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Originally published online as doi:10.1189/jlb.0706442 on February 19, 2008

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(Journal of Leukocyte Biology. 2008;83:1155-1164.)
© 2008 by Society for Leukocyte Biology

The secretory leukocyte protease inhibitor (SLPI) and the secondary granule protein lactoferrin are synthesized in myelocytes, colocalize in subcellular fractions of neutrophils, and are coreleased by activated neutrophils

Lars C. Jacobsen*, Ole E. Sørensen{dagger}, Jack B. Cowland*, Niels Borregaard* and Kim Theilgaard-Mönch*,1

* The Granulocyte Research Laboratory, Department of Hematology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark; and
{dagger} Division for Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden

1Correspondence: The Granulocyte Research Laboratory, Department of Hematology-9322, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, 2100 Copenhagen-Ø, Denmark. E-mail: k_theilgaard_moench{at}hotmail.com


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ABSTRACT
 
The secretory leukocyte protease inhibitor (SLPI) re-establishes homeostasis at sites of infection by virtue of its ability to exert antimicrobial activity, to suppress LPS-induced cellular immune responses, and to reduce tissue damage through inhibition of serine proteases released by polymorphonuclear neutrophil granulocytes (PMNs). Microarray analysis of bone marrow (BM) populations highly enriched in promyelocytes, myelocytes/metamyelocytes (MYs), and BM neutrophils demonstrates a transient, high mRNA expression of SLPI and genuine secondary granule proteins (GPs) in MYs. Consistent with this finding, immunostaining of BM cells showed SLPI and the secondary GP lactoferrin (LF) to be present in cells from the myelocyte stage and throughout neutrophil differentiation. Subcellular fractionation studies demonstrated the colocalization of SLPI and LF in subcellular fractions highly enriched in secondary granules. Finally, exocytosis studies demonstrated a corelease of SLPI and LF within minutes of activation. Collectively, these findings strongly indicate that SLPI is localized in secondary granules of PMNs. However, the amount of SLPI detected in PMNs is low compared with primary keratinocytes stimulated by growth factors involved in wound healing. This implicates that neutrophil-derived SLPI might not contribute essentially to re-establishment of homeostasis at sites of infection but rather, exert physiologically relevant intracellular activities. These might include the protection of secondary GPs against proteolytic activation and/or degradation by proteases, which might be dislocated to secondary granules at minute amounts as a consequence of spillover.

Key Words: granulocytes • elastase • wound healing


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INTRODUCTION
 
Polymorphonuclear neutrophil granulocytes (PMNs; neutrophils) constitute 50–60% of blood leukocytes and are key effectors of the innate immune system. PMNs are the first cells to be recruited to sites of infection or injury, where they phagocytose, kill, and degrade invading bacteria, fungi, or protozoa using reactive oxygen intermediates as well as antibiotic proteins and proteases stored in distinct granule subsets [1 , 2 ].

Granule proteins (GPs) are key effectors of the neutrophil immune response. They are traditionally classified according to their subcellular localization in primary (azurophil), secondary (specific), and tertiary [gelatinase (GEL)] granules, which are defined by their respective marker proteins myeloperoxidase (MPO), lactoferrin (LF), and GEL [1 , 2 ]. This classification of granule subsets has proven to be physiologically meaningful, as primary, secondary, and tertiary granules differ essentially with respect to protein content and extent of protein release to the extracellular environment (tertiary > secondary >>> primary granules) [1 , 2 ].

A hallmark of neutrophil differentiation is the sequential formation of primary, secondary, and tertiary granules and their constituent GPs. Primary granules and their associated GPs are formed in promyelocytes (PMs), secondary granules and their GPs are formed in myelocytes/metamyelocytes (MYs), and tertiary granules and their GPs are formed in bone marrow (BM) neutrophils (bm-PMNs) [3 , 4 ]. These findings have fostered the targeting-by-timing hypothesis, which states that targeting of GPs to a distinct granule subset is determined by the time of their synthesis rather than by a distinct sorting mechanism [3 4 5 ].

Human neutrophil elastase (ELA2), cathepsin G (CTSG), and proteinase 3 are serine proteases localized in primary granules of PMNs [1 , 6 ]. Following phagocytosis of microorganisms by PMNs, the majority of their primary granules fuses with the phagosome and releases proteases and antimicrobial substances to exert antimicrobial activity. However, upon activation of neutrophils, small numbers of primary granules also fuse with the cell membrane and release their content into the extracellular environment [7 ]. The serine proteases exocytosed by PMNs degrade a variety of extracellular matrix (ECM) proteins and thus, support migration of PMNs toward microorganisms at sites of infection [8 ]. In addition, serine proteases have been demonstrated to cleave proforms of antimicrobial proteins and other proteases that are stored in secondary and tertiary granules and are released by PMNs at sites of infection [9 , 10 ]. Finally, serine proteases play pivotal roles in disorders such as chronic obstructive pulmonary disease, cystic fibrosis, and rheumatoid arthritis by virtue of their ability to degrade ECM proteins and activate proinflammatory cytokines and protease-activated receptors [11 12 13 14 15 16 ]. To regulate the activity of exocytosed serine proteases and to restore homeostasis, the liver responds to infection by production of acute-phase proteins, including protease inbibitors such as {alpha}1-antitrypsin [17 ]. In addition, protease inhibitors are secreted locally at sites of infection by the surrounding tissue and effector cells of the innate immune system [18 19 20 21 22 ].

Secretory leukocyte protease inhibitor (SLPI), also known as antileukoproteinase, is a nonglycosylated, two-domain protein of 11.7 kDa, whose major function is to regulate/inhibit the activity of a wide range of proteases including ELA2, CTSG, chymotrypsin, and trypsin at sites of infection. Hence, SLPI is synthesized locally by secretory cells, namely by Clara and goblet cells of the respiratory epithelium, by dermal keratinocytes, and by macrophages, B-cells, and PMNs [22 23 24 25 26 27 28 ]. The COOH-terminal domain residue Leu-72 has been shown to be important for the protease-inhibitory function of SLPI [28 , 29 ], and the NH3-terminal domain (residues Ser-1 to Asp-49) has been shown to have antibacterial activity [24 ]. Furthermore, there is evidence that SLPI regulates LPS-induced activation of NF-kcyB by inhibiting degradation of IkcyB{alpha} and IkcyBβ [30 ]. Finally, SLPI has been shown to inhibit the ELA2-dependent conversion of proepithelin to epithelin and thereby, sustain a high level of proepithelin, which enhances wound healing by promotion of epithelial cell growth [29 ].

Thus, the net effect of SLPI at sites of infection is to re-establish homeostasis by reducing serine protease-mediated tissue damage, propagating antimicrobial activity, and suppressing LPS-induced cellular immune responses.

Recently, SLPI has been reported to be a major cytosolic protein in PMNs [22 ]. We here provide evidence that SLPI is not a major cytosolic protein of PMNs but rather, is synthesized at the MY stage during neutrophil differentiation and stored at low amounts in secondary granules of PMNs.


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MATERIALS AND METHODS
 
Cell isolation
Blood and BM samples were collected from healthy volunteers following informed consent, according to the ethics committees of the cities of Copenhagen and Frederiksberg. Peripheral blood neutrophils (pb-PMNs) were isolated from freshly collected blood samples by density centrifugation and subsequent hypotonic lysis of erythrocytes as described previously [31 ]. Populations highly enriched in PMs, MYs, and bm-PMNs were isolated from human BM samples by three-layer density gradient centrifugation and subsequent immunomagnetic depletion of nongranulocytic cells as described previously [32 ]. 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, Shandon cytocentrifuge, Thermo Electron, Waltham, MA, USA). 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.

Keratinocyte culture
Primary keratinocytes were maintained in culture and stimulated as described previously [33 ]. Primary keratinocytes (Cascade Biologics, Portland, OR, USA) were grown in serum-free keratinocyte medium supplemented with transferrin, human epidermal growth factor (hEGF; 100 ng/ml), 0.5 mg/ml hydrocortisone, gentamicin, amphotericin B, epinephrine, and insulin (KG®-2 BulletKit, Cambrex Walkersville, MD, USA). Two days before stimulation with hEGF, insulin was withdrawn from the medium. Cell cultures were stimulated with TGF-{alpha} (50 ng/ml) and insulin-like growth factor-1 (IGF-1; 100 ng/ml) 24 h after complete confluence was reached. After 48 h of stimulation, cells and medium were collected for analysis.

Human skin wounds
Samples from human skin wounds were obtained using protocols approved by the Ethics Committee of Lund University (Sweden) as described previously [33 ]. Briefly, skin wounds were generated by a punch biopsy on the upper arm of healthy male volunteers after informed consent. After 4 days, new punch biopsies were taken from the edges of the initial biopsies and subjected to immunohistochemical staining.

Exocytosis studies
Exocytosis studies were performed essentially as previosly described [34 ]. Briefly, isolated neutrophils were resuspended at a density of 3 x 107 cells/ml in Krebs-Ringer phosphate buffer with glucose (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 exocytosis, 3 ml cell suspension was preincubated for 5 min at 37°C and subsequently incubated for another 15 min at 37°C following addition of PMA (final concentration 2.5 µg/ml, Sigma-Aldrich, St. Louis, MO, USA). Cell suspension (1 ml) incubated for 20 min on ice served as control. The stimulation process was stopped by immediate centrifugation at 4°C. The supernatant containing exocytosed GPs and the cell pellet were collected separately for subcellular fractionation and/or analysis. Supernatants, cell pellets, and subcellular fractions prepared from cell pellets were resuspended in 2 x Laemmli buffer or ELISA sample buffer for subsequent Western blot and ELISA analysis, respectively. The percentage of released SLPI was calculated as the amount of SLPI in the supernatant divided by the total amount of SLPI in the supernatant and the pellet x 100.

Sequencing of SLPI transcripts
To analyze the sequence of SLPI transcripts synthesized during terminal granulocytic differentiation, total RNA was isolated from a BM population highly enriched in MYs using Trizol (Invitrogen, San Diego, CA, USA). Subsequently, first-strand cDNA was generated by RT 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 PCR using two pairs of primer covering the complete coding sequence of SLPI (GenBank locus: NM_003055)—SLPI 5'-end primer pair: forward primer, AGA GTC ACT CCT GCC TTC AC; reverse primer, CAA CTG GCA CTT CTT GAA AGC; and SLPI 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).

Immunocytochemical and immunohistochemical staining
Cytospins of purified BM populations and pb-PMNs were fixed in TBS (50 mM Tris, 150 mM NaCl, pH 7.6)/4% formaldehyde (37% stock, Sigma-Alrich) at room temperature for 20 min, washed in TBS, and permeabilized in TBS/1% Triton X-100 (Sigma-Aldrich) at room temperature for 30 min. Subsequently, cytospins were washed in TBS/1% BSA, and unspecific binding was blocked by incubation in TBS/1% BSA (Sigma-Aldrich) at room temperature for 30 min. Then, cytospins were probed at room temperature for 1 h with the following primary antibodies diluted in TBS/1% BSA: goat anti-human SLPI (5 µg/ml, AF1274, R&D Systems Europe Ltd., Abingdon, UK), goat anti-human LF (5 µg/ml, Nordic Immunological Laboratories, Tilburg, Netherlands), and control goat IgG (5 µg/ml, AB-108-C, R&D Systems Europe Ltd.). Cytospins were stained using the R&D Systems Europe Ltd. cell and tissue-staining kit (CTS009). Cytospins probed with rabbit anti-human GEL antibody [35 ] were stained using the Dako (Glostrup, Denmark) EnVision System AP. The cytospins were counterstained with hematoxylin for 3 min, washed in tap water for 1 min, and examined by microscopy (BX51 microscope, DP70 photo system, Olympus, Hamburg, Germany).

For immunohistochemical staining of human skin, biopsies were fixed in 10% formalin, followed by dehydration, and embedding in paraffin. Sections of 5 µm thicknesses were placed on polylysine-coated glass slides, deparaffinized in xylene, and rehydrated in graded alcohols. The slides were then trypsinated for 15 min in 0.05 M Tris (pH 7.4) with 0.5 mg/ml trypsin and 0.5 mg/ml CaCl2 or treated with Dako antigen-retrieval solution for 40 min at 97°C. The slides were probed with polyclonal goat anti-human SLPI antibody, diluted 1:1000 in TBS/1% BSA/0.05% Tween 20 (Sigma-Aldrich)/0.01% thimerosal, and incubated for 24 h at room temperature. After three washes of 20 min each in TBS/0.05% Tween 20, the slides were incubated with alkaline phosphatase-conjugated rabbit anti-goat IgG (Pierce Biotechnology, Rockford, IL, USA), diluted 1:1000 in the same buffer as the primary antibody, and incubated for another 24 h at room temperature followed by three washes of 20 min each. Color was developed with Fast Red chromogen (Sigma-Aldrich) in Tris buffer, and the slides were counterstained with hematoxylin (EM Science, Lawrence, KS, USA).

Subcellular fractionation
Neutrophils isolated from freshly collected peripheral blood were incubated in saline/5 mM diisopropylfluorophosphate (Aldrich Chemical Co., Milwaukee, WI, USA) 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 Na2ATP, 3.5 mM MgCl2, 10 mM PIPES, pH 7.2) containing 0.5 mM PMSF (Sigma-Aldrich). Cells were disrupted by nitrogen cavitation at 600 pounds per square inch (psi) [36 ]. Nuclei and residual, intact cells were pelleted (400 g, 15 min). Postnuclear supernatant (S1; 10 ml) was applied carefully 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 (1.131 g/ml, Amersham Bioscience, Uppsala, Sweden) to disruption buffer/0.5 mM PMSF (Sigma-Aldrich) [36 ]. 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 β1-band enriched in secondary granules, the β2-band enriched in tertiary granules, and the {gamma}-band enriched in cell membranes and secretory vesicles containing plasma proteins. The top part of the gradient, also termed the S2 fraction, contains the cytosolic proteins. For some experiments, the four bands and the S2 fraction were aspirated manually (2 ml). To sediment the Percoll, samples were centrifuged at 100,000 g for 1.5 h at 4°C, and the supernatant containing the cellular material was resuspended in PBS. Alternatively, fractions of 1 ml were successively aspirated from the bottom of the three-layer gradient. Fractions (450 µl from each) were centrifuged for 20 min at 28 psi in an airfuge (Beckman, Palo Alto, CA, USA) to sediment the Percoll, and the cellular material was collected and resuspended in PBS. Samples were then subjected to ELISA analysis or mixed with an equal volume of 2 x Laemmli buffer for Western blot analysis.

Western blot analysis
Supernatants containing proteins exocytosed by neutrophils, cell pellets, and subcellular fractions, prepared from cell pellets, were dissolved in 2 x Laemmli buffer (0.125 M Tris-base, pH 6.8, 20% v/v glycerol, 4% v/v SDS, bromophenol blue, 2% v/v 2-ME) and boiled for 5 min. Protein lysates were separated on SDS polyacrylamide gels (7–14%) and transferred to nitrocellulose membranes (Amersham Bioscience) by electroblotting. Subsequently, the membranes were incubated in blocking solution [PBS/2% v/v skim milk powder (Merck, Darmstadt, Germany)/0.05% Tween 20 (Sigma-Aldrich)] for 1 h at room temperature, prior to incubation with the following primary antibodies: goat anti-human SLPI (1:100, AF1274, R&D Systems Europe Ltd.), rabbit anti-human MPO (1:1000, A0398, Dako), rabbit anti-human LF (1:10,000, gift from Dako), mouse anti-6xHis (1:500, ab18184, Abcam, Cambridge, UK) in blocking solution, overnight at 4°C, followed by a secondary, HRP-conjugated swine anti-rabbit antibody (1:1000, P0217, Dako), rabbit anti-goat antibody (1:1000, P0449 Dako), or goat anti-mouse antibody (1:1000, P0447 Dako). The immune complexes were visualized by ECL (Amersham Bioscience).

The specificity of the SLPI Western blot analysis was tested by preincubation of goat anti-human SLPI antibody (5 µg/ml) for 1 h at room temperature with an equal volume of recombinant human SLPI (37.5 µg/ml, 1274-PI, R&D Systems Europe Ltd.) before probing of Western blot membranes.

ELISA
To analyze the content of SLPI, subcellular fractions were mixed 1:20 with ELISA sample buffer and subjected to ELISA analysis, according to the manufacturer’s instructions (DP100, R&D Systems Europe Ltd.). The content of SLPI in selected samples was confirmed by application of a second commercial anti-human SLPI ELISA kit (HK316, Hycult Biotechnology, The Netherlands). In addition, the contents of MPO, LF, and GEL in each subcellular fraction were determined by ELISA analyses, as described previously [37 , 38 ].

Real-time RT-PCR
Expression of SLPI transcripts in PMs, MYs, bm-PMNs, and pb-PMNs was assessed as described previously [39 ]. Briefly, first-strand cDNA was generated by RT of 1 µg total RNA at 42°C for 1 h using an oligo(dT)24 primer and SuperScript II as described by the manufacturer. First-strand cDNA was subjected to real-time PCR analysis using TaqMan® gene expression assays (SLPI, Hs000268204_m1; β-actin, Hs99999903_ml) and the Applied Biosystems (Foster City, CA, USA) 7500 Real-Time PCR system, according to the manufacturer’s instructions. Expression levels of SLPI were normalized to the constitutively expressed housekeeping gene β-actin.

Real-time RT-PCR was performed to compare the levels of SLPI transcripts in nontransfected keratinocytes and keratinocytes transfected with plasmids constitutively expressing His-tagged, full-length human SLPI (SLPI His-tag) and human SLPI lacking the candidate 25 amino acid (AA) signal peptide ({Delta}1-25SLPI His-tag). For this, total RNA was isolated (Trizol, Invitrogen), treated with RNase-free DNase (Fermentas, Burlington, Canada) to remove traces of plasmid DNA, and subjected to cDNA synthesis (iScript cDNA synthesis kit, Bio-Rad, Hercules, CA, USA). Real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad) and the following primer pairs: GAPDH: 5'-TGG TAT CGT GGA AGG ACT C-3', 5'-AGT AGA GGC AGG GAT GAT G-3'; SLPI: 5'-GCC AAT GTT TGA TGC TTA AC-3', 5'-AGT CTC AGG GTG GAA AGG-3'. (SLPI primers were placed downstream of the signal peptide sequence within the coding sequence.). Real-time PCR was performed using an iCycler, and data were analyzed by the iCycler iQ optical system software (Bio-Rad). Expression levels of SLPI were normalized to the constitutively expressed housekeeping gene GAPDH.

SLPI constructs and transient transfection assays
cDNA constructs for full-length SLPI, and SLPI lacking the putative 25-AA signal peptide ({Delta}1-25SLPI) were generated by RT of total RNA purified from a BM population highly enriched in MYs (SuperScript II, Life Technologies, Rockville, MD, USA) and subsequent PCR using the following primers: full-length human SLPI cDNA: forward primer, 5'-AC GAA TCC GCC ACC ATG AAG TCC AGC GGC CTC T-3'; human SLPI lacking a 25-AA signal peptide: forward primer, 5'-AC GAA TTC GCC ACC ATG TCT GGA AAG TCC TTC AAA GCT-3'; common reverse primer including His-tag, 5' TG CTC GAG TCA ATG ATG ATG ATG ATG ATG AGC TTT CAC AGG GGA AAC GC-3'.

The SLPI His-tag and the {Delta}1-25SLPI His-tag cDNAs were inserted into the polylinker (EcoRI and ExoI restriction sites) of the pcDNA3.1 plasmid (Invitrogen) using Quick ligase, according to the manufacturer’s instructions (Qiagen). Correct insertion of SLPI His-tag and the {Delta}1-25SLPI His-tag cDNAs into the pcDNA3.1 plasmid was confirmed by sequencing (MWG Biotech).

Primary keratinocytes were grown to confluence in six-well plates, and 1 µg cDNA constructs were transfected using Effectene as described by the manufacturer (Qiagen). After 6 h of incubation, the medium was discarded, and cells were incubated for another 48 h with fresh medium. Then, medium and one-half of the cells were subjected to TCA precipitation of proteins and subsequent Western blot analysis. The other half of the cells was subjected to RNA purification and subsequent real-time RT-PCR to assess levels of SLPI transcripts in keratinocytes as described above.


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RESULTS
 
SLPI is synthesized in MYs
Primary, secondary, and tertiary granules and their constituent GPs are formed sequentially during neutrophil differentiation, namely in PMs, MYs, and bm-PMNs, respectively [40 , 41 ]. Thus, the targeting of GPs to distinct granule subsets is determined by the time of their synthesis rather than by a specific sorting mechanism (targeting-by-timing hypothesis) [5 ]. We have recently monitored the global change in gene expression during neutrophil differentiation by comprehensive microarray analysis of populations highly enriched in PMs, MYs, bm-PMNs, and pb-PMNs [32 ]. This analysis demonstrated a transient, high expression of SLPI transcripts and the genuine, secondary GP LF in MYs (Fig. 1A ), whereas transcripts for the genuine, tertiary GP GEL were expressed at high levels even in bm-PMNs and pb-PMNs (Fig. 1A) . Real-time RT-PCR confirmed the transient, high expression of SLPI transcripts in MYs (data not shown). To test whether SLPI, like LF, is present in cells from the MY stage and throughout neutrophil differentiation, we performed immunocytochemistry of populations highly enriched in PMs, MYs, bm-PMNs, and pb-PMNs using antibodies raised against SLPI, LF, and GEL (Fig. 1B) . This analysis indeed demonstrated that SLPI, like LF, is present in cells from the MY stage and throughout neutrophil differentiation.


Figure 1
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  		Figure 1. SLPI, LF, and GEL are expressed in cells from the myelocyte stage and throughout neutrophil 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 SLPI, LF (marker for secondary granules), and GEL (marker for tertiary granules) during granulocytic differentiation (mean±SD, n=3) [32 ]. The microarray expression profile for SLPI was confirmed by real-time RT-PCR (data not shown). (B) Immunocytochemical staining of BM and peripheral blood populations. The staining demonstrates the cytoplasmic localization of SLPI, LF, and GEL from the myelocyte stage throughout granulocytic differentiation. BM populations highly enriched in PMs stained with goat anti-human SLPI or rabbit anti-human LF and GEL antibodies were all negative. Populations stained with irrelevant goat (control) or rabbit (data not shown) IgG antibodies were negative.

Subcellular localization of SLPI in neutrophils
Subcellular fractions of PMNs were isolated by three-layer Percoll density gradient centrifugation. With this method, fractions highly enriched in primary, secondary, and tertiary granules as well as secretory vesicles are readily identified based on their high contents of distinct marker proteins. Fractions 1–6 contain primary granules with high density and a high content MPO. Secondary granules with intermediate density and a high content of LF localize in fractions 10–16. Finally, tertiary granules, with low density and a high content of GEL localize in fractions 16–19. ELISA analysis revealed the highest content of SLPI and LF in fractions 10–16 (Fig. 2A ). Consistent with this finding, Western blot analysis demonstrated the highest content of SLPI and LF in pooled, subcellular β-fractions highly enriched in secondary and tertiary granules (Fig. 2B) . These findings demonstrate the subcellular colocalization of SLPI and LF in organelles of equal density (Fig. 2A) .


Figure 2
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  		Figure 2. SLPI and LF colocalize in subcellular fractions of neutrophils. (A) Resting PMNs (solid lines) or PMNs stimulated with PMA (2.5 µg/ml; dotted lines) were subjected to subcellular fractionation and ELISA. The absolute protein concentrations in each subcellular fraction are shown for MPO (marker for primary granules), LF (marker for secondary granules), SLPI, and GEL (marker for tertiary granules). (B) Pooled subcellular fractions (manually collected) highly enriched in primary granules ({alpha}-fraction), secondary and tertiary granules (β-fraction), secretory vesicles and plasma membranes ({gamma}-fraction), and cytosol (S2) were purified from PMNs (3x108) and analyzed by Western blotting using antibodies raised against human MPO, LF, and SLPI. The loaded cellular equivalent on the Western blot from which each of the subcellular fractions was purified corresponds to 3.1 x 107 cells. It is important to note that the MPO and LF signals were strong and therefore, visualized already after 15 s of exposure, whereas the weak SLPI signal was visualized after 15 min of exposure to obtain a robust signal. Collectively, ELISA and Western blot analysis are consistent with the presence of low amounts of SLPI in a subcellular fraction highly enriched in secondary GPs.

Neutrophils exocytose SLPI in response to activation
PMNs exocytose their granules in response to activation by inflammatory mediators. We have previously shown that PMA stimulates the release of the major part of secondary and tertiary GPs and only releases a minor fraction of primary GPs [1 ].

Stimulation of PMNs by PMA significantly reduced the content of SLPI and LF in subcellular fractions enriched in secondary granules (fractions 10–16, Fig. 2A ) but did not essentially reduce the content of MPO in fractions enriched in primary granules (fractions 1–6, Fig. 2A ). Consistent with these findings, ELISA analysis of supernatants and cell pellets collected following PMA stimulation of PMNs revealed a marked corelease of SLPI (68% release) and LF (60% release) but no significant release of MPO (5%; Fig. 3 ). Hence, the observed corelease of SLPI and LF is in agreement with the colocalization of SLPI and LF in secondary granules.


Figure 3
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  		Figure 3. Neutrophils corelease SLPI and LF in response to activation. Purified PMNs were stimulated by PMA (2.5 µg/ml) for 15 min. The contents of MPO (marker for primary granules), SLPI, and LF (marker for secondary granules) were assayed by ELISA in cell pellets and in supernatants containing exocytosed GPs. The relative (%) and the absolute protein concentrations of MPO, SLPI, and LF released by PMA-stimulated neutrophils are shown. The percentage of released GPs is calculated as the amount of protein detected in the supernatant divided by the total amount of protein detected in the supernatant and pellet x 100.

SLPI synthesized in myelocytes contains a signal peptide
Signal peptides represent intrinsic signals for protein transport and localization within the cell. Hence, proteins that are secreted to the extracellular environment or stored in intracellular organelles such as neutrophil granules generally contain signal peptides, whereas cytosolic proteins typically lack signal peptides.

A most recent study reported that resting PMNs contain substantial amounts of SLPI in the cytosol but not in granules [22 ]. As this finding argues against the presence of a signal peptide in neutrophil SLPI and contrasts our finding of SLPI being located in secondary granules, we investigated whether neutrophil SLPI contains a signal peptide.

Sequence analysis of SLPI transcripts synthesized in MYs revealed an mRNA sequence identical to that reported in the Gene Bank database (Accession No. NM_003064). We next calculated the probability for the presence of a signal peptide in the corresponding SLPI AA sequence (Accession No. NP003055.1) using the SignalP 3.0 server program (www.cbs.dtu.dk/services/SignalP-3.0; Fig. 4 ) [42 ]. This analysis revealed a signal peptide probability of 0.999 for SLPI synthesized in MYs (Fig. 4) . Subsequent analysis of the GPs MPO, LF, and GEL revealed signal peptide probabilities similar to that of SLPI (Fig. 4) . In contrast, analysis of the cytosolic proteins LDHA and β-actin revealed no signal peptide probability for either of the proteins (Fig. 4) . In agreement with previous studies, the SignalP 3.0 analysis identified a high cleavage-site probability for SLPI between AA25 and AA26 [43 ]. To define the functional significance of the predicted signal peptide sequence AA1–25, we performed transient transfection assays using primary keratinocytes and plasmids constitutively expressing His-tagged, full-length human SLPI (SLPI His-tag) or human SLPI lacking the candidate 25-AA signal peptide ({Delta}1-25SLPI His-tag). Quantitative PCR demonstrated a fourfold higher expression of SLPI transcripts in keratinocytes transfected with plasmids constitutively expressing SLPI His-tag or human SLPI lacking the candidate 25-AA signal peptide ({Delta}1-25SLPI His-tag) compared with nontransfected keratinocytes only expressing endogenous SLPI (Fig. 4) . Consistent with the SignalP 3.0 analysis, keratinocytes only secreted full-length SLPI His-tag into the medium but not {Delta}1-25SLPI His-tag (Fig. 4) . Despite the expression of SLPI transcripts in transfected keratinocytes at comparable levels, we were only able to detect SLPI His-tag protein intracellularly and in the medium by Western blot analysis but not {Delta}1-25SLPI His-tag protein. These findings were confirmed in three independent experiments. The latter indicates that the {Delta}1-25 peptide represents a signal peptide, which is not only critical for the secretion of SLPI but also for its synthesis and/or its protection against intracellular degradation.


Figure 4
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  		Figure 4. SignalP 3.0 analysis predicts the presence of a signal peptide in neutrophil-derived SLPI. (A) mRNA isolated from BM populations highly enriched in MYs was subjected to sequence analysis. The corresponding AA sequence was submitted to the SignalP 3.0 server program (www.cbs.dtu.dk/services/SignalP-3.0) to calculate a signal peptide probability and maximum cleavage site probability for SLPI. The S-score reports the probability for each AA in the submitted sequence of being part of the signal peptide; the C-score reports the probability of a cleavage site; and the Y-score assigns a cleavage site where the slope of the S-score is steep, and there is a high C-score. NN, Neural networks. (B) Table representing the signal peptide probabilities and cleavage-site probabilities calculated by the SignalP 3.0 server program for SLPI synthesized in MYs, the GPs MPO (NP_000250), LF (NP_002334.2), and GEL (NP_004985.2), and the cytosolic proteins lactate dehydrogenase A (LDHA; NP_005557.1) and β-actin (NP_001092.1). (C) The functional significance of the predicted signal peptide sequence AA1–25 was evaluated by transient transfection of primary keratinocytes with plasmids constitutively expressing His-tagged, full-length human SLPI (SLPI His-tag) or His-tagged SLPI lacking the candidate 25-AA signal peptide ({Delta}1-25SLPI His-tag). Nontransfected keratinocytes served as control. Real-time RT-PCR was performed using DNase-treated RNA to measure the transcript levels for SLPI in keratinocytes constitutively expressing SLPI His-Tag and {Delta}1-25SLPI His-tag relative to endogenous SLPI expression (i.e., SLPI transcript levels in nontransfected keratinocytes). SLPI transcript levels in keratinocytes were normalized using the GAPDH housekeeping gene. Western blot analysis of cell lysates and medium was performed 3 days after transfection using anti-His antibodies. Consistent with the SignalP 3.0 analysis, keratinocytes only secreted full-length SLPI into the medium but not SLPI lacking the predicted 25-AA signal peptide.

Overall, these findings indicate the presence of a bona fide signal peptide in full-length SLPI synthesized in MYs and thus, corroborate our findings that SLPI is targeted to and stored in secondary granules of PMNs. These findings also argue against the cytosolic localization of SLPI in PMNs described previously.

Validation of ELISA and Western blot assays
Analysis of the SLPI content in resting PMNs using a commercial SLPI ELISA kit (R&D Systems Europe Ltd.) revealed low amounts of SLPI in pooled, subcellular fractions as well as whole cell lysates (1.23–1.96 ng/107 cells; Fig. 5 ). As these data were not consistent with recent ELISA analyses demonstrating that resting PMNs contain substantial amounts of SLPI in the cytosol (7549–8850 ng/107 cells) [22 ], we validated our ELISA data by several means. First, we confirmed the low content of SLPI in pooled subcellular fractions and cell lysates from resting PMNs using a second commercial SLPI ELISA kit including anti-human SLPI antibodies different from those in the ELISA kit used first (Hycult Biotechnology; Fig. 5 ). Second, we evaluated the performance of the SLPI ELISA kit by analyzing another SLPI-producing cell type, namely keratinocytes. This analysis demonstrated that primary human keratinocytes cultured in vitro contained substantially higher amounts of SLPI than resting PMNs (Fig. 5) . In addition, ELISA analysis of primary keratinocytes stimulated by growth factors involved in wound healing, namely TGF-{alpha} and IGF-1, resulted in a 5.5-fold increase of their SLPI content (Fig. 5) . The ELISA data about primary human keratinocytes were confirmed by immunohistochemical analysis of human skin biopsies, which indeed demonstrated a weak SLPI staining of suprabasal keratinocytes in normal skin and a strong SLPI staining of keratinocytes at skin-wound margins (Fig. 5) .


Figure 5
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  		Figure 5. Comparison of SLPI content in neutrophils and primary keratinocytes. (A) SLPI contents were measured by ELISA in whole cell lysates of resting PMNs and pooled subcellular fractions (manually collected) enriched in primary granules ({alpha}-fraction), secondary and tertiary granules (β-fraction), and secretory vesicles and plasma membranes ({gamma}-fraction). The recovery rate for SLPI and LF in manually collected {alpha}-, β-, and {gamma}-fractions as compared with whole cell lysates is 34%, 38%, and 38%, respectively. This is consistent with a typical recovery rate for secondary GPs in manually collected, subcellular {alpha}-, β-, and {gamma}-fractions between 30% and 50%. In addition, SLPI contents were measured by ELISA in a cell lysate of resting keratinocytes as well as in the supernatant and cell lysate of keratinocytes stimulated by TGF-{alpha} (50 ng/ml) and IGF-1 (100 ng/ml) for 48 h. (B) Immunohistochemical staining of human skin biopsies using a goat anti-human SLPI antibody. The staining demonstrates a weak SLPI staining of suprabasal keratinocytes in normal skin (see arrows) and a strong SLPI staining of keratinocytes at skin-wound margins (see arrows).

Finally, we validated the specificity of the polyclonal goat anti-human SLPI antibody (R&D Systems Europe Ltd.) applied in the present study in ELISA and Western blot assays. For this, we performed Western blot analyses of recombinant human SLPI (control), whole cell lysates of primary human keratinocytes, and pooled β-fractions highly enriched in secondary GPs, with or without prior blocking of the goat anti-human SLPI antibody by recombinant human SLPI. These Western blot analyses detected one specific band of identical size in all samples without prior blocking of the antibody and no bands in any of the samples following blocking of the antibody (Fig. 6 ). Hence, these blocking experiments indicate that the goat anti-human SLPI antibody applied in our study has a high specificity for SLPI in neutrophils and primary keratinocytes and does not cross-react with other proteins.


Figure 6
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  		Figure 6. Highly specific detection of SLPI in neutrophils and keratinocytes by Western blot analysis. Recombinant human (rh) SLPI (10 ng), cell lysates of primary human keratinocytes (2x104 cells), and pooled, subcellular β-fractions (isolated from 3.1x107 PMNs) were subjected to Western blot analysis using a goat anti-human SLPI antibody. Prior blocking of the goat anti-human SLPI antibody by recombinant human SLPI before probing of Western blot membranes reveals no cross-reactivity but only a highly specific detection of SLPI in keratinocytes and PMNs.

Overall, these experiments strongly indicate that resting PMNs contain low amounts of SLPI stored in secondary granules rather than large amounts of SLPI located in the cytosol.


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DISCUSSION
 
The present study strongly supports the localization of SLPI in secondary granules of human PMNs by several means. First, SLPI transcripts were transiently increased in MYs during neutrophil differentiation. Consistent with this finding, SLPI protein was detected in cells from the myelocyte stage and throughout neutrophil differentiation by immunocytochemistry. In addition, subcellular fractionation studies demonstrated colocalization of SLPI and LF in subcellular fractions highly enriched in secondary granules. Finally, neutrophils coreleased SLPI and LF immediately in response to PMA, a stimulus, which promotes exocytosis of secretory vesicles and secondary/tertiary GPs but not of primary GPs. This corelease of SLPI and LF by PMNs within minutes of activation strongly supports a localization of SLPI within the granule matrix rather than the granule membrane or the cytosol. Indeed, the latter is supported by our findings that SLPI, like LF, contains a functional signal peptide. It is, however, important to point out that SLPI, despite several attempts, was not detected in PMNs by immunoelectron microscopy, most likely as a result of the limited sensitivity of this technique. Nevertheless, we argue that SLPI, which is present in PMNs at low levels and therefore cannot be detected by immunoelectron microscopy but fulfills criteria discussed above, should be defined as secondary GP. In a broader perspective, our findings support a new, operational definition for matrix proteins of secondary granules that are low-abundant and cannot be visualized by immunoelectron or confocal microscopy based on the following four criteria: colocalization with LF in subcellular fractions of PMNs; corelease with LF by PMNs in response to PMA and/or other inflammatory stimuli; expression profile similar to LF during terminal granulocytic differentiation (i.e., transiently high expressed in MYs); presence of a signal peptide.

The presence of SLPI in PMNs is partly consistent with a study by Sallenave et al. [22 ]. However, our findings differ substantially from those reported by Sallenave et al. [22 ], as we were unable to detect large amounts of SLPI in PMNs located in the cytosol but rather, detected small amounts of SLPI in PMNs stored in secondary granules.

Because of this major discrepancy with respect to content and location of SLPI in PMNs, we performed several experiments to validate our findings thoroughly.

As GPs generally have signal peptides, we investigated whether SLPI transcripts synthesized in MYs encoded for a SLPI protein containing a signal peptide. This analysis revealed that SLPI synthesized in myeloid cells contains a functional signal peptide, which indeed supports the subcellular localization of SLPI in secondary granules rather than the cytosol. We next confirmed our initial ELISA data demonstrating a low content of SLPI in PMNs by parallel analysis of pooled, subcellular fractions and whole cell lysates from resting PMNs using the initial SLPI ELISA kit as well as a second commercial SLPI ELISA kit. Finally, blocking experiments demonstrated that the polyclonal goat anti-human SLPI antibody applied in the present study in ELISA and Western blot assays has a high specificity for SLPI contained in PMNs as well as primary keratinocytes and does not cross-react with other proteins. Hence, all performed validation experiments supported our initial findings of small amounts of SLPI stored in secondary granules of PMNs. Of interest, Sallenave et al. [22 ] demonstrated that the rabbit anti-SLPI antibody applied in their study not only bound to SLPI (14 kDa) but also to a 30-kDa protein contained in PMNs (see Fig. 1B , Lane 8 and 9, Sallenave et al. [22 ]). Indeed, Sallenave et al. [22 ] argued that this finding might represent cross-reactivity of the rabbit anti-SLPI antibody applied in their study, with a 30-kDa protein of unknown origin contained in PMNs. As discussed above, the goat anti-SLPI antibody applied in the present study did not cross-react with neutrophil proteins other than SLPI. In this context, the cross-reactivity of the rabbit anti-SLPI antibody might account for the substantially higher content of SLPI in PMNs detected in the Sallenave study compared with ours.

Various studies have demonstrated the importance of serine protease inhibitors in regulating the activities of serine proteases released by leukocytes during inflammation. Thus, SLPI localized in secondary granules and released by activated PMNs might serve as an immediate source of serine protease inhibitors at sites of infection. To estimate to what extent PMNs contribute to the total SLPI activity at sites of infection such as skin wounds, we compared the content of SLPI in resting PMNs (as activated PMNs already have released some SLPI) and primary keratinocytes. This analysis revealed that whole cell lysates from resting PMNs contained low amounts of SLPI compared with unstimulated, primary keratinocytes cultured in vitro. Moreover, primary keratinocytes increased their SLPI content significantly in vitro, i.e., in response to growth factors involved in wound healing, as well as in vivo in a human skin wound model. These findings indicate that SLPI contained in PMNs does not contribute essentially to the SLPI activity at sites of infections such as skin wounds. In other words, the low amount of SLPI contained in secondary granules and released into phagolysosmes or to the extracellular environment by activated PMNs is unlikely to exert substantial antibacterial activity or inhibit the vast amount of serine proteases released from primary granules into phagolysosomes and to the extracellular environment. Serine protease activity is critical for neutrophil functions such as migration, degradation of phagocytosed microorganism, and activation of antimicrobial and proteolytic GPs released from secondary and tertiary granules. In this context, the low amount of SLPI in secondary granules seems physiologically relevant, inasmuch as high levels of SLPI released from secondary granules into the phagolysosome and the extracellular environment would inhibit serine protease activity substantially and therefore, interfere with this critical component of the neutrophil immune response.

However, the low amount of SLPI contained in secondary granules might indeed exert physiologically relevant, intracellular activities. These might include the protection of secondary GPs against activation and/or degradation by proteases and in particular, serine proteases, which as a result of spillover, might be targeted to secondary granules at minute amounts during neutrophil differentiation and in resting PMNs.

One might therefore speculate that the low amount of SLPI in secondary granules represents a fail-safe mechanism that might suffice to protect secondary GPs against dislocated proteases but on the other hand, does not interfere essentially with serine protease activity once PMNs get activated and execute their immune response.

Collectively, this study defines SLPI as a novel, secondary GP of PMNs, which challenges recent findings of PMNs containing large amounts of SLPI located in the cytosol.


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ACKNOWLEDGEMENTS
 
This work was supported in part by the following foundations: The Danish Medical Research Council, the Danish Cancer Research Foundation, the Novo Nordisk Foundation, the Amalie Jørgensens Memorial Foundation, the Gangsted Foundation, and the Lundbeck Foundation. L. C. J. is the recipient of a scholarship from the Lundbeck Foundation. O. E. S. is the recipient of a senior fellowship form the Novo Nordisk Foundation, and K. T-M. is the recipient of a scholarship from the Danish Medical Research Council. We thank Charlotte Horn for expert technical assistance and our colleagues Lene Udby and Carsten Niemann for critical review of the manuscript.

Received July 13, 2006; revised January 14, 2008; accepted January 14, 2008.


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