Quantification of immunogold labelling in two populations of dendritic cells: a study on endogenous protease inhibitor

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1 Quantification of immunogold labelling in two populations of dendritic cells: a study on endogenous protease inhibitor T. Zavašnik-Bergant 1 1 Department of Biochemistry, Molecular and Structural Biology, Jožef Stefan Institute, Jamova c. 39, SI-1000 Ljubljana, Slovenia The preparation of ultrathin cryosections represents a method where cell proteins preserve their antigenicity well which further enables the application of specific antibodies as probes for efficient immunogold labelling prior to transmission electron microscopy (TEM). As a representation of this technique and consecutive quantification of gold label distribution, a small human protease inhibitor named cystatin C has been quantified in different organelles of human dendritic cells. The same sets of cell compartments were compared for immunogold labelling with anti-cystatin antibody and Protein A-gold in immature and mature dendritic cells. In professional antigen-presenting cells proteolytic capability is crucial for efficient but controlled degradation of antigen to antigenic peptides. Therefore, the understanding of inhibitory role of cystatin C in regulation of proteolytic activity of lysosomal enzymes is of importance in these immune cells. Keywords dendritic cell; immune cell; cystatin; cryosection; immunogold; electron microscopy; quantification 1. Introduction Quantitative immunoelectron microscopy involves determination of gold label distributions followed by drawing comparisons and interpreting differences and analogy, respectively, between compartments in the same sample of cells or between different experimental groups of cells, such as treated/untreated, differentiated/non-differentiated, activated/non-activated, immature/mature, viable/apoptotic, apoptotic/necrotic cells etc. When choosing an appropriate sample preparation there are issues that should be addressed and attained as much as possible: (1) the distribution of antigen should be preserved, (2) its antigenicity should not be compromised by fixative, and (3) antibody used for specific labelling should be able to access the antigen. In this way immunolocalized molecules can be accurately correlated to observed subcellular compartments within labelled sections. The application of antibody or other affinity probe to ultrathin sections of cells prior to high resolution TEM is an important tool in all biological sciences. Preservation of the structure of antigen that is being labelled is important, especially when specific (primary) antibody, followed by secondary immunoreagent (such as secondary antibody or Protein A with bound colloidal gold), has been applied to point out the location and distribution of labelled protein. Antibodies have the ability to recognize and discriminate among very similar proteins and are often used as highly specific probes for identification, evaluation and quantification of selected cellular proteins within a multitude of other cell components. The preparation of ultrathin cryosections represents a method where cell proteins preserve their antigenicity well which further enables the application of specific antibodies as probes for efficient immunogold labelling prior to TEM observation [1, 2]. In this study a small protease inhibitor named cystatin C, a single-chain protein consisting of 120 amino acids with a molecular weight of 13 kda and a known modulator of proteolytic activity of lysosomal enzymes, has been quantified in different organelles of special phagocytic and antigen-presenting cells of the immune system, i.e. in dendritic cells. Cystatins are important natural reversible inhibitors of lysosomal proteolytic enzymes (cysteine proteases). They are wide spread in many organisms and are involved in various biological processes where they regulate proteolytical degradation (proteolysis) [3]. Precise understanding of their regulatory roles on proteolytic enzymes will provide a valuable insight how proteolysis could be modulated to treat a specific disease [4, 5]. Cystatin-type inhibitors from ticks represent another intriguing example of modulatory role of cystatins. Ticks are parasitic arthropods that feed on the blood of their hosts (humans, animals). Ticks secrete cystatins together with other components of tick saliva when biting the host. It has been suggested that secreted tick cystatins can interfere with antigen degradation and antigenic peptide presentations in antigen-presenting cells in skin (i.e. in distinct population of dendritic cells) and in this way interfering with efficient immune response of tick host [6]. Dendritic cells play a pivotal role in the adaptive immunity by initiation of efficient T-cell mediated immune response. Dendritic cells circulate through tissues like scavengers. Their maturation occurs as they migrate from peripheral tissues (skin, for example), where they search for antigen, to lymphoid organs, where they present captured and processed antigen (as antigenic peptides bound to major histocompatibility complex class II molecules (MHC II) on the surface of dendritic cells) to specific T cells [7]. Here the distribution of endogenous protease inhibitor cystatin C has been highlighted in human dendritic cells. The purpose was to find differences in immunogold labelling of protease inhibitor cystatin C between cell compartments (organelles) depending on the maturation of these immune cells FORMATEX 358

2 2. Material and methods 2.1 Fixation of cells, pellet formation and cryoprotection Human immature dendritic cells were generated from freshly isolated blood monocytes and further stimulated with TNF-alpha to mature dendritic cells as previously described [8]. Cells were fixed with paraformaldehyde (Sigma- Aldrich) added directly into the culture medium up to the final concentration of 4%. Fixation took 2 hours at room temperature. Mixture of growth medium and fixative was removed with a series of centrifugation steps. After final centrifugation step a pellet of cells was embedded in 10% gelatine (Merck) in 0.1 M phosphate buffer. Gelatine was solidified on ice for 30 min and the pellet with cells excised from the tube and cut into small cubes (1 mm 3 ). Cubes with cells were immersed in 2.6 M sucrose (used here as a cryoprotectant) in 0.1 M phosphate buffer and infiltrated overnight at 4 ºC. Finally, cryoprotected cells were mounted on specimen carriers (special aluminium pins) and put into liquid nitrogen for preservation and storage (Figure 1, Figure 2). 2.2 Preparation of ultrathin cryosections Trimming and sectioning were performed on Leica EM FC6 ultramicrotome for cryosectioning. Frozen specimen blocks were trimmed at 90 C using diamond trimming knife (Diatome). Ultrathin cryosections (70 nm) were cut at 120 C using diamond cryo-immuno knife (35 angle, Diatome). Sections were collected and thawed on droplets of pick-up solution, i.e. 1:1 mixture of 2.3 M sucrose (Merck) in 0.1 M phosphate buffer and 2% methyl cellulose (Sigma-Aldrich) in water. Retrieved ultrathin sections of cells were put on a thin layer of carbon-coated Formvar film (TAAB Lab.) spread over hexagonal Cu grids (Agar) and stored at 4 C. 2.3 Immunogold labelling Sections on grids were put on melted 2% gelatine in 0.1 M phosphate buffer for 20 minutes at 37 C (to remove residual gelatine used as supporting matrix around cells, Figure 1). Grids were transferred to droplets of buffer where grids were floating on top of the droplets with sections facing the droplets. Sections were rinsed with 20 mm glycine in PBS (to block residual aldehyde groups) and 1% BSA in PBS (to diminish non-specific binding of antibodies and Protein A- gold, respectively). For immunogold labelling sections were incubated with rabbit anti-cystatin C polyclonal antibody in blocking solution. Described immunolabelling procedure was performed by series of incubation/rinsing steps (Figure 3). Sections were incubated with primary antibody (on 25 μl droplets) for 30 min. PBS was used for extensive rinsing after each incubation step. After the removal of an excess of antibodies sections were incubated with Protein A gold (with 10-nm colloidal gold particles) for 20 min and according to the producer s instructions (Cell Microscopy Centre, University Medical Centre Utrecht). Controls omitting the primary antibody were also performed. 2.4 Contrasting the sections with uranyl acetate Additional extensive rinsing of grids (with immunogold labelled sections) on droplets of ultra pure water was performed prior to uranyl acetate staining (to remove traces of salts and thereby prevent precipitation of uranyl acetate). Sections were contrasted with 0.4% uranyl acetate (SPI-Chem TM uranyl acetate, Spi Supplies), ph 4, in solution with 1.8% methyl cellulose (Sigma). Uranyl acetate contrasted the proteins but not the lipids (membranes). 2.5 Transmission electron microscopy Immunogold-labelled sections were observed with Philips CM120 BioTWIN transmission electron microscope at 100 kv. Micrographs were taken using KeenView digital camera and item software. 2.6 Quantification of labelling Quantification of immunogold labelling patterns in the same sets of compartments (or organelles) in two different groups of cells (immature and mature dendritic cells) was performed for labelled inhibitor cystatin C. Random sampling of specimen was followed by counting of 400 gold particles on three grids for each sample [9]. The distributions of gold particles in different compartments were compared by contingency table analysis with statistical degrees of freedom for chi-squared values (X 2 ) being determined by the number of compartments and the number of experimental groups of cells. Compartmental chi-squared values making substantial contributions to the total chi-squared values identified where the main differences between groups resided. 2.7 Confocal microscopy Dendritic cells were also labelled for confocal fluorescence microscopy. Same anti-cystatin C antibody was applied as described above together with goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (fluorophore emitting 2012 FORMATEX 359

3 Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.) green fluorescence). Dendritic cells in suspension were cytocentrifuged (StatSpin Cytofuge) onto poly-l-lysine (Sigma)coated glass slides. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) in 0.1 M HEPES buffer for 2 hours at room temperature and permeabilized with 0.1% Triton X-100 (detergent) for additional 5 min. After this and all subsequent steps, cells were rinsed in PBS. ph of all fixatives, immunoreagents, buffers and rinsing solutions was adjusted to ph 7.4. Non-specific binding of primary and secondary antibody was blocked with 3% BSA (Sigma). Cells were labelled with rabbit polyclonal antibody against human cystatin C. Primary antibody was added for 2 hours at 37 ºC. Another incubation for 45 min at 37 ºC followed with secondary Alexa Fluor 488-labelled goat anti-rabbit IgG antibody (Invitrogen Molecular Probes). Controls omitting the primary and/or secondary antibody were also performed. Labelled dendritic cells on glass slides were mounted with ProLong AntiFade Kit (Invitrogen Molecular Probes) to thin cover glass squares. Confocal microscopy was performed using a confocal laser scanning microscope LSM 510 (objective 60, N.A. = 1.4). Fluorophore Alexa Fluor 488 was excited with argon laser at 488 nm. Emission signal was filtered using BP nm. Carl Zeiss LSM image software was used. Fig. 1 Pellet formation and cryoprotection. Pellet was made of fixed cells after centrifugation (A). Solidification of gelatine around and between the cells forming the pellet (A, B) kept the cells within the pellet for later preparation steps. Next the pellet with cells (but not the surrounding gelatine) was cut into small blocks (C, D, G) and immersed in sucrose solution for cryoprotection (E). Cryoprotected cells were mounted on specimen carriers (F, G) and put into liquid nitrogen for preservation and storage until ultracryomicrotomy (further preparation of ultrathin cryosections). Fig. 2 Micrograph of ultrathin section of dendritic cells showing how cells are placed inside the pellet. Fine intracellular ultrastructure with diverse vesicle populations and cell organelles, respectively, can be observed. As well as multiple plasma membrane extensions, typical morphological characteristics of these phagocytic immune cells, are clearly visible. Bar: 5 μm FORMATEX 360

4 a) Fig. 3 Schematic presentation of immunogold labelling of prepared ultrathin sections of dendritic cells (a) for transmission electron microscopy (TEM) observation. b) Prior to labelling ultrathin sections were first incubated in blocking solution (b) such as bovine serum albumin (BSA) to prevent non-specific binding of primary antibody (c). c) Unbound antibody was removed in the next step (d) and sections were incubated with Protein A- gold (e) which bound to Fc portion of primary antibody and in this way marked the position of a target antigen (f). d) e) f) 2012 FORMATEX 361

5 Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.) 3. Results and discussion A high-resolution technique has been applied to observe cell organelles and search for immunogold labelled cystatin C. Described method consisted of preparation of ultrathin cryosections from fixed and frozen cells, their immunogold labelling, quantification of attached gold particles by TEM (Table 1) and statistical analysis of obtained data (Table 2). It was reasonable, prior to immunogold electron microscopy, to detect the antigen of interest also with confocal fluorescence microscopy (Figure 4) in order to acquire general observation about its distribution; here presumably maturation dependent in two populations of investigated dendritic cells. a) b) c) d) Fig. 4 Confocal fluorescence microscopy of labelled cystatin C in immature (a, b) and mature (c, d) dendritic cells. Fixed dendritic cells were labelled with rabbit cystatin C polyclonal antibody (b, d), followed by secondary goat anti-rabbit antibody conjugated to Alexa Fluor 488 fluorophore. Fluorescence signal of labelled cystatin C has revealed its approximate position (localization). In immature dendritic cells strong fluorescence signal was observed in Golgi (b), whereas in mature dendritic cell population green fluorescence of labelled endogenous cystatin C was mostly confined to cell periphery. DIC images of cells are also shown (a, c). Magnification: 64. Protease inhibitor cystatin C has been quantified in different organelles of human dendritic cells (Table 1). The same sets of cell compartments were compared for immunogold labelling with anti-cystatin antibody and Protein A-gold in immature and mature dendritic cells. Random sampling of specimen was strictly considered and was followed by 2012 FORMATEX 362

6 Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.) counting of gold particles associated with particular cell organelles (compartments). An example of immunogold labelled cell structure (Golgi) is shown Figure 5. a) b) Fig. 5 Transmission electron microscopy (TEM) of Golgi with immunogold labelled cystatin C in immature (a) and mature (b) dendritic cells. Fixed cells were cryosectioned and ultrathin sections retrieved and labelled with rabbit anti-cystatin C polyclonal antibody and Protein A-gold (with 10 nm colloidal gold particles). Uranyl acetate stained cellular proteins (they appear dimmed) whereas membranes remained unstained and appear white on presented micrographs. Gold particles indicated the position of labelled cystatin C highly present in Golgi cisternae of immature (a) but not mature (b) dendritic cells. Bar: 200 nm. Strong immunogold labelling of Golgi was observed in immature dendritic cells whereas in mature dendritic cells Golgi remained practically unlabelled after incubation with specific antibody (Figure 4, Figure 5). Gold counts and 2012 FORMATEX 363

7 results of calculations for Golgi and other compartments (organelles) are provided in Table 2. High number of gold particles was associated with Golgi, endoplasmic reticulum and nuclear membrane indicating high expression of cystatin C in immature dendritic cells. On the other hand, in mature dendritic cells the number of gold particles was increased in multivesicular bodies and small vesicles near the plasma membrane. High content of cystatin C in Golgi in immature dendritic cell population (Figure 4b) but not in mature dendritic cells was also confirmed with confocal microscopy. Fluorescence signal of labelled cystatin C in mature dendritic cells was mostly confined to cell periphery (i.e. near the plasma membrane) as shown in Figure 4d. Table 1 Distribution of 10-nm gold particles in immature and mature dendritic cells after labelling with anti-cystatin C antibody and Protein A-gold (shown as percentage of the total count for each cell population). Cystatin C Immature dendritic cells Mature dendritic cells nucleus nuclear membrane 1.5 <0.5 endoplasmic reticulum Golgi small vesicles multivesicular bodies multilamelar bodies vesicles near plasma membrane plasma membrane cytoplasm mitochondria unknown structures, other Table 2 Labelling distributions of inhibitor cystatin C and Chi-squared (X 2 ) test. Values represent observed and expected (in brackets) numbers of 10-nm gold particles in immature and mature dendritic cells. Chi-squared values Immature Mature Row Immature Mature Compartments dendritic cells dendritic cells totals dendritic cells dendritic cells nucleus 8 (8.0) 8 (8.0) 16 <0.01 <0.01 nuclear membrane 6 (3.5) 1 (3.5) endoplasmic reticulum 34 (18.0) 2 (18.0) Golgi 122 (63.0) 4 (63.0) small vesicles 156 (168.0) 180 (168.0) multivesicular bodies 32 (41.0) 50 (41.0) multilamelar bodies 2 (3.0) 4 (3.0) vesicles near plasma membran 12 (53.0) 94 (53.0) plasma membrane 8 (6.0) 4 (6.0) cytoplasm 10 (11.0) 12 (11.0) mitochondria 6 (19.5) 33 (19.5) unknown structures, other 4 (6.0) 8 (6.0) Column totals * *For a total chi-squared value (X 2 ) of and 11 degrees of freedom (P<0.001) these two distributions are significantly different. Of 400 gold particles counted in immature dendritic cells 122 were associated with Golgi. In contrast, only 4 were observed on Golgi in mature dendritic cells. By contingency table analysis, the predicted number of gold particles on a given compartment in a given cell group is given by [(column total row total)/grand total] where the grand total is the sum of the column (or row) totals. Consequently, the predicted number of gold particles associated to Golgi in immature dendritic cells, for example, is calculated to be [(400x126)/800] or 63 (Table 2). For each compartment and cell type, the partial chi-squared value is given by [(observed golds expected golds) 2 / expected golds]. Therefore, the partial chisquared value for Golgi in immature dendritic cells is equivalent to [(122 63) 2 /63] or Table 2 shows that the 2012 FORMATEX 364

8 total chi-squared value (X 2 ) is For 11 degrees of freedom [(2 1 columns) by (12 1 rows)] this indicates that the null hypothesis of no difference between groups must be rejected (P < 0.001). The distribution of cystatin C is significantly different in immature and mature dendritic cells and the gold counts are consistent with a shift towards greater-than-predicted labelling of Golgi in immature dendritic cells (Table 2). Furthermore, the partial (or compartmental) chi-squared values indicate that immature dendritic cells have more-thanexpected gold particles on Golgi, endoplasmic reticulum and nuclear membrane (Table 2). On the contrary, partial chisquared values indicate that mature dendritic cells have fewer-than-expected gold particles on Golgi but more-thanexpected gold particles on the vesicles near the plasma membrane, small vesicles and multivesicular bodies (Table 2). Probably non-specific labelling of mitochondria, particularly in mature dendritic cells, has been observed where at this point no biologically meaningful explanation can be provided. We have confirmed that in human immature dendritic cells cystatin C content was elevated compared to mature dendritic cell population. In this study, cystatin C high content in Golgi (Figure 4, Figure 5) as well as its presence in endoplasmatic reticulum and nuclear membrane, continuous with the endoplasmic reticulum, indicate the elevated expression of cystatin C in immature dendritic cell population but not in mature dendritic cells. Furthermore, the transport of cystatin C was shown from Golgi towards the cell membrane, supported by the decrease of cystatin C in Golgi and endoplasmic reticulum and increase in labelling of different populations of transport vesicles (small vesicles, vesicles near plasma membrane) and multivesicular bodies. In dendritic cells limited antigen degradation, performed by proteolytic enzymes (such as lysosomal cathepsins) and their control by intracellular protease inhibitors (such as cystatins), represents a crucial step in generating antigenic peptides prior to MHC II antigen presentation to helper T cells [7]. Multivesicular bodies are MHC II loading compartments that have been reported positive for lysosomal cathepsins (proposed targets enzyme of cystatin C) in both, immature and mature dendritic cells [8]. Furthermore, as described here, labelling of multivesicular bodies for cystatin C was higher-than-expected in mature but not immature dendritic cells indicating higher content of cystatin C inside these antigen-loading compartments in mature dendritic cells compared to immature population. Therefore, maturation dependent distribution of endogenous cystatin C supports its intracellular regulatory potential towards present proteolytic enzymes along the endocytic pathway of human dendritic cells. 4. Conclusions Generation of ultrathin cryosections together with immunogold electron microscopy and specific antibody application has been successfully used for quantification of gold label distributions in various cell compartments. Preparation of cryosections of human dendritic cells, their immunogold labelling with specific anti-cystatin C antibody and transmission electron microscopy were successfully applied to quantify protease inhibitor cystatin C in different organelles of immature and mature dendritic cells. Furthermore, increased expression of cystatin C has been confirmed in immature dendritic cells. On the other hand, different cellular distribution of cystatin C upon dendritic cell maturation has been associated to potential modulatory role of cystatin C in regulating lysosomal proteolytic enzymes. Acknowledgements Experiment was performed at Electron Microscopy Core Facility (EMBL Heidelberg, Germany) and granted by EMBO short-term fellowship to T.Z.B. References [1] Tokuyasu KT. Immunocytochemistry of ultrathin frozen sections. Histochemistry Journal. 1980;12: [2] Slot JW, Geuze HJ. Cryosectioning and immunolabelling. Nature Protocols. 2007;2: [3] Zavašnik-Bergant T. Cystatin protease inhibitors and immune functions. Frontiers in Bioscience. 2008;13: [4] Zavašnik-Bergant T, Turk B. Cysteine proteases: destruction ability versus immunomodulation capacity in immune cells. Biological Chemistry. 2007;388: [5] Conus S, Simon HU. Cathepsins and their involvement in immune responses. 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