This Article Abstract Full Text (PDF) All Versions of this Article: jhc.5A6792.2006v1 54/8/877    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bach, J.-P. Articles by Schrader, M. Search for Related Content PubMed PubMed Citation Articles by Bach, J.-P. Articles by Schrader, M. Social Bookmarking                      What's this?

The Secretory Granule Protein Syncollin Localizes to HL-60 Cells and Neutrophils

Jan-Philipp Bach, Heike Borta, Waltraud Ackermann, Floriane Faust, Oliver Borchers and Michael Schrader

Department of Cell Biology and Cell Pathology, University of Marburg, Marburg, Germany

Correspondence to: Michael Schrader, Department of Cell Biology and Cell Pathology, University of Marburg, Robert-Koch Str. 6, D-35037 Marburg, Germany. E-mail: schrader{at}mailer.uni-marburg.de

	Summary

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 
The secretory granule protein syncollin was first identified in the exocrine pancreas where a population of the protein is associated with the luminal surface of the zymogen granule membrane. In this study we provide first morphological and biochemical evidence that, in addition to its pancreatic localization, syncollin is also present in neutrophilic granulocytes of rat and human origin. By immunohistological studies, syncollin was detected in neutrophilic granulocytes of the spleen. Furthermore, syncollin is expressed by the promyelocytic HL-60 cells, where it is stored in azurophilic granules and in a vesicular compartment. These findings were confirmed by fractionation experiments and immunoelectron microscopy. Treatment with a phorbol ester triggered the release of syncollin indicating that in HL-60 cells it is a secretory protein that can be mobilized upon stimulation. A putative role for syncollin in host defense is discussed. (J Histochem Cytochem 54:877–888, 2006)

Key Words: pancreas • granule biogenesis • neutrophil • secretion • HL-60 • antimicrobial

	Introduction

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 
POLYMORPHONUCLEAR LEUKOCYTES (neutrophils) play an important role in early host defense against invading bacteria, fungi, and protozoa. In response to infection, neutrophils are recruited from the bloodstream and migrate toward inflammatory foci. Exocytosis of granules and secretory vesicles plays a crucial role in neutrophil functions (reviewed in Faurschou and Borregaard 2003). Based on their protein content, neutrophil granules have traditionally been divided into several types, e.g., azurophilic (primary), specific (secondary), and gelatinase (tertiary) granules and secretory vesicles (Sengelov 1996; Borregaard and Cowland 1997). The various subsets of granules contain a multitude of antimicrobial proteins, proteases, components of the respiratory burst oxidase, and membrane-bound receptors. The regulated exocytosis of these granules enables the neutrophil to deliver the potentially cytotoxic granule proteins in a targeted manner. Much interest has been focused on the various granule types and their constituents to identify novel antibiotics for clinical use and to understand the molecular basis of neutrophil-mediated inflammatory disorders.

The small 13-kDa protein syncollin was first identified as a component of zymogen granules derived from exocrine pancreatic acinar cells (Edwardson et al. 1997). A thorough biochemical characterization of pancreatic syncollin revealed several unusual properties. Syncollin can form oligomeric structures, most likely hexamers, and is predominantly associated with the luminal surface of the zymogen granule membrane (An et al. 2000; Hodel et al. 2001; Geisse et al. 2002). In contrast to typical peripheral membrane proteins, it resists salt washing of the granule membrane but is removed under alkaline conditions when it becomes monomerized (An et al. 2000). Cross-linking studies revealed an association of syncollin with GP-2, the major glycoprotein present in the granule membrane. Both proteins are associated with detergent-insoluble, cholesterol-enriched complexes, indicating that they are present in lipid microdomains (Kalus et al. 2002). Interestingly, purified syncollin binds to liposomes in a pH- and cholesterol-dependent manner, and cholesterol can be coimmunoprecipitated with syncollin (Hodel et al. 2001). The notion that syncollin exists as a homo-oligomer in conjunction with its lipid interaction has led to the suggestion that syncollin might have pore-forming properties. In support of this idea, the oligomer has been shown to form a doughnut-shaped, pore-like structure, both when bound to a solid support and in association with a lipid bilayer (Geisse et al. 2002). Furthermore, purified native syncollin was able to permeabilize both liposomes (Geisse et al. 2002) and erythrocytes (Wasle et al. 2004).

Despite this thorough biochemical characterization, the physiological role of syncollin is still a matter of debate. Previous studies of syncollin knockout (KO) mice revealed abnormalities on the secretory pathway in the exocrine pancreas and a higher susceptability for a hormone-induced pancreatitis, but no changes in secretion of digestive enzymes (Antonin et al. 2002). In contrast, Wäsle et al. (2005) found that pancreatic secretion in KO mice was significantly compromised and concluded that syncollin is required for efficient exocytosis in pancreatic acinar cells.

In this study we present evidence that syncollin is also present in neutrophilic granulocytes of rat and human origin. We show that syncollin is expressed and secreted by promyelocytic HL-60 cells where it is found to be partially associated with myeloperoxidase (MPO)-containing secretory granules. An additional function for syncollin in host defense is discussed.

	Materials and Methods

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 
Antibodies
Antibodies used were as follows: 87.1, a mouse monoclonal antibody against recombinant syncollin (An et al. 2000); anti-syncollin B and FG, two polyclonal antibodies raised against recombinant syncollin (B) (Hodel and Edwardson 2000) or a C-terminal peptide (FG) (Edwardson et al. 1997) (kindly provided by J.M. Edwardson, Department of Pharmacology, University of Cambridge, Cambridge, UK); rabbit polyclonal (kindly provided by A. Hasilik, University of Marburg, Marburg, Germany), and mouse monoclonal (PeliCluster; Amsterdam, The Netherlands) antibodies against MPO; RP-1, a mouse monoclonal antibody against rat neutrophils (BD Biosciences-Pharmingen; San Diego, CA); and CD63 Ab-1, a mouse monoclonal against CD63 (Lab Vision Corp.; Fremont, CA). Species-specific anti-IgG antibodies conjugated to horseradish peroxidase (HRP) or to the fluorophores TRITC and Alexa Fluor 488 were obtained from BioRad (Richmond, CA) or Molecular Probes Europe (Leiden, The Netherlands).

Animals and Tissues
Animals were handled according to German law for the protection of animals, with the permission of the local authorities. Male Wistar rats (Charles River; Sulzfeld, Germany) weighing 200–230 g were killed by exsanguination. Tissues were dissected from the animal, immediately frozen in liquid nitrogen, and stored at –80C until use.

Cell Culture, Neutrophil Isolation, and Stimulation of Cells
The human promyelocytic HL-60 cell line (kindly provided by A. Hasilik, University of Marburg, Marburg, Germany) was cultured in RPMI medium containing 10% fetal calf serum (FCS). The human mast cell line HMC-I (kindly provided by J.H. Butterfield, Allergic Diseases and Internal Medicine, Mayo Clinic, Rochester, MI) was grown in Iscove's modified Dulbecco's medium containing 10% FCS and 1.2 mM -thioglycerol. RBL cells (a rat mast cell line kindly provided by U. Lippert, Department of Dermatology, University of Goettingen, Goettingen, Germany) were cultured in DMEM containing 15% FCS and 1.2 mM sodium pyruvate. The mouse macrophage cell line RAW-264.7 (kindly provided by K. Heeg, University of Marburg) was grown in DMEM containing 15% FCS. HepG2 and AR42J cells were cultured in DMEM containing 10% FCS. All media were supplemented with 2 g/liter sodium bicarbonate, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from PAA Laboratories; Cölbe, Germany), and cells were maintained at 37C in a humidified atmosphere containing 5% CO₂. Human neutrophils (>80% pure, as verified by light and fluorescence microscopy) were isolated from the blood of healthy volunteers by density gradient centrifugation over Ficoll–Hypaque. Neutrophils were separated from erythrocytes by sedimentation in 1.5% dextran and subsequent hypotonic lysis of the remaining erythrocytes. Bone marrow was isolated from the tibia and femur of Wistar rats. Isolated monocytes from human blood (95% purity) were kindly provided by A. Kaufmann (University of Marburg). For stimulation experiments, HL-60 cells were either incubated in serum-free medium (Panserin 401; PAN Biotech GmbH, Aidenbach, Germany) or resuspended in a Na⁺-based solution [127 mM NaCl, 1.2 mM KH₂PO₄, 5.4 mM KCl, 0.8 mM MgSO₄, 1.8 mM CaCl₂, 5.6 mM glucose, 10 mM N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES), pH 7.3]. Cells were incubated in the absence or the presence of phorbol 12-myristate 13-acetate (PMA; 100 nM) for different time intervals at 37C. Cells were spun down afterwards, and the pellet and supernatant fractions were analyzed by immunoblotting or by ELISA. Lactate dehydrogenase (LDH) activity was assayed using commercially available test kits (Boehringer Mannheim; Mannheim, Germany).

Enzyme-linked Immunosorbent Assay
Microtiter plates were coated by overnight incubation at 4C with dilutions of experimental media. Plates were washed six times with phosphate-buffered saline (PBS) (116.4 mM NaCl, 4.9 mM Na₂HPO₄, 1.7 mM KH₂PO₄, pH 7.3) containing 0.05% Tween 20 and were thereafter blocked with 200 µl/well washing buffer containing 1% bovine serum albumin (BSA) for 2 hr at room temperature (RT). This was followed by incubation with antibodies against syncollin (B) and MPO (pcRb) (100 µl/well, dilution 1:1000) for 1 hr at RT. Bound antibody was detected using the appropriate secondary antibody coupled to HRP (100 µl/well, dilution 1:2000) and a chromogenic substrate solution (100 µg/ml tetramethylbenzidine in 100 mM sodium acetate, pH 6.0, 2 µl/well 30% H₂O₂). The reaction was stopped after 10–15 min by adding 1 M H₂SO₄. Each incubation step was followed by a washing step.

Indirect Immunofluorescence and Deconvolution
Cryostat sections of rat tissues and cultured cells were fixed with 4% paraformaldehyde in PBS, pH 7.4, or with cold acetone. Non-adherent cells were carefully spun down on poly-L-lysine-coated coverslips after fixation (500 g, 3 min). Samples were permeabilized with 0.2% Triton X-100, blocked with 1% BSA, and incubated with primary and secondary antibodies as described (Schrader et al. 1996). Appropriate controls were performed without primary antibodies and with anti-syncollin antibodies preincubated with isolated syncollin. Nuclei were stained with Hoechst 33,258. Samples were examined using a Leitz Diaplan (Leica; Wetzlar, Germany) or an Olympus BX-61 microscope (Olympus Optical Co. GmbH; Hamburg, Germany) equipped with the appropriate filter combinations and photographed on Kodak TMY film (Kodak; Stuttgart, Germany) or digitalized using an F-view II CCD camera (Soft Imaging System GmbH; Münster, Germany). Deconvolution studies were performed with a Soft Imaging System using an Olympus BX-61 microscope for optical sectioning. Deconvolved images were taken at 200-nm intervals with a x100 Plan-Neofluar objective with a 1.35 numerical aperture through focus. Digital images were optimized for contrast and brightness using Adobe Photoshop (Palo Alto, Oberkochen, CA) software.

Electron Microscopy
For routine electron microscopy, cultured cells were fixed with Ito-fixative (Ito and Karnovsky 1968) for 30 min at RT. After postfixation with 1% K₄Fe(CN)₆-reduced osmium tetroxide (1 hr at 4C), samples were stained with 0.3% uranyl acetate. For cytochemical localization of MPO, HL-60 cells were incubated in DAB medium (0.2% 3,3'-diaminobenzidine, 0.15% H₂O₂ in 0.1 M cacodylate buffer, pH 7.35) for 1 hr at RT followed by postfixation in 1% K₄Fe(CN)₆-reduced osmium tetroxide and 0.3% uranyl acetate. Samples were dehydrated in a graded series of alcohol and embedded in Epon 812 (Polysciences Ltd.; Eppenheim, Germany). For immunostaining, samples were fixed in 0.1 M cacodylate buffer, pH 7.35, containing 1% paraformaldehyde (Serva; Heidelberg, Germany). The samples were dehydrated in a graded series of alcohol, embedded in Lowicryl K4M (Polysciences Ltd.), and polymerized at –20C and UV light (360 nm) for 48 hr. Thin sections (70 nm) were incubated with the polyclonal antibodies B or FG directed against syncollin in a dilution of 1:200 to 1:500 and visualized using 10-nm protein A–gold solution (kindly provided by Dr. J. Slot, University of Utrecht, The Netherlands) at a dilution of 1:60 or 1:70, both in 0.5% BSA in PBS. Appropriate controls were performed with anti-syncollin antibodies preincubated with isolated syncollin. Sections were stained with uranyl acetate/lead citrate and analyzed using a Zeiss EM 109 electron microscope (Zeiss; Oberkochen, Germany).

Isolation of Zymogen Granules and Purification of Syncollin
Zymogen granules were isolated as described previously (Dartsch et al. 1998) from the pancreas of male Wistar rats (200–230 g) (Charles River) that were fasted overnight. The following buffer was used for homogenization: 0.25 M sucrose, 5 mM 2-N-morpholino-ethanesulfonic acid, pH 6.25, 0.1 mM Mg SO₄, 1 mM dithiothreitol, 10 µM Foy-305 (Sanol Schwarz; Monheim, Germany), 2.5 mM Trasylol (Bayer; Leverkusen, Germany), and 0.1 mM phenylmethylsulfonyl fluoride. Granules were resuspended in 50 mM HEPES, pH 8.0, and lysed by freezing and thawing. Granule membranes and content were separated by centrifugation at 100,000 x g for 30 min. Syncollin was purified as described recently (An et al. 2000). Briefly, freshly isolated granule membranes were washed with 0.6 M KI for 30 min at 4C with gentle agitation. The membranes were then recovered by centrifugation at 21,000 x g for 20 min and washed with 0.1 M Na₂CO₃, again for 30 min at 4C. The membranes were pelleted by centrifugation at 21,000 x g for 30 min. The supernatant was recovered and dialyzed overnight against 50 mM HEPES, pH 7.6. The resulting precipitate was collected by centrifugation at 21,000 x g for 5 min. The pellet was either dissolved in 50–100 µl of 0.5% taurodeoxycholate in 50 mM HEPES, pH 7.6, or resuspended in 50 mM HEPES, pH 8.0.

Subcellular Fractionation
Subcellular fractionation of HL-60 cells was performed by density centrifugation on OptiPrep (Axis-Shield; Oslo, Norway) gradients. Cells were homogenized in disruption buffer (250 mM saccharose, 10 mM HEPES, pH 7.4, 0.3 mM Na-EDTA, 10 µM Foy-305 (Sanol Schwarz), 2.5 mM Trasylol (Bayer), 0.1 mM phenylmethylsulfonyl fluoride) using a Potter S homogenizer (Braun; Melsungen, Germany). Nuclei and unbroken cells were sedimented by centrifugation at 500 x g for 5 min at 4C. The postnuclear supernatant (PNS) was further separated in a flotation gradient to isolate secretory vesicles from plasma membranes (Dahlgren et al. 1995). The PNS (7 ml) was mixed with a heavy OptiPrep solution (7 ml, 1.12 g/ml). The mixture was layered under 14 ml light OptiPrep solution (1.05 g/ml). Five ml heavy OptiPrep solution was applied to the bottom of the tube. Relaxation buffer (5 ml) was applied on top of the gradient. The gradient was centrifuged at 37,000 x g for 35 min at 4C using a fixed-angle Beckman JA-20 rotor (Beckman Instruments, Inc.; Palo Alto, CA). After centrifugation, 1-ml fractions were collected from the bottom of the tube. Proteins were precipitated with 10% (w/v) trichloracetic acid (TCA) and analyzed by gel electrophoresis and immunoblotting.

Gel Electrophoresis and Immunoblotting
Cells were either solubilized in lysis buffer [25 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.5% deoxycholate, and 0.5% Triton X-100 supplemented with 1 mM phenylmethylsulfonyl fluoride, 10 µM Foy-305 (Sanol Schwarz), and 2.5 mM Trasylol (Bayer)] for 30 min at 4C or were homogenized as described above. Protein samples were boiled in SDS sample buffer containing DTT, separated by SDS-PAGE, transferred to nitrocellulose (Schleicher and Schüll; Dassel, Germany) using a semi-dry apparatus, and analyzed by immunoblotting. INSTA-Blot ready-to-use membranes were obtained from Imgenex (San Diego, CA). A membrane contained 10 µg per lane of different rat tissue lysates, which were separated on 12.5% acrylamide gels and transferred to Immobilon PVDF membranes. Immunoblots were processed using HRP-conjugated secondary antibodies and enhanced chemiluminescence reagents (Amersham; Arlington Heights, IL). For quantification, immunoblots were scanned and processed using Pcbas software (Raytest; Straubenhardt, Germany).

Isolation of RNA, Reverse Transcription, and PCR
RT-PCR was performed to amplify parts of the coding sequence of rat (GenBank accession number NM_139086) (Edwardson et al. 1997), mouse (GenBank accession number NM_026716), and human (GenBank accession number BC039541) syncollin. Total RNA was isolated using the RNeasy Protect Mini Kit (Qiagen; Hilden, Germany), and reverse transcribed using an oligo(dT) primer and AMV reverse transcriptase (Clontech; Mountain View, CA) at 42C. PCR was performed with 100 ng of template using the following primer pairs: 5'-CAC TTG CGC CAA GCT CTA TGA-3' (forward primer hs syncollin.up); 5'-GCA GTA GAG CGC GGA GAT AGC-3' (reverse primer hs syncollin.down); 5'-TCC CCC CGG GAC CAT GGC TTG TCC AGT GCC CGC A-3' (forward primer rat syncollin.up); 5'-AGC AAT AGC ACT TGC AGT AGA-3' (reverse primer rat syncollin. down); 5'-CCC ACT GCT GCT GGC TTT A-3' (forward primer mouse syncollin.up); 5'-TTG CAG TAG AGG GCA GAG ATG-3' (reverse primer mouse syncollin.down). Samples were analyzed by agarose gel electrophoresis.

RNA Quantitation by Northern- and Dot-blot RNase Protection Assay
For Northern blots, 15 µg of total RNA was subjected to denaturing agarose gel electrophoresis followed by overnight capillary blotting. ³²P-labeled probes were generated by random primed labeling of syncollin-specific cDNA fragments using a Hexalabel DNA labeling kit (Fermentas; St. Leon-Rot, Germany). The probe was purified via a Microspin G50 column (Amersham) to exclude unbound radioactivity. Blots were prehybridized in ultrasensitive hybridization buffer (Ultrahyb; Ambion, Austin, TX) for 1 hr at 42C. Hybridization was performed at 42C overnight. For quantitative analysis of mRNA, a dot-blot RNase protection assay was used (Zhan et al. 1997). Briefly, 1–5 µg RNA was directly applied in a constant volume onto a nylon membrane (Qiagen; Chatsworth, CA), hybridized with the above probe, and incubated with RNase A (1 µg/ml). The membranes were exposed to autoradiographic film (Biomax; Kodak) and quantified by densitometric analysis. The methylene blue-stained 18S rRNA band was used to normalize the amount of loaded RNA and to correct for minor loading variations.

Statistical Analysis of Data
Significant differences between experimental groups were detected by ANOVA for unpaired variables using Microsoft Excel. Data are presented as means ± SD, with an unpaired t-test used to determine statistical differences; p values <0.05 are considered as significant, and p values <0.01 are considered as highly significant.

	Results

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 
Immunohistological Localization of Syncollin
Syncollin was first identified as a secretory granule protein of the rat pancreas (Edwardson et al. 1997) but was also reported to be expressed in the small intestine, colon, spleen, and parotid gland using in situ hybridization or PCR techniques (Tan and Hooi 2000; Imai et al. 2001). To identify the cells expressing syncollin in the spleen, we first performed immunohistological studies using rat tissues (Figure 1 ). Because the localization of syncollin to the exocrine pancreas has been confirmed in several studies (Edwardson et al. 1997; An et al. 2000; Hodel et al. 2001; Antonin et al. 2002; Kalus et al. 2002; Wasle et al. 2005), rat pancreas served as a positive control. A specific granular staining of the apical region of the acinar cells was obtained when the polyclonal antibodies B and FG specific for rat syncollin were used (Figures 1A and 1B). The endocrine pancreas with the islets of Langerhans, or interlobular septa, blood vessels, and connective tissues showed no labeling. We observed that staining of cryosections with syncollin antibody B required paraformaldehyde fixation, whereas staining with antibody FG was improved by acetone fixation. In the spleen, single cells or small groups of cells were stained by the syncollin antibodies showing a fine granulovesicular staining pattern (Figures 1C and 1D). Interestingly, the syncollin-positive cells all had lobulated nuclei indicating that they were neutrophilic polymorphonuclear leukocytes. This notion was confirmed by costaining of the cells with a monoclonal antibody specific for rat neutrophilic granulocytes (Figures 1E and 1F). Other leukocytes such as B- or T-lymphocytes of the white pulp were not positive for syncollin. In controls where the immunoreactivity of the syncollin antibodies was blocked by preincubation with isolated syncollin, no labeling of subcellular structures was observed.

View larger version (40K): [in this window] [in a new window]   Figure 1 Immunohistological localization of syncollin. Cryosections of rat pancreas (A,B) and spleen (C–H) were incubated with polyclonal antibodies specific for syncollin (B,D,E,H). Nuclei were stained with Hoechst 33,258 (A,C,G). Arrows in C,D point to neutrophilic polymorphonuclear leukocytes that are positive for syncollin. For better orientation, some polymorph nulclei have been encircled in D. (E,F) Costaining of the syncollin-positive cells in the spleen (E) with a monoclonal antibody specific for rat neutrophilic granulocytes (F). Nuclei are marked with asterisks. (D) Control where the immunoreactivity of the syncollin antibody has been blocked with antigen. Bars: A,B = 10 µm; C,D,G,H = 20 µm; E,F = 5 µm.

View larger version (24K): [in this window] [in a new window]   Figure 2 Tissue-specific distribution and expression of syncollin in the rat. (A) Lysates of different rat tissues were separated on 12.5% acrylamide gels, and immunoblots were performed with a specific monoclonal antibody against syncollin (87.1). The positions of molecular mass markers (in kDa) are indicated on the right. (B) Total RNA was isolated from different rat tissues and pancreatic AR42J cells. RT-PCR was performed with syncollin-specific primer pairs, and the PCR products were separated by agarose gel electrophoresis on 1% agarose gels. Note that the PCR data are not quantitative.

View larger version (16K): [in this window] [in a new window]   Figure 3 Northern- and dot-blot RNase protection assay for syncollin mRNA. (A) Northern analysis showing rat syncollin mRNA in total RNA samples (15 µg) from rat pancreas (Lane 1) and bone marrow (Lane 2). Total RNA was separated by denaturing agarose gel electrophoresis, transferred to nylon membranes, and probed with a radioactively labeled fragment of syncollin cDNA. The abundance of 18S rRNA in each lane is shown separately in the lower panel. (B) Dot-blot RNase protection assay for quantification of syncollin mRNA in total RNA samples from rat pancreas (1), bone marrow (2), and skeletal muscle (3), which served as a negative control. Equal amounts of total RNA (1–5 µg/dot) were directly dotted in a constant volume onto a nylon membrane and hybridized followed by RNase digestion of non-hybridized RNA. Signals obtained from several dot blots were quantified by densitometry and represent relative values for comparison. The 18S rRNA was used to normalize the amount of loaded RNA and to correct for minor loading variations.

View larger version (17K): [in this window] [in a new window]   Figure 4 Syncollin is expressed by human neutrophils. Purified syncollin (A) (Lane 1; 3 µg/lane) and a postnuclear supernatant of HL-60 cells (A) (Lane 2; 30 µg/lane) or of isolated neutrophilic granulocytes from human blood (B) (60 µg/lane) were separated on 12.5% acrylamide gels, and immunoblots were performed with a specific monoclonal antibody against syncollin (87.1). Potential homo-oligomers of syncollin are labeled with asterisks. The positions of molecular mass markers (in kDa) are indicated on the right. The immunoblot in B has been overexposed due to the low amount of syncollin. (C) Total RNA was isolated from HL-60 cells, isolated monocytes from human blood, RBL (rat basophilic granulocytes), HepG2 (human hepatoblastoma cells), HMC-1 (human mast cells), and RAW-264.7 cells (mouse macrophage cells). cDNA from human (or rat; see Figure 2) pancreas was used as a positive control. RT-PCR was performed with species-specific syncollin primer pairs, and the PCR products were separated by agarose gel electrophoresis on 1% agarose gels. Note that the PCR data are not quantitative.

View larger version (102K): [in this window] [in a new window]   Figure 5 Subcellular localization of syncollin in HL-60 cells. (A–H) Immunofluorescence microscopy of HL-60 cells (A–E) and isolated human neutrophils (hGC) (F–H) incubated with syncollin antibody B (Sync) (A–C,F) and a monoclonal antibody to myeloperoxidase (MPO) (D,G). (C–H) Deconvolved images out of a stack of images taken at 200-nm intervals of the immunofluorescence preparations. Arrows highlight some regions of colocalization. Bar = 5 µm.

View larger version (51K): [in this window] [in a new window]   Figure 6 Ultrastructural localization of syncollin in HL-60 cells. (A) Ultrastructure of granules (Gr; asterisks) in HL-60 cells processed for routine electron microscopy. (B) Diaminobenzidine (DAB) cytochemistry for MPO in HL-60 cells. (C–E) HL-60 cells were processed for immunogold electron microscopy, incubated with the polyclonal anti-syncollin antibody B, and visualized using 10-nm protein A–gold. (D) Control where the immunoreactivity of the syncollin antibody has been blocked with antigen. Arrows point to gold particles on azurophilic granules. Bar = 500 nm.

View larger version (23K): [in this window] [in a new window]   Figure 7 Localization of syncollin in subcellular fractions from HL-60 cells. (A) HL-60 cells were homogenized and fractionated in a flotation gradient (see Materials and Methods). Immunoblots of the fractions were probed with antibodies to syncollin (Sync) (87.1), MPO, and CD63. (B) The resulting immunoblots were quantified by densitometry.

View larger version (12K): [in this window] [in a new window]   Figure 8 Release of syncollin from HL-60 cells. (A,B) HL-60 cells were incubated in the absence of a stimulus or in the presence of phorbol 12-myristate 13-acetate (PMA; 100 nM) at 37C. (A) HL-60 cells were stimulated in a serum-free culture medium. After 4 hr, the medium was collected and proteins were precipitated by trichloroacetic assay. Equal amounts of protein (200 µg/lane) were further analyzed by SDS-PAGE (12.5% acrylamide) and immunoblotting, using antibodies against syncollin (87.1) and MPO. Background staining of BSA (due to its high concentration in the medium) (asterisks) indicates equal loading. Lactose dehydrogenase activity was measured to control for cell breakage. (B) HL-60 cells were treated at 37C with PMA for 30 min or were incubated for 30 min at 37C or 4C without stimulation in a Na+-based solution. The release of syncollin and MPO was determined by an indirect ELISA. Data represent the relative release of syncollin and MPO (compared with the total amount of these proteins in the medium and cell lysates) and are the mean ± SD of four experiments, each performed in duplicate (p<0.01).

	Discussion

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

A recent study has presented the proteome of granules isolated from human polymorphonuclear neutrophils from peripheral blood (Lominadze et al. 2005). Unfortunately, syncollin has so far not been detected by this group. This might be due to the low amount of syncollin present in azurophilic granules from human neutrophils. As indicated, we have chosen rat tissues and HL-60 cells for our studies because syncollin appeared to be less prominent in neutrophils isolated from human blood. It is currently not clear if and how the expression of syncollin in neutrophils is regulated and if such a regulation shows variations in rodents and humans. It is possible that the level of syncollin is upregulated when the neutrophils leave the circulation and enter the lymphatic organs or during the course of an infection. Furthermore, the detection of cationic syncollin on 2D gels is hampered by the fact that MPO, which is a very prominent cationic protein of azurophilic granules, forms a huge spot on 2D gels that covers most of the basic region.

Our data confirm that syncollin is not exclusively expressed by pancreatic acinar cells. In addition to neutrophils, it appears to be present (although in lower abundance relative to rat pancreas) in the duodenum and colon (Tan and Hooi 2000), in the parotid gland (Imai et al. 2001), and according to a recent report in lacrimal acinar cells of the rabbit (Jerdeva et al. 2003). In most cases, the secretory products of these cell types are packaged in secretory granules or vesicles and are released in a regulated manner.

The physiological role of syncollin is still unclear. However, evidence has been presented that it is required for efficient exocytosis in the pancreatic acinar cell, particularly in compound exocytosis (Wasle et al. 2005). As azurophilic granules in neutrophils are regarded as regulated, secretetory organelles (Cieutat et al. 1998), syncollin might also play a role in granule exocytosis of neutrophils. However, syncollin is a secretory protein and does not localize to all but only to a subset of azurophilic granules in HL-60 cells arguing somewhat against a prominent function in neutrophil exocytosis. An interesting, alternative function might be a role for syncollin in host defense. Neutrophils, and especially azurophilic granules, contain several biologically active molecules. Microbicidal polypeptides, such as defensins, lysozyme, bactericidal permeability-increasing protein, and MPO are mostly cationic at physiological pH and thus are able to bind to the negatively charged surface of bacteria. Interestingly, syncollin is also a cationic protein with a theoretical isoelectric point of 8.6. Furthermore, pancreatic syncollin has been shown to interact with lipids and to form oligomeric, doughnut-shaped structures that might have pore-forming properties (Geisse et al. 2002). Purified native syncollin from rat pancreas was able to permeabilize both liposomes (Geisse et al. 2002) and erythrocytes (Wasle et al. 2004). Based on the biochemical properties and the expression pattern of syncollin, it is tempting to speculate that it might fulfill different functions before and after secretion. As shown recently by proteomic studies (Gronborg et al. 2004), syncollin is also a component of the pancreatic juice. In the pancreatic acinar cell where it is best studied, it might be involved in the exocytosis of zymogen granules (Wasle et al. 2005) and afterwards might fulfill a protective or defensive function within the pancreatic duct and the intestinal lumen. This hypothesis would nicely fit the observation that syncollin is expressed by pancreatic, parotic, and lacrimal acinar cells as well as by intestinal epithelial cells.

To examine a possible microbicidal role of syncollin, we have studied the effect of isolated native syncollin from rat pancreas on the growth of different aerobic (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa) and anaerobic (Bacteroides fragilis, Prevotella buccalis, Fusobacterium nucleatum, Veillonella sp.) bacteria usually found in the intestinal tract using minimal inhibitory concentration and 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays. In these preliminary experiments we could not detect a microbicidal effect of syncollin (unpublished observation). Although isolated syncollin was able to permeabilize liposomes and erythrocytes (Geisse et al. 2002; Wasle et al. 2004), we cannot exclude that additional factors are required for efficient permeabilization of bacterial membranes that are not/no longer present in our syncollin preparation. It is likely that potential pore-forming properties of syncollin will be regulated. A potential binding partner of syncollin in the pancreas is GP-2, the major glycosylphosphatidylinositol (GPI)-anchored glycoprotein within zymogen granule membranes (Kalus et al. 2002). The physiological role of GP-2 is also a matter of debate (Scheele et al. 1994; Schmidt et al. 2001), but based on studies with GP-2 KO mice an extracellular function has been suggested (Yu et al. 2004). Interestingly, uromodulin/THP, a homolog of GP-2 in the kidney, which is also secreted after cleavage of its GPI-anchor, is suggested to function in host cell defense against infection by binding to E. coli and preventing bacterial adherence to the host cell (Bates et al. 2004; Mo et al. 2004). However, an additional function in the regulation of transporters has recently been proposed (Bachmann et al. 2005). Without doubt, further work is required to elucidate the functions and the importance of syncollin during and after acinar and neutrophil secretion.

	Acknowledgments

 
This study was partially supported by the Jürgen Manchot foundation (Düsseldorf, Germany) (MS,HB).

The authors thank all the researchers mentioned in Materials and Methods who have kindly provided antibodies, cell lines, or other reagents. We are grateful to K. Heeg and S. Zimmermann (Department of Medical Microbiology, University of Marburg, Marburg, Germany) for examining the putative microbicidal role of syncollin, and we thank G. Schneider, B. Agricola, and V. Kramer for excellent technical assistance.

	Footnotes

 
Received for publication July 26, 2005; accepted February 22, 2006

	Literature Cited

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

 

An SJ, Hansen NJ, Hodel A, Jahn R, Edwardson JM (2000) Analysis of the association of syncollin with the membrane of the pancreatic zymogen granule. J Biol Chem 275:11306–11311[Abstract/Free Full Text]

Antonin W, Wagner M, Riedel D, Brose N, Jahn R (2002) Loss of the zymogen granule protein syncollin affects pancreatic protein synthesis and transport but not secretion. Mol Cell Biol 22:1545–1554[Abstract/Free Full Text]

Bachmann S, Mutig K, Bates J, Welker P, Geist B, Gross V, Luft FC, et al. (2005) Renal effects of Tamm-Horsfall protein (uromodulin) deficiency in mice. Am J Physiol Renal Physiol 288:F559–567[Abstract/Free Full Text]

Bates JM, Raffi HM, Prasadan K, Mascarenhas R, Laszik Z, Maeda N, Hultgren SJ, et al. (2004) Tamm-Horsfall protein knockout mice are more prone to urinary tract infection: rapid communication. Kidney Int 65:791–797[CrossRef][Medline]

Borregaard N, Cowland JB (1997) Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89:3503–3521[Free Full Text]

Cieutat AM, Lobel P, August JT, Kjeldsen L, Sengelov H, Borregaard N, Bainton DF (1998) Azurophilic granules of human neutrophilic leukocytes are deficient in lysosome-associated membrane proteins but retain the mannose 6-phosphate recognition marker. Blood 91:1044–1058[Abstract/Free Full Text]

Dahlgren C, Carlsson SR, Karlsson A, Lundqvist H, Sjolin C (1995) The lysosomal membrane glycoproteins Lamp-1 and Lamp-2 are present in mobilizable organelles, but are absent from the azurophil granules of human neutrophils. Biochem J 311:667–674

Dartsch H, Kleene R, Kern HF (1998) In vitro condensation-sorting of enzyme proteins isolated from rat pancreatic acinar cells. Eur J Cell Biol 75:211–222[Medline]

Edwardson JM, An S, Jahn R (1997) The secretory granule protein syncollin binds to syntaxin in a Ca2(+)- sensitive manner. Cell 90:325–333[CrossRef][Medline]

Faurschou M, Borregaard N (2003) Neutrophil granules and secretory vesicles in inflammation. Microbes Infect 5:1317–1327[CrossRef][Medline]

Geisse NA, Wasle B, Saslowsky DE, Henderson RM, Edwardson JM (2002) Syncollin homo-oligomers associate with lipid bilayers in the form of doughnut-shaped structures. J Membr Biol 189:83–92[CrossRef][Medline]

Gronborg M, Bunkenborg J, Kristiansen TZ, Jensen ON, Yeo CJ, Hruban RH, Maitra A, et al. (2004) Comprehensive proteomic analysis of human pancreatic juice. J Proteome Res 3:1042–1055[CrossRef][Medline]

Hodel A, An SJ, Hansen NJ, Lawrence J, Wasle B, Schrader M, Edwardson JM (2001) Cholesterol-dependent interaction of syncollin with the membrane of the pancreatic zymogen granule. Biochem J 356:843–850[CrossRef][Medline]

Hodel A, Edwardson JM (2000) Targeting of the zymogen-granule protein syncollin in AR42J and AtT-20 cells. Biochem J 350:637–643

Imai A, Nashida T, Shimomura H (2001) mRNA expression of membrane-fusion-related proteins in rat parotid gland. Arch Oral Biol 46:955–962[CrossRef][Medline]

Ito S, Karnovsky MJ (1968) Formaldehyd-glutaraldehyd fixatives containing trinitro compounds. J Cell Biol 39:1680–1690

Jerdeva GV, Yarber FA, Mircheff AK, Hamm-Alvarez SF (2003) Association of syncollin with secretory vesicles in primary rabbit lacrimal acinar cells. Mol Biol Cell 14(suppl):251a

Kalus I, Hodel A, Koch A, Kleene R, Edwardson JM, Schrader M (2002) Interaction of syncollin with GP-2, the major membrane protein of pancreatic zymogen granules, and association with lipid microdomains. Biochem J 362:433–442[CrossRef][Medline]

Lominadze G, Powell DW, Luerman GC, Link AJ, Ward RA, McLeish KR (2005) Proteomic analysis of human neutrophil granules. Mol Cell Proteomics 4:1503–1521[Abstract/Free Full Text]

Mo L, Zhu XH, Huang HY, Shapiro E, Hasty DL, Wu XR (2004) Ablation of the Tamm-Horsfall protein gene increases susceptibility of mice to bladder colonization by type 1-fimbriated Escherichia coli. Am J Physiol Renal Physiol 286:F795–802[Abstract/Free Full Text]

Scheele GA, Fukuoka S, Freedman SD (1994) Role of the GP2/THP family of GPI-anchored proteins in membrane trafficking during regulated exocrine secretion. Pancreas 9:139–149[Medline]

Schmidt K, Schrader M, Kern HF, Kleene R (2001) Regulated apical secretion of zymogens in rat pancreas: involvement of the GPI-anchored glycoprotein GP-2, the lectin ZG16p and cholesterol-glycosphingolipid enriched microdomains. J Biol Chem 276:14315–14323[Abstract/Free Full Text]

Schrader M, Burkhardt JK, Baumgart E, Luers G, Spring H, Volkl A, Fahimi HD (1996) Interaction of microtubules with peroxisomes. Tubular and spherical peroxisomes in HepG2 cells and their alterations induced by microtubule-active drugs. Eur J Cell Biol 69:24–35[Medline]

Sengelov H (1996) Secretory vesicles of human neutrophils. Eur J Haematol Suppl 58:1–24[Medline]

Tan S, Hooi SC (2000) Syncollin is differentially expressed in rat proximal small intestine and regulated by feeding behavior. Am J Physiol Gastrointest Liver Physiol 278:G308–320[Abstract/Free Full Text]

Wasle B, Hays LB, Rhodes CJ, Edwardson JM (2004) Syncollin inhibits regulated corticotropin secretion from AtT-20 cells through a reduction in the secretory vesicle population. Biochem J 380:897–905[CrossRef][Medline]

Wasle B, Turvey M, Larina O, Thorn P, Skepper J, Morton AJ, Edwardson JM (2005) Syncollin is required for efficient zymogen granule exocytosis. Biochem J 385:721–727[CrossRef][Medline]

Yu S, Michie SA, Lowe AW (2004) Absence of the major zymogen granule membrane protein, GP2, does not affect pancreatic morphology or secretion. J Biol Chem 279:50274–50279[Abstract/Free Full Text]

Zhan J, Fahimi HD, Voelkl A (1997) Sensitive nonradioactive dot blot/ribonuclease protection assay for quantitative determination of mRNA. Biotechniques 22:500–505[Medline]

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. Zhang, M.-L. Bang, D. S. Gokhin, Y. Lu, L. Cui, X. Li, Y. Gu, N. D. Dalton, M. C. Scimia, K. L. Peterson, et al. Syncoilin is required for generating maximum isometric stress in skeletal muscle but dispensable for muscle cytoarchitecture Am J Physiol Cell Physiol, May 1, 2008; 294(5): C1175 - C1182. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jhc.5A6792.2006v1 54/8/877    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bach, J.-P. Articles by Schrader, M. Search for Related Content PubMed PubMed Citation Articles by Bach, J.-P. Articles by Schrader, M. Social Bookmarking                      What's this?

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact