Enrichment and analysis of secretory lysosomes from lymphocyte populations
© Schmidt et al; licensee BioMed Central Ltd. 2009
Received: 26 March 2009
Accepted: 29 July 2009
Published: 29 July 2009
In specialized cells, such as mast cells, macrophages, T lymphocytes and Natural Killer cells in the immune system and for instance melanocytes in the skin, secretory lysosomes (SL) have evolved as bifunctional organelles that combine degradative and secretory properties. Mutations in lysosomal storage, transport or sorting molecules are associated with severe immunodeficiencies, autoimmunity and (partial) albinism. In order to analyze the function and content of secretory lysosomes in different cell populations, an efficient enrichment of these organelles is mandatory.
Based on a combination of differential and density gradient centrifugation steps, we provide a protocol to enrich intact SL from expanded hematopoietic cells, here T lymphocytes and Natural Killer cells. Individual fractions were initially characterized by Western blotting using antibodies against an array of marker proteins for intracellular compartments. As indicated by the presence of LAMP-3 (CD63) and FasL (CD178), we obtained a selective enrichment of SL in one of the resulting organelle fractions. The robustness and reproducibility of the applied separation protocol was examined by a high-resolution proteome analysis of individual SL preparations of different donors by 2D difference gel electrophoresis (2D-DIGE).
The provided protocol is readily applicable to enrich and isolate intact secretory vesicles from individual cell populations. It can be used to compare SL of normal and transformed cell lines or primary cell populations from healthy donors and patients with lysosomal storage or transport diseases, or from corresponding mutant mice. A subsequent proteome analysis allows the characterization of molecules involved in lysosomal maturation and cytotoxic effector function at high-resolution.
Cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells deliver their cytolytic effector molecules via so-called secretory lysosomes (SL). These organelles contain a number of cytotoxic proteins and undergo regulated secretion in response to external stimuli, for example to eliminate virus-infected and tumorogenic cells by the induction of apoptosis. In most eukaryotic cells, lysosomes are acidic organelles equipped with hydrolases to degrade and recycle endogenous or exogenous biomolecules . Interestingly, in various hematopoietic cell lineages associated with secretory effector functions (CTL, NK, mast cells and macrophages), a subset of lysosomes gained a second function as a storage and transport vehicle for effector molecules [2, 3]. Similarly, outside the immune system, melanocytes utilize a comparable organelle to regulate pigmentation .
In mast cells, histamine and serotonin are the key factors associated with SL, whereas in macrophages, for example MHC molecules and cytokines are stored and transported via this compartment . As mentioned, SL of mature T and NK cells are characterized by the presence of cytotoxic effector molecules including perforin , granzymes , granulysin , and the TNF family protein FasL (CD178) [7, 8]. Interestingly, the 'packaging' of individual effector molecules is quite different. Granzymes and perforins appear to be stored at least in part in association with carrier proteins (e.g. serglycins) in the vesicular lumen to form dense core particles . In contrast, the FasL is a transmembrane protein that in fact might be 'directly' involved in the transport of SL during organelle maturation and effector responses. During maturation, FasL is transported to SL by SH3 domain proteins interacting with its proline-rich C-terminus. Subsequently, FasL might participate in connecting SL to the actin-cytoskeleton via its interaction with Nck and associated actin-regulatory proteins [10–12].
Genetic diseases based on defects of the secretory machinery such as the Chédiak-Higashi Syndrome (CHS), the Hermansky-Pudlak Syndrome (HPS) or the Griscelli Syndrome (GS) are characterized by a combination of immunodeficiency and albinism, reflecting the close relationship of the SL-related secretory pathway in hematopoietic cells and melanocytes as non-hematopoietic cells . Although the genetic defects have been mostly deciphered, their influence on the overall protein composition of secretory lysosomes is not completely understood. Of note, a number of mouse strains are available that reflect the pathophysiology in patients. As an example, LYST-deficient mice are the murine counterpart of CHS and thus suffer from immunodeficiency and also carry a respective fur phenotype ('beige mouse'). Cells of such mice harbor giant lysosomes, indicating that the lysosomal maturation and storage might still be operational, while exocytosis and recycling are severely impaired .
For the detailed analysis of the proteomic profile of SL, it is important to efficiently enrich or purify this compartment from a limited number of cells. In 1978, a first density gradient centrifugation protocol was developed to purify organelles from rat hepatocytes using a non-ionic X-ray contrast compound termed Metrizamide . Compared to traditional sucrose gradients, the advantage of Metrizamide was a much lower osmolarity that enabled adequate separation of lysosomes and mitochondria without artificially altering their density as for instance observed with Triton WR1339  or dextran . In the meantime, several non-ionic iodinated density gradient media have been developed, which are often derivatives of metrizoic acid, for example Nycodenz and its dimer Iodixanol (commercially available as OptiPrep™, a 60% solution of Iodixanol-5,5'- [(2-hydroxy-1,3-propanediyl)-bis(acetylamino)]bis [N, N'-bis(2,3-dihydroxypropyl-2,4,6-triiodo-1,3-benzenecarboxamide). The major advantage of Iodixanol in this context is its ability to form iso-osmotic solutions at high densities (up to 1.32 g/ml) . In 1994, Graham et al. first described the usage of Iodixanol as density gradient media and provided a first set of subcellular isolation protocols [18, 19].
Another standard gradient medium that also offers lower osmolarity and viscosity is Percoll™. It consists of colloidal silica coated with polyvinylpyrrolidone (PVP). In the last three decades, Percoll™ gradients were not only used to separate lymphocyte subpopulations but also to isolate cytotoxic granules of NK [20, 21] and T cell lines [21, 22]. Recently, several Percoll-based isolation protocols were compared with regard to the functionality of effector molecules in the obtained fractions in cytotoxicity assays . A major disadvantage of Percoll™ as a separation medium is that silica often has to be removed from the obtained fractions because it might interfere with downstream applications. Unfortunately, however, such washing steps are mostly accompanied by the loss of organelle integrity.
Here, we describe a rapid and reliable Iodixanol-based subcellular fractionation protocol that allows the substantial enrichment of intact secretory vesicles for example from in vitro expanded lymphocyte populations. Importantly, the obtained fractions are compatible with standard analyses such as Western blotting and even with CyDye labeling for subsequent high-resolution two-dimensional difference gel electrophoresis .
Electron micrographs of isolated vesicles
2D-DIGE of enriched secretory granules
Peptide mass fingerprinting of selected spots
Selected proteins identified by peptide mass fingerprinting.
MHC class II alpha chain
capping protein (actin filament), gelsolin-like
chaperonin containing TCP1, subunit 8
capping protein (actin filament), gelsolin-like
Similarity of individual SL preparations
In the present study we describe a reliable protocol for the enrichment of intact secretory vesicles for the subsequent detailed analysis of this subcellular compartment. Combinations of differential and density gradient centrifugation procedures are widely used to isolate organelle fractions. Iodinated, low osmotic media are advantageous in this context not only because of their low osmolarity but also because they lack silica, which might interfere with organelle integrity and subsequent analyses. Since the density of lysosomes might vary depending on the cell or tissue type, an optimization of gradient sizes and concentrations, as well as centrifugation setting is required to obtain reproducible results. We optimized this protocol for the rapid and reproducible enrichment of secretory lysosomes of lymphocyte subpopulations. Using this methodology, we already defined the SL proteome of NK cell lines and primary NK cells . In the present study, we tested the reproducibility and reliability of the procedure by comparing purified SL fractions of polyclonal activated T cell populations (PHA blasts) and subpopulations (CD4+ T cells) of different donors by subsequent Western blotting and high-resolution 2D difference gel electrophoresis. We show that the SL fractions of T cells from healthy donors are very similar with regard to the overall protein content, with only minor differences in MHC molecules or associated transporter proteins or chaperons. Thus, this methodology is apparently ideal as basis for the detailed investigation of consequences of genetic alterations associated with lysosomal transport and storage diseases.
One initial key step of organelle preparation is the disruption of the cells. We applied a mild dounce homogenization and empirically determined the number of strokes for optimal cell opening with minimal damage to organelles and nuclei by trypan blue staining. The first centrifugation step efficiently removed intact cells, nuclei (data not shown) and associated plasma membrane fragments (Figure 2). Since mitochondria are more sensitive to hydrostatic pressure , bottom loading of density gradients caused an increase in their density relative to that of lysosomes. As reported before, we also noted that in iodinated density gradient media such as Iodixanol, lysosomes tend to better separate from mitochondria or peroxisomes than in silica-based percoll media .
The interphases containing individually enriched organelles can be removed from the top of the gradient or by perforation of the centrifugation tube with a suitable needle. We routinely collected the samples from the top of the tubes. In our study, the individual fractions were analyzed by Western blotting for the presence of marker proteins for secretory lysosomes (CD63 (LAMP-3), CD107a (LAMP-1) and CD178 (FasL)), mitochondria (Cox IV), peroxisomes (catalase), ER (Bip/Grp78), Golgi (Golgin84) and early endosomes (EEA-1). Although contaminations of lysosomal fractions with mitochondria often hamper analyses of lysosomes obtained by density gradient centrifugation, the use of a discontinuous Iodixanol® separation gradient resulted in a clear separation of Cox IV containing mitochondria (fraction 5) and secretory lysosomes characterized by the presence of LAMP-1, CD63 and FasL (fraction 2). Besides the enrichment of FasL and lysosomal membrane proteins, which is indicative for the enrichment of SL in fraction 2, we observed a rather high abundance of other cytotoxic effector molecules (including granzyme B, perforin and granulysin) in fractions 4 and 6 (Figure 2). The presence of effector molecules in separate intact electron dense fraction 6 granules (Figure 3) was unexpected. Employing the separation method described here, we are currently investigating and comparing the composition of the two vesicular structures in different T and NK cell populations in more detail. We think that the distinct SL/granules actually form two separate compartments that might arise from an initial multivesicular complex during vesicular maturation. Fractions 2 and 6 not only differ substantially in the content of effector molecules but also in proteins that regulate cytoskeletal association (Schmidt et al., unpublished). Of note, since the described purification protocol does not imply nucleases and results in intact granules as shown by electron microscopy (Figure 3), enriched SL (and also fraction 6 granules) should be suitable to assay their capacity to directly induce target cell apoptosis by standard DNA fragmentation or chromium release assays as described before by Trapani and colleagues .
Importantly, the relatively high number of cells (we routinely started with 400 × 106) needed as an input for the enrichment of secretory vesicles is easily achievable if one wants to analyze leukemic cell lines or murine or human T cell or NK cell populations. To demonstrate the reproducibility and reliability of this technique, we isolated enriched secretory lysosomes of PHA stimulated lymphocytes of two different donors and compared these fractions by 2D-difference gel electrophoresis. This direct comparison at high-resolution revealed an almost identical spot pattern (96.5% at a threshold of 1.7), indicating that the presence and also the abundance of the approximately 1000 individual proteins in these enriched fractions are highly conserved in biological replicates. To stress this point: if one compares the different fractions from one donor, the protein content is completely different (21.4% comparable proteins in fractions 2 and 5 at a threshold of 1.7).
Since we were interested in the few differences between the two samples, we picked four selected spots and identified the proteins by mass spectrometry. Although only 200 μg of total protein were separated on the respective gels, even low abundant spots (#1, #3) were identified. It is important to mention that spot picking was performed from Flamingo Pink stained gels since we and others noted that CyDye minimal labeling results in significant alterations of protein masses, especially of low molecular weight proteins . Since only 3% of a given protein is labeled by the employed minimal labeling procedure, 97% of the protein might be actually located below the initially detected protein spot. Nonetheless, the subsequent PMF analyses revealed differences in MHC class II molecules, gelsolin-like capping protein isoforms and in chaperonin containing TCP1, subunit 8 between the two samples (Figure 5). Without going into further details, these or related proteins have been associated with secretory vesicles in other studies before . In a recent study, we provided a detailed analysis of the proteome of enriched SL from different NK cell lines and primary NK cells . We identified roughly 400 protein spots representing 237 individual proteins. As a proof of principle, although in NK cells the overall protein content of isolated SL was also very similar, we described a heterogeneous profile of functionally relevant proteins in leukemic and primary NK cells.
The reliability of the presented protocol is further stressed by the comparison of SL from CD4+ T cells isolated from three different donors. Of note, most CD4+ T cells also gain cytotoxic potential associated with SL when expanded in vitro . As expected from the initial 2D-DIGE experiments on PHA blasts, also standard 2D gel electrophoresis followed by Flamingo Pink staining and indirect comparison of individual gels using Image Master software revealed a very high degree of similarity between the individual fraction 2 preparations (Figure 6). This notion is highly relevant since future proteome analyses need to be standardized and suitable to compare appropriate numbers of samples for statistical analyses .
We describe a reliable, reproducible and easy to perform method for the enrichment of secretory lysosomes. We demonstrate that enriched SL fractions of individual donors or lymphocyte subpopulations are almost identical in their overall protein content. We thus provide a standard protocol for the future comparison of this important compartment in different T and NK cell subpopulations, in untransformed and transformed cells, and in T and NK cell populations of patients or mice suffering from genetically caused lysosomal storage diseases. Furthermore, the protocol might be easily adapted to investigate lysosomes of other cellular origin, including macrophages, mast cells or melanocytes.
Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coat preparations by Ficoll (1.077 g/ml) density gradient centrifugation. CD4+, CD8+ or NK cells were further purified by magnetic-activated cell sorting from PBMC (MACS Cell Isolation Kits, Miltenyi, Bergisch Gladbach, Germany) and adjusted to 106 cells/ml in RPMI 1640 supplemented with 10% FCS for CD4+ and CD8+ or with 10% heat-inactivated pooled AB human serum for NK cells. All isolated cells were expanded in the presence of a feeder cocktail of irradiated EBV-transformed B cells and PBMCs. For the generation of PHA blasts, PBMC were adjusted to 106 cells/ml in RPMI 1640 supplemented with 5% heat-inactivated FCS and 0.5 μg/ml phytohemagglutinin A (PHA) (Remel, Lenexa, KS, USA). After 4 days of stimulation, dead cells were removed by Ficoll gradient centrifugation. All cell cultures were maintained at a cell density of 1.5–2.0 × 106 cells/ml for up to 3 weeks in the presence of 1000 U/ml human recombinant interleukin 2 (rIL-2, Chiron GmbH, Marburg, Germany) for NK cells and 100 U/ml for CD4+ and CD8+ T cells, respectively. All culture media contained penicillin (50 U/ml) and streptomycin (50 μg/ml). Cell culture was performed at 37°C in a humidified atmosphere with 5% CO2.
Total protein and protein concentrations in individual fractions obtained after differential and density centrifugation.
enriched organelles (input)
Transmission electron microscopy
Enriched granules of fractions 2 and 6 were fixed with 2.5% glutaraldehyde in PBS at 4°C overnight, washed in PBS, postfixed in 2% OsO4, dehydrated in ethanol, and embedded in araldite (Sigma). Thin sections were mounted on formvar-coated grids and double-stained with a saturated solution of uranyl acetate in 70% methanol and with lead citrate. The grids were examined with a Zeiss EM 900 transmission electron microscope.
SDS-PAGE and Western blotting
For Western blot analysis, 4 μg of protein per fraction were separated by SDS-PAGE on 4–12% gradient Bis-Tris gels (Invitrogen, Karlsruhe, Germany). After blotting to 0.2 μm nitrocellulose membranes (Biometra, Goettingen, Germany), protein loading and efficacy of transfer was checked by reversible staining with Ponceau S (not shown). Membranes were routinely blocked with 5% non-fat dry milk powder or bovine serum albumin in TBST (TBS + 0.05% Tween), respectively. The fractions were analyzed with anti-FasL monoclonal antibody (mAb) clone G-247.4 (1/1000, BD Bioscience, San Diego, CA, USA), anti-LAMP-1 mAb clone 25 (1/500, BD Biosciences), anti-CD63 mAb clone MEM-259 (1/1000, Acris, Hiddenhausen, Germany), anti-Perforin mAb clone B-D48 (1/500, Abcam, Cambridge, UK), anti-Vti1b mAb clone 7 (1/1000, BD Biosciences), anti-Golgin84 mAb clone 26 (1/250, BD Biosciences), anti-CoxIV mAb clone 10G8D12C12 (1/1000, MitoScience, Eugene, OR, USA), anti-granzyme B mAb clone 2C5/F5 (1/2000, BD Biosciences), anti-EEA-1 mAb clone 14 (1/2500, BD Biosciences), anti-Bip/Grp78 mAb clone 40 (1/250, BD Biosciences), anti-pan-Cadherin mAb clone 22744 (1/1000, Abcam), and horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (1/7500, GE Healthcare, Uppsala, Sweden). For reprobing, membranes were stripped in stripping solution (100 mM 2-mercaptoethanol, 2% SDS, 60 mM Tris) for 30 min at 56°C. The polyclonal antibodies anti-catalase (1/5000, Abcam) and anti-granulysin (1/1000, RnD Systems, Minneapolis, MN, USA) were detected with HRP-conjugated goat anti-rabbit antibodies and donkey anti-goat, respectively. ECL (enhanced chemiluminescence) reagents (GE Healthcare) were used for chemiluminescent detection using Hyper Film (GE Healthcare).
Pelleted fractions were lyzed on ice for at least 30 min with 30 μl lysis buffer (pH 8.5) containing 7 M urea, 2 M thiourea, 30 mM Tris and 4% CHAPS . The supernatant was removed after centrifugation for 20 min at 20000 × g at 4°C. 50 μg of protein per sample were labeled on ice with 400 pmol of Cy3 or Cy5 (CyDyes, GE Healthcare) for 30 min in the dark. After quenching the labeling reaction with 1 μl lysine (10 mM) for 10 min on ice, the differentially labeled samples were pooled with 50 μg of unlabeled protein of each sample and mixed with a double volume of rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2% (v/v) IPG buffer pH 3–11 and 2% (w/v) DTT). The mixed samples were applied by cup-loading onto 24 cm IPG non-linear pH 3–11 gel strips, which had been rehydrated at 20°C for at least 10 h in 450 μl DeStreak solution (GE Healthcare) with 0.5% (v/v) IPG buffer pH 3–11 NL. Isoelectric focusing (IEF) was carried out for a total of 50–55 kVh at 20°C in five steps with a gradual increase of voltage (150 V for 3 h, 300 V for 4 h, gradient from 300 V to 1000 V for 6 h, gradient from 1000 V to 10000 V for 3.5 h and 10000 V ~3 h) using the IPGphor 3 system (GE Healthcare). For the second dimension, the IPG strips were first reduced for 15 min in SDS-equilibrium buffer (6 M urea, 50 mM Tris-HCl, pH 8.8, 30% glycerol, 2% SDS, trace of bromophenol blue and 0.5% (w/v) DTT) and then carbamidomethylated for 15 min in the same buffer containing 4.5% (w/v) iodoacetamide instead of DTT. The second dimension was performed on 12.5% polyacrylamide gels (20 × 24 cm) using the Ettan Dalt six system (GE Healthcare) at 10 mA per gel for 1 h, and then with an increased current of 15 mA per gel until the bromophenol blue band reached the end of the gel cassette. Electrophoresis was carried out under continuous cooling at 20°C.
Image analysis and spot picking
The in-gel fluorescence was scanned on a Typhoon Trio (GE Healthcare) at appropriate wavelengths to detect Cy3 and Cy5 specific emission corresponding to the protein concentration in every single spot . The intragel spot detection of the multiplexed gel images was performed using differential in-gel analysis (DIA) included in the DeCyder 6.5 software (GE Healthcare). The images for each gel were merged; spot boundaries were detected followed by a normalization of the spot volumes revealing differential spots. The gels were removed from the glass plates and stained with Flamingo Pink (Bio Rad) according to the manufactures instructions. The post-stained gels were mounted on a non-backed gel frame, scanned and analyzed using Image Master 6.0 (GE Healthcare). Selected spots were picked by an Ettan spotpicker (GE Healthcare) equipped with a 2 mm picking head according to the manufacturer's instructions. The picked gels were again scanned to verify the correct location of the cut spots.
In-gel tryptic digestion and mass spectrometry
Gel pieces were washed twice with HPLC grade water (Roth, Karlsruhe, Germany) and dehydrated in a first step with 25 mM ammonium bicarbonate (ABC) in 50% acetonitrile (ACN) and shrunk in pure ACN as a second step, according to . The dry gel pieces were then rehydrated with 100 ng sequencing-grade trypsin (Serva, Heidelberg, Germany) in 10 μl of 5 mM ABC. After rehydration, 10 μl of 5 mM ABC were added. For tryptic in-gel digestion, samples were incubated overnight at 37°C. To extract the peptides after the incubation period, 20 μl of 0.3% trifluoroacetic acid (TFA) in ACN were added and the samples were sonicated in a water bath (Sonorex Super RK100; Bandelin, Berlin, Germany) for 15 min. The liquid phases were collected, lyophilized, redissolved in 0.5 to 1 μl MALDI matrix solution (3,2 mg/ml α-cyanohydroxycinnamic acid (Sigma) dissolved in 65% ACN/0,1% TFA) and spotted onto a stainless steel 192-well MALDI plate and air-dried. The samples were analyzed by peptide mass finger printing in reflectron mode using the 4700 Proteomics Analyzer mass spectrometer (Applied Biosystems, Framingham, MA, USA). For MS analyses, typically 750 shots were accumulated for each spot. The peptide mass spectra were processed by internal calibration with autolytic fragments of porcine trypsin using the GPS Explorer software version 3.6 (Applied Biosystems) and searched against human proteins in the NCBI database (191694 entries, 2007/02/28, Homo sapiens) using MASCOT V2.0 (Matrix Sciences, London, UK).
Database searches with MASCOT were performed using the following parameters: the modification on cysteine residues by amidomethylation was set as obligate, methionine oxidation was considered as a potential modification; the maximum number of missed tryptic cleavages was one; the monoisotopic masses were considered and the mass tolerance was set to ± 50 ppm.
This work forms part of the PhD thesis of HS. We gratefully acknowledge the financial support by the Medical Faculty of the Christian-Albrechts University, the Cluster of Excellence Inflammation@Interfaces and the Innovationsfond Schleswig-Holstein.
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