute a major first line of defense at sites of infection/injury, we propose that neutrophil-derived AGP exerts immunomodulatory activities locally during the initial phase of the immune response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell isolation
Polymorphonuclear neutrophils were isolated from freshly collected peripheral blood (pb-PMNs) or buffy coats by density centrifugation and subsequent hypotonic lysis of erythrocytes, as described previously [19 ]. Bone marrow (BM) samples were collected from healthy volunteers following informed consent, according to the ethical committees of the cities of Copenhagen and Frederiksberg. Populations highly enriched in promyelocytes (PMs), myelocytes/metamyelocytes (MYs), and BM PMNs (bm-PMNs) were isolated from human BM samples by two-layer density centrifugation and subsequent immunomagnetic depletion of nongranulocytic cells, essentially as described previously [20 , 21 ]. To minimize changes in gene expression as a result of cellular activation, all steps of the immunomagnetic sorting were performed immediately after cell collection at ≤4°C, i.e., on ice, in a cold room, or a cooled centrifuge, using nonpyrogenic reagents and plastic ware.

Cytospins were prepared by centrifugation of 1–2 x 105 isolated cells onto glass slides [300 rpm, 10 min, room temperature (RT)]. The purity of isolated BM populations and neutrophils was assessed by microscopy of Wright Giemsa-stained cytospins. Cell numbers were assessed using an improved Neubauer hemocytometer.

Sequencing of AGP transcripts
To define the sequence of AGP transcripts synthesized during terminal granulocytic differentiation, total RNA was isolated from a BM population highly enriched in MYs using TRIzol (Invitrogen, San Diego, CA). Subsequently, first-strand cDNA was generated by reverse transcription of 5 µg total RNA at 42°C for 1 h using an oligo(dT) 24 primer and Superscript II, as described by the manufacturer (Invitrogen). First-strand cDNA was then subjected to polymerase chain reaction (PCR) using the two primer pairs covering the complete coding sequence of AGP (GenBank locus: NM_000607). AGP 5'-end primer pair: forward primer, GGT GAC TGC ACC CTG CAG; reverse primer, TCG TTC ACG TCA AAA GCA AC. AGP 3'-end primer pair: forward primer, AGT GCA ATC TAT AAC ACC ACC T; reverse primer, GCA AGT GAG GGA AAA AGC TG. The resultant PCR products were gel-purified (Qiaex II gel extraction kit, Qiagen, Hilden, Germany) and sequenced (MWG Biotech, Ebersberg, Germany).

Immunocytochemistry
Cytospins of purified BM populations and pb-PMNs were fixed in Tris-buffered saline (TBS; 50 mM Tris, 150 mM NaCl, pH 7.6)/4% formaldehyde (37% stock, Sigma Chemical Co., St. Louis, MO) at RT for 20 min, washed in TBS, and permeabilized in TBS/1% Triton X-100 (Sigma Chemical Co.) at RT for 30 min. Subsequently, cytospins were washed in TBS/1% bovine serum albumin (BSA), and unspecific binding was blocked by incubation in TBS/1% BSA (Sigma Chemical Co.) at RT for 30 min. Then, cytospins were probed at RT for 30 min with the following primary antibodies diluted in TBS/0.25% BSA: rabbit anti-human orosomucoid (AGP, 1:500, A0011, DakoCytomation, Glostrup, Denmark), rabbit-anti-human lactoferrin (1:1000, gift from DakoCytomation), and control rabbit immunoglobulin G (IgG; 1:500, X0936, DakoCytomation). The cytospins were washed twice in TBS, incubated at RT for 30 min with alkaline phosphatase-conjugated anti-rabbit/mouse polymer, washed twice in TBS, and stained with Fast-Red, as recommended by the manufacturer (Dual Envison System-AP, DakoCytomation). Finally, cytospins were washed in running tap water for 10 min, counterstained 1 min in Mayer’s hematoxylin, washed again in running tap water for 3 min, and mounted.

Exocytosis studies
Isolated neutrophils were resuspended at a density of 3 x 107 cells/ml in Krebs-Ringer phosphate buffer with glucose (KRG; 130 mM NaCl, 5 mM KCl, 1.27 mM MgSO4, 0.95 mM CaCl2, 10 mM NaH2PO4/Na2HPO4, 5 mM glucose, pH 7.4). For stimulation of exocytosis, 1 ml cell suspension was preincubated for 5 min at 37°C and subsequently stimulated for 15 min at 37°C by ionomycin (1 µM, Calbiochem, La Jolla, CA) or phorbol-12-myristate 13-acetate (PMA; 2.5 µg/ml, Sigma Chemical Co.). Cell suspension (1 ml), incubated for 20 min on ice, served as control. Stimulation was stopped by addition of 1 ml ice-cold KRG. Subsequently, cells were pelleted immediately to collect the supernatant containing exocytosed granule proteins for Western blot analysis.

Immunoelectron microscopy
Neutrophils isolated from peripheral blood were fixed for 24 h in 0.1 M PHEM buffer (pH 6.9) containing 4% paraformaldehyde and subjected to cryosectioning as described previously [22 ]. Cryosections of 45 nm were cut at –125°C using an ultracryomicrotome (Leica AG, Vienna, Austria) equipped with diamond knives (Drukker, Cuijk, The Netherlands) and transferred onto formvar-coated copper grids with a mixture of sucrose and methylcellulose [23 ]. The copper grids were placed on 35-mm Petri dishes containing 2% gelatine. Double-immunolabeling was performed as described by Slot et al. [24 ]. Briefly, cryosections were labeled with polyclonal rabbit anti-human orosomucoid (AGP) antibodies (DakoCytomation) followed by a protein A-conjugated colloidal gold probe (15 nm diameter). Subsequently, cryosections were labeled using polyclonal rabbit anti-human lactoferrin antibodies (Cappel Laboratories, Cochranville, PA) and a protein A-conjugated colloidal gold probe (10 nm diameter). Controls were labeled with an irrelevant rabbit IgG antibody. After immunolabeling, the cryosections were embedded in methylcellulose/uranyl acetate and examined by electron microscopy (Philips CM 10, Eindhoven, The Netherlands).

Subcellular fractionation
Neutrophils isolated from buffy coats were incubated in saline/5 mM diisopropylfluorohosphate (Aldrich Chemical Co., Milwaukee, WI) for 5 min, pelleted (200 g, 6 min), and 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 PIPES, pH 7.2) containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF; Sigma Chemical Co.). Cells were disrupted by nitrogen cavitation at 600 pounds per square inch (psi) [25 ]. Nuclei and intact cells were pelleted (400 g, 15 min). Postnuclear supernatant (S1; 10 ml) was carefully applied on top of a Percoll gradient, including three layers of 9 ml, with densities of 1.05, 1.09, and 1.12 g/ml. Gradients were generated by adding precalculated amounts of Percoll (range 1.129–1.131 g/ml, Amersham Bioscience, Uppsala, Sweden) to disruption buffer/0.5 mM PMSF (Sigma Chemical Co.) to obtain densities of 1.05, 1.09, and 1.12 g/ml [25 ]. The three-layer gradient topped by the S1 was centrifuged at 37,000 g for 30 min for subcellular fractionation. This resulted in four major bands: the {alpha}-band enriched in primary granules, the ß1-band enriched in secondary granules, the ß2-band enriched in tertiary granules, and the {gamma}-band enriched in cell membranes and secretory vesicles containing plasma proteins. Fractions of 1 ml were aspirated from the bottom of the three-layer gradient. From each fraction, 450 µl was centrifuged for 20 min at 28 psi in an airfuge (Beckman, Paolo Alto, CA) to sediment the Percoll. The supernatant containing the cellular material was resuspended in phosphate-buffered saline and subjected to enzyme-linked immunosorbent assay (ELISA) analysis as described previously or mixed with an equal volume of 2x Laemmli buffer for subsequent Western blot analysis [26 ].

AGP, from pooled subcellular fractions containing secondary granules (20 µl pooled ß-fractions) and from plasma (10 µl), was deglycosylated by peptide N-glycosidase F (PNGase-F) treatment for 3 h at 37°C, according to the manufacturer (New England Biolabs, Beverly, MA).

Western blot analysis
Samples were diluted with an equal volume of 2x Laemmli buffer and denatured at 100°C for 10 min [27 ]. Samples were then electrophoresed on 8%, 10%, or 12% sodium dodecyl sulfate polyacrylamide gels (BDH Laboratory Supplies, Poole, UK) and transferred to nitrocellulose membranes (Amersham Bioscience) by electroblotting. Subsequently, the membranes were incubated as indicated with primary rabbit-anti-human orosomucoid (AGP, 1:1000, A0011, DakoCytomation), myeloperoxidase (MPO; 1:1000, A0398, DakoCytomation), and lactoferrin (1:10,000, gift from DakoCytomation), followed by a secondary horseradish peroxidase-conjugated swine anti-rabbit antibody (1:1000, P0217, DakoCytomation). Binding of antibodies was visualized by enhanced chemiluminescence (Amersham Bioscience).

32Dcl3-CCAAT/enhancer-binding protein-{varepsilon}-estradiol receptor (C/EBP{varepsilon}-ER)TM cells
The murine 32Dcl3 cell line, generated from diploid myeloid progenitors, proliferates in the presence of interleukin (IL)-3 and can differentiate into mature neutrophils within 10–12 days upon granulocyte-colony stimulating factor stimulation [28 ]. 32Dcl3 cells were transduced with a retroviral vector constitutively expressing the C/EBP{varepsilon}-ERTM fusion protein, which is maintained in the cytoplasma and only translocates to the nucleus to exert C/EBP{varepsilon} activity in the presence of the estrogen derivative 4-hydroxy-tamoxifen (4-HT).

32Dcl3 cells were maintained in Iscove’s modified Dulbecco’s medium (Invitrogen) containing 10% heat-inactivated calf serum, 1 ng/ml murine IL-3 (StemCell Technologies, Vancouver, BC, Canada), and 100 units/ml penicillin/100 µg/ml streptomycin (Invitrogen).

The C/EBP{varepsilon}-ERTM cDNA construct was prepared by linking the full-length human C/EBP{varepsilon} cDNA in-frame to a modified tamoxifen-responsive estrogen receptor ligand-binding domain (murine ERTM; amino acids 281–599) [29 ]. The C/EBP{varepsilon}-ERTM and ERTM cDNA was inserted into the polylinker of the pBabePuro retroviral vector (Nolan lab homepage, Garry P. Nolan, Stanford University, CA, http://www.stanford.edu/group/nolan/index.html). The pBabePuro-C/EBP{varepsilon}-ERTM vector and the pBabePuro-ERTM control vector were transfected into the ecotrophic packaging cell line Phoenix NX by calcium-phosphate precipitation. After 24 h, 32Dcl3 cells were cocultured with transfected Phoenix cells for another 48 h in 32D medium plus 4 µg/ml polybrene (Nolan lab). Subsequently, 32Dcl3 cells were selected in puromycin (2 µg/ml), and subclones were generated by transfer of single cells into 96-well dishes using an automated Quixell transfer device (Stoelting, Wood Dale, IL) [19 ].

To define whether C/EBP{varepsilon} can transactivate AGP expression in myeloid progenitors, C/EBP{varepsilon} activity was induced in 32Dcl3-C/EBP{varepsilon}-ERTM cells by addition of 200 nM 4-HT (Sigma Chemical Co.) to the 32D medium. 32Dcl3-ERTM cells induced by 4-HT served as control. Total RNA was isolated from 32D cells using TRIzol (Invitrogen) before and after 4-HT induction at indicated time-points. Subsequently, AGP expression was assayed by real-time reverse transcriptase-PCR.

Real-time reverse transcriptase-PCR
Expression of AGP transcripts in 32Dcl3-C/EBP{varepsilon}-ERTM and 32Dcl3-ERTM cells was assessed as described previously [30 ]. Briefly, first-strand cDNA was generated by reverse transcription of 5 µg total RNA at 42°C for 1 h using an oligo(dT)24 primer and Superscript II as described by the manufacturer (Invitrogen). First-strand cDNA was then subjected to real-time PCR using the following primers and probes: ß-actin forward primer, GAA GTC CCT CAC CCT CCC A; ß-actin reverse primer, GGC ATG GAC GCG ACC A; ß-actin probe, AAG CCA CCC CCA CTC CTA AGA GGA GG; AGP forward primer, TCT CTG AAC TCC GAG GGC TG; AGP reverse primer, GAG ACA GAA TCA AAG TGC ACA GGA; AGP probe, CCA CAG GCT CAC CAA ACC CCA CC. The constitutively expressed housekeeping gene ß-actin was used to normalize AGP expression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
AGP is synthesized in myclocytes
Differentiation of PMs via MYs into mature neutrophils is characterized by the sequential formation of primary, secondary, and tertiary granules and their constituting granule proteins [1 , 2 ]. According to the targeting-by-timing hypothesis, granule proteins are targeted to a distinct subset of granules dependent on the time of their synthesis [31 ]. Hence, primary, secondary, and tertiary granule proteins are readily indentified by their unique gene expression profiles during granulocytic differentiation. We recently monitored the expression profiles for 17,020 genes in populations highly enriched in PMs, MYs, bm-PMNs, and pb-PMNs. This analysis demonstrated a transient high expression of AGP transcripts in MYs.

Notably, AGP expression was initiated later than that of the secondary granule protein lactoferrin (Fig. 1A ) but most important, also terminated earlier than that of the tertiary granule protein gelatinase, which in contrast to AGP, was still expressed at high levels in pb-PMNs (Fig. 1A) [21 , 32 ]. To define the sequence of AGP transcripts highly expressed during terminal granulocytic differentiation, RNA was isolated from a human BM population highly enriched in MYs and subjected to sequence analysis. This analysis revealed that AGP transcripts synthesized in MYs were 100% identical to AGP transcripts synthesized in the liver (GeneBank locus NM_000607).



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  		Figure 1. AGP and lactoferrin are expressed in cells from the MY stage and throughout granulocytic differentiation. BM populations, highly enriched in PMs, MYs, bm-PMNs, and pb-PMNs, were isolated from healthy individuals. (A) Total RNA was purified from BM and peripheral blood populations and subjected to microarray analysis to monitor the expression profiles for AGP, lactoferrin (LF; marker for secondary granules), and gelatinase (GEL; marker for tertiary granules) transcripts during granulocytic differentiation (mean±SD, n=3) [21 ]. (B) Immunocytochemical staining of BM and peripheral blood populations using rabbit anti-human AGP and LF antibodies demonstrates the cytoplasmic localization (red color) of AGP and LF from the MY stage throughout granulocytic differentiation. BM populations highly enriched in PMs stained with rabbit anti-human AGP or LF antibodies and populations stained with irrelevant rabbit IgG antibodies were all negative.

 
Immunocytochemistry of populations highly enriched in PMs, MYs, bm-PMNs, or pb-PMNs demonstrated that AGP, like the secondary granule protein lactoferrin, is highly expressed at the protein level from the MY stage throughout granulocytic differentiation (Fig. 1B) . Hence, the mRNA and protein expression profiles indicate that AGP is a secondary granule protein candidate.

AGP and lactoferrin colocalize in secondary granules of neutrophils
To define the subcellular localization of AGP, subcellular fractions from disrupted neutrophils were isolated by three-layer density gradient centrifugation. Subsequently, the contents of AGP and four marker proteins for distinct granule subsets and secretory vesicles were assessed in each of the subcellular fractions by Western blot analysis and ELISAs, respectively. As shown in Figure 2 , the majority of AGP protein was detected in subcellular fractions with high contents of lactoferrin, a marker protein for secondary granules. To further substantiate this finding, we performed double-labeling immunogold electron microscopy on intact neutrophils using antibodies raised against AGP and lactoferrin. As depicted in Figure 3 , AGP and lactoferrin colocalized in secondary granules, and it is important that AGP was not present at detectable levels in granules other than those containing lactoferrin. Overall, these findings strongly indicate that AGP is a genuine secondary granule protein.



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  		Figure 2. AGP and lactoferrin colocalize in subcellular fractions of neutrophils. Subcellular fractions were isolated from purified pb-PMNs and assayed by ELISA and Western blot analysis. Depicted lines indicate subcellular fractions containing concentrations >50 µg/ml MPO (marker for primary granules) and LF (marker for secondary granules), >5 µg/ml GEL (marker for tertiary granules), and >0.5 µg/ml albumin (ALB; marker for secretory vesicles). Bars indicate fractions with peak concentrations of MPO, LF, GEL, and ALB. Two forms of AGP were detected by Western blot analysis: An abundant and highly glycosylated form of 45–60 kDa colocalized in fractions containing the secondary granule protein lactoferrin, and a scarce form of 41 kDa colocalized in fractions containing albumin, a protein endocytosed from plasma and stored in secretory vesicles of neutrophils. MW, Molecular weight.

 


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  		Figure 3. AGP and lactoferrin colocalize in secondary granules of neutrophils. Cryosections of pb-PMNs were labeled with rabbit anti-human AGP antibodies followed by a 15-nm protein A-gold probe (left). In addition, cryosections (right) were double-labeled, first with a rabbit anti-human AGP antibody, followed by a 15-nm protein A-gold probe and subsequently, with a rabbit anti-human lactoferrin antibody and a 10-nm protein A-gold probe. Immunoelectron microscopy demonstrates the ultrastructural colocalization of AGP and LF in secondary granules.

 
Neutrophil-derived AGP localized in secondary granules is more glycosylated than liver-derived AGP in plasma
AGP is a plasma protein of 41 kDa with a carbohydrate content of 40%. The carbohydrate part of AGP consists of five di-, tri-, or tetra-antennary N-linked glycans. The heterogeneity among the glycans linked to AGP generates 12–20 glycoforms of AGP in plasma [33 ].

Of interest, two forms of AGP were detected in neutrophils by Western blot analysis. An abundant form with a high MW of 45–60 kDa was detected in subcellular fractions highly enriched in lactoferrin, a marker protein for secondary granules (Figs. 2 and 4 , ß1 and ß2fractions). In contrast, a scarce form with a low MW of ~41 kDa was detected in subcellular fractions highly enriched in albumin, an endocytosed plasma protein localized in secretory vesicles of neutrophils (Figs. 2 and 4 , {gamma} fractions).



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  		Figure 4. AGP located in secondary granules of neutrophils is highly glycosylated. Pooled subcellular fractions isolated from pb-PMNs as well as plasma samples were analyzed by Western blot analysis using rabbit anti-human AGP antibody. Western blots to the left demonstrate pooled subcellular fractions highly enriched in primary granule proteins ({alpha} fraction), secondary/tertiary granule proteins (ß1and ß2fractions), and secretory vesicles containing mainly plasma proteins ({gamma} fractions). These Western blots show that AGP present in ß1 and ß2 fractions has a higher MW (45–60 kDa) than the plasma-derived AGP (41 kDa) in secretory vesicles ({gamma} fractions). Western blots to the right demonstrate that AGP from secondary granules (pooled ß fractions) and plasma samples have the expected MW of 20 kDa following complete N-deglycosylation (degly.) by PNGase-F. Note that incomplete deglycosylation of plasma sample results in bands of 25 kDa (AGP with one N-linked glycan) and 30 kDa (AGP with two N-linked glycans).

 
To assess whether the high MW of AGP localized in secondary granules was a result of N-glycosylation, pooled subcellular ß-fractions were subjected to N-deglycosylation by PNGase-F. Subsequent Western blot analysis demonstrated a complete deglycosylation of AGP from pooled subcellular ß fractions, resulting in an AGP band with the expected MW of 20 kDa. Moreover, PNGase-F treatment of plasma resulted in complete as well as partial deglycosylation of AGP, as demonstrated by the three AGP bands with the expected MW of 20 kDa (complete deglycosylated AGP) or 25 kDA (AGP containing one residual N-linked glycan) and 30 kDa (AGP containing two residual N-linked glycans), respectively (Fig. 4) .

Overall, these findings demonstrate that neutrophils contain two forms of AGP, an abundant and highly glycosylated form, which is generated in MYs and stored in secondary granules, and a scarce form with a lower MW, endocytosed from plasma and stored in secretory vesicles.

AGP is exocytosed by activated neutrophils
Neutrophils migrate to sites of infection/injury and respond to inflammatory stimuli by exocytosis of effector proteins stored in granules and secretory vesicles. Exocytosis of granule proteins can be induced in vitro by stimulation of neutrophils with PMA and Ca2+ ionophores such as ionomycin. We have previously shown that PMA induces the release of proteins from secretory vesicles and secondary/tertiary granules but not primary granules, whereas ionomycin induces the release of proteins from secretory vesicles and all three types of granules [26 , 34 ].

In the present study, stimulation of neutrophils by PMA as well as by ionomycin for 15 min resulted in a marked release of lactoferrin and AGP with a MW of 41 kDa and 45–60 kDa (Fig. 5 ). These observations support the above findings that AGP is stored in secretory vesicles and secondary granules.



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  		Figure 5. AGP is exocytosed by activated neutrophils. Purified pb-PMNs were stimulated by the calcium ionophore ionomycin and PMA for 15 min, and the content of LF and AGP in the supernatant was detected by Western blot analysis. Ionomycin- and PMA-activated neutrophils released LF and AGP, whereas nonstimulated neutrophils did not.

 
AGP is not expressed in blood cells from a specific granule deficiency (SGD) patient
SGD is a rare congenital disease characterized by severe recurrent bacterial infections. Neutrophils from SGD patients display atypical bi-lobed nuclei and have an abnormal function as a result of the lack of secondary/tertiary (specific) granules and their constituting granule proteins [35 , 36 ]. However, they express primary granule proteins such as MPO at normal levels. In this context, we reasoned that being a secondary granule protein, highly glycosylated AGP would not be expressed in blood cells from SGD patients. To test this hypothesis, we obtained blood samples from an Asian girl with the diagnosis of SGD (M. Gharib, N. Borregaard, A. F. Gombart, P. D. Arkwright, unpublished data). Following delivery, she presented with postnatal sepsis, delayed separation of the umbilical cord, and recurrent opportunistic infections including diarrhea. She required frequent courses of intravenous broad-spectrum antibiotic treatment and has recently undergone successful matched, unrelated allogeneic stem cell transplantation. Peripheral blood smears demonstrated hypo- to agranular neutrophils with Pelger-Huet anomaly, i.e., bi-lobed nuclei.

Neither lactoferrin nor the highly glycosylated neutrophil-derived AGP protein was detected by Western blot analysis in an erythrocyte-depleted blood sample collected from the SGD patient but were present in a control sample collected from a healthy individual (Fig. 6 ). In contrast, the primary granule protein MPO was detected in both samples (Fig. 6) .



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  		Figure 6. AGP and lactoferrin are not expressed in blood cells from a patient suffering from SGD. Blood samples from a SGD patient and a healthy individual (control) were erythrocyte-depleted, and lysates, including total nuclear cells (neutrophils and mononuclear cells) and residual plasma, were subjected to Western blot analyses using rabbit anti-human MPO, LF, and AGP antibodies. Western blot analyses demonstrated that blood cells from the SGD patient express the primary granule protein (GP) MPO but not the secondary GP LF nor the highly glycosylated AGP (AGP with a high MW of 45–60 kDa). Contamination of cell lysates with residual plasma resulted in the detection of plasma AGP with a low MW (41 kDa) in the control sample and the sample obtained from the SGD patient.

 
AGP is induced by C/EBP{varepsilon}
The myeloid transcription factor C/EBP{varepsilon} has been defined as a key regulator of terminal granulocytic differentiation, i.e., the transition from PMs into mature neutrophils [37 ]. In support of this notion, functional loss of C/EBP{varepsilon} in mice or in patients suffering from SGD results in the generation of dysfunctional neutrophils lacking secondary/tertiary (specific) and tertiary granule proteins [35 , 36 , 38 ]. Consistent with these findings, C/EBP{varepsilon} has been shown to induce expression of the secondary granule protein lactoferrin in vitro [39 ].

To investigate whether C/EBP{varepsilon} can induce expression of AGP in vitro, we generated a myeloid 32Dcl3 cell line constitutively expressing a fusion protein containing the C/EBP{varepsilon} wild-type protein and the ligand-binding domain of the estrogen receptor (32Dcl3-C/EBP{varepsilon}-ERTM). With this cell line, the C/EBP{varepsilon}-ERTM fusion protein is maintained in the cytoplasma and only translocates to the nucleus to exert C/EBP{varepsilon} activity in the presence of the estrogen derivative 4-HT. Hence, the transcriptional activity of C/EBP{varepsilon} can be analyzed in an inducible manner. A 32Dcl3 cell line, expressing only the ligand-binding domain of the estrogen receptor, served as control (32Dcl3-ERTM).

Induction of 32Dcl3-C/EBP{varepsilon}-ERTM cells by 4-HT resulted in a 4.2-fold increase of AGP transcript levels within 8 h, and after 4 days of 4-HT induction, AGP transcript levels were increased maximally, i.e., 7.8–fold higher compared with baseline (Fig. 7 ). Induction of 32Dcl3-ERTM control cells by 4-HT did not result in any significant increase of AGP transcript levels compared with baseline (0.3- to 1.5-fold change). These findings indicate that expression of AGP in myeloid cells, like the secondary granule protein lactoferrin, is partially regulated by C/EBP{varepsilon}.



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  		Figure 7. C/EBP{varepsilon} induces AGP expression in myeloid cells. Myeloid 32Dcl3 cells, constitutively expressing a fusion protein containing the C/EBP{varepsilon} wild-type and the ligand-binding domain of the estrogen receptor (32Dcl3-C/EBP{varepsilon}-ERTM), were induced with 4-HT, resulting in nuclear translocation of C/EBP{varepsilon}-ERTM. AGP mRNA levels were measured relative to ß-actin levels by RT real-time PCR at the indicated time-points. Changes of AGP mRNA levels at indicated time-points following 4-HT induction were calculated relative to the AGP mRNA level before 4-HT induction (0 h; mean±SD, n=3). Myeloid 32Dcl3 cells constitutively expressing the ligand-binding domain of the estrogen receptor (32Dcl3-ERTM) served as control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study identifies AGP as a genuine secondary granule protein of human neutrophils by several means. First, AGP transcripts were increased transiently in MYs during granulocytic differentiation. Consistent with this finding, AGP protein was detected in cells from the MY stage and throughout granulocytic differentiation by immunocytochemistry. In addition, AGP colocalized with lactoferrin in secondary granules, as demonstrated by immunoelectron microscopy and subcellular fractionation of neutrophils. Moreover, neutrophils released AGP in response to PMA, a stimulus that promotes exocytosis of secretory vesicles and secondary/tertiary granule proteins but not of primary granule proteins [26 , 34 ]. Finally, AGP and lactoferrin were not detected in blood cells from a SGD patient.

Although the expression profile of AGP transcripts during granulocytic differentiation demonstrated that AGP synthesis was initiated later than that of the secondary granule protein lactoferrin, it was still terminated earlier than that of the tertiary granule protein gelatinase. In spite of these minor differences between AGP and lactoferrin mRNA expression profiles, our findings indicate that AGP is a secondary rather than a tertiary granule protein.

Of interest, our study identified two forms of AGP in neutrophils: an abundant and highly glycosylated form of 45–60 kDa, which is generated in MYs and stored in secondary granules, and a scarce form of 41 kDa, which is endocytosed from plasma and stored in secretory vesicles. In agreement with our observation, Poland et al. [40 ] recently identified a highly glycosylated, 47-kDa form of AGP in seminal plasma, which supposedly originated from the prostate. Hence, our findings support the idea that the glycosylation pattern of AGP depends on its cellular origin. As the glycosylation pattern of AGP has functional implications [41 ], future studies are needed to delineate the functional properties of the highly glycosylated AGP contained in secondary granules of neutrophils.

The proinflammatory cytokines IL-1 and IL-6 as well as glucocorticoids are major regulators of AGP expression in the liver [42 , 43 ]. IL-1 and IL-6 are strong inducers of C/EBPß, which in turn mediates AGP expression through C/EBP binding sites located in a distal response element (–5 kb) [43 ]. In addition, glucocorticoids mediate AGP expression through glucocorticoid receptors, which recruit C/EBPß, and maintain binding to a proximal AGP promoter (–155 to –65 kb) containing a glucocorticoid responsive element flanked by C/EBP binding sites [44 ]. In the present study, we showed that C/EBP{varepsilon}, a C/EBP family member that transactivates the secondary granule protein lactoferrin in myeloid cells [39] , also induces the expression of AGP in a myeloid cell line. In context with our previous observation that C/EBP{varepsilon} mRNA and protein transiently increase in MYs when AGP is synthesized [21] , these findings strongly indicate that C/EBP{varepsilon} at least partially regulates AGP expression during granulocytic differentiation.

Currently, AGP is mostly thought of as an APP synthesized by the liver to re-establish systemic homeostasis during infections by propagating various anti-inflammatory activities and nonspecific bacterial resistance. However, more recent data have demonstrated induction of AGP expression in alveolar macrophages and type II alveolar epithelial cells by immunoregulatory mediators, suggesting that AGP modulates the immune response locally [17 , 18 ]. Consistent with these findings, our study indicates that neutrophil-derived AGP exerts immunomodulatory activities locally. In support of this notion, we demonstrate that AGP, like other secondary granule proteins such as lactoferrin and human cathelicidin antimicrobial peptide 18 (hCAP18), is released by neutrophils in response to PMA activation. In context with our previous observations that lactoferrin and hCAP18 are released by neutrophils following in vivo exudation into skin lesions [45 , 46 ], the latter findings suggest that neutrophils might serve as the primary, local source of AGP at sites of infection/injury.

Of interest, Tsukahara et al. [47 ] recently demonstrated that LPS stimulation of pb-PMNs in vitro induced a significant increase of AGP transcript levels.

In context with the present study, this finding by Tsukahara et al. [47 ] points to a model where AGP is prestored in secondary granules and released by neutrophils to exert immediate, local immunomodulatory and antimicrobial activities, before de novo-synthesized AGP from various cell sources, including neutrophils and liver cells, successively increases and takes over.


   ACKNOWLEDGEMENTS
 
This work was supported in part by the following foundations: the Novo Nordisk Foundation, the Amalie Jørgensens Memorial Foundation, the Danish Cancer Research Foundation, the Danish Medical Research Council, the Gangsted Foundation, and the Lundbeck Foundation. K. T-M. is the recipient of a scholarship from the IMK Foundation and Rigshospitalet. The expert technical assistance of Charlotte Horn and Marianne Lodahl is highly appreciated. We thank our colleagues Malene D. Bjerregaard, Pia Klausen, Jack Cowland, and Anders Holmer for critical review of the manuscript.

Received January 24, 2005; revised April 6, 2005; accepted May 5, 2005.


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