ARTICLE |
Correspondence to: Karin Öllinger, Division of Pathology II, Faculty of Health Sciences, Linköping, Sweden.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We describe a pre-embedding immunocytochemical method for visualization of the lysosomal enzyme cathepsin D in cultured cells. The protein was demonstrated at both light and electron microscopic levels by neutral-pH silver enhancement of ultrasmall (0.8-nm) gold particles conjugated to the antibodies. The best morphological preservation and the highest labeling density were achieved by initial fixation for 20 min at 4C in 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde (GA) in 0.15 M sodium cacodylate buffer, followed by permeabilization in sodium borohydride. Three cell types were used: human foreskin fibroblasts, histocytic lymphoma (J-774) cells, and primary rat heart myocytes. In all three, cathepsin D was demonstrated in lysosome-like structures. The rat heart myocytes were also exposed to the redox cycling substance naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) to induce oxidative stress. This was done for such a short period of time that the cells initially did not show any signs of morphological damage and retained normal plasma membrane stability, although an early and clear redistribution of cathepsin D from membrane-bound structures to the cytosol was apparent. This redistribution was followed by cell degeneration and, eventually, by cell death. (J Histochem Cytochem 46:411-418, 1998)
Key Words: immunocytochemistry, pre-embedding, cathepsins, ultrasmall gold, cultured cells, oxidative stress
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunocytochemical techniques for demonstration of specific antigens have been successfully applied at both light and electron microscopic levels (
Cathepsin D is a lysosomal aspartic protease that is present in almost all animal cells (
We propose that, during oxidative stress, increased amounts of hydrogen peroxide are produced that can diffuse freely and may therefore cross the lysosomal membrane and spread within the acidic vacuolar apparatus. Inside this compartment, the acidic milieu and the occurrence of reducing compounds (e.g., cysteine) would promote iron reduction and Fenton-like chemistry. This would give rise to hydroxyl radicals that could destabilize the lysosomal membranes (e.g., through peroxidation) and thereby cause leakage of lysosomal contents to the cytosol (
To study the actual localization of lysosomal enzymes in normal cells and in cells exposed to oxidative stress, we developed a pre-embedding method to visualize the lysosomal enzyme cathepsin D. This was done using ultrasmall gold particles and silver enhancement. To show the applicability of the method, three different types of cultured cells were used: human foreskin fibroblasts, an established line of histocytic lymphoma (J-774) cells, and primary rat heart myocytes. The labeling density and morphological preservation at different concentrations of paraformaldehyde (PFA) and glutaraldehyde (GA) were evaluated. Using TEM (transmission electron microscopy), we also found that cathepsin D in myocyte lysosomes was partially released into the cytosol during exposure to oxidative stress and that only later did cell degeneration and death occur.
![]() |
Material and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chemicals
Glutamine, penicillin-G, streptomycin, Eagle's minimal essential medium (EMEM), Ham's F-10 medium, fetal calf serum, and dialyzed calf serum were purchased from GIBCO (Paisley, UK). Hydroquinone was obtained from BDH Ltd (Poole, UK); silver lactate, paraformaldehyde, and Epon-812 from Fluka AG (Buchs, Switzerland); and glutaraldehyde (vacuum distilled) from Agar Scientific (Essex, UK). Naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) came from Aldrich Chemie (Steinheim, Germany). Collagen (Type I), sodium borohydride, sodium cacodylate, citric acid, and sodium citrate were supplied by Sigma (St Louis, MO). Gelatin (coldwater fish skin gelatin) and goat anti-rabbit IgG tagged with 0.8-nm gold particles were bought from Aurion (Wageningen, The Netherlands). Cathepsin D antibodies and the normal rabbit immunoglobulin fraction were from Dakopatts (Älvsjö, Sweden).
Cells and Culture Conditions
Human foreskin fibroblasts, AG-1518 (passages 15-25), were cultured in EMEM supplemented with 2 mM glutamine, 50 IU/ml penicillin-G, 50 µg/ml streptomycin, and 10% fetal calf serum.The established murine histocytic lymphoma cell line (J-774 cells) was cultured in Ham's F-10 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 50 IU/ml penicillin-G, and 50 µg/ml streptomycin.
These two cell lines were incubated in humidified air with 5% CO2 at 37C and were subcultured once a week. Before an experiment (24 hr), the cells were trypsinized (fibroblasts) or scraped (J-774 cells) and seeded into 35-mm Petri dishes (Costar; Cambridge, MA) at a density of 10,000 and 30,000 cells/cm2 for fibroblasts and J-774 cells, respectively. For LM (light microscopy), round glass coverslips (diameter 22 mm) were placed in the culture dishes before cell plating.
Heart myocytes were prepared from 2- to 3-day-old male and female SpragueDawley rats as previously described (
Exposure to Naphthazarin
Myocytes were exposed to naphthazarin dissolved in 99% ethanol added in pre-warmed (37°C) Hank's balanced saline solution, pH 7.4 (final ethanol concentration 0.25%) and incubated at 37°C. Viability (i.e., plasma membrane integrity) was studied by analysis of lactate dehydrogenase (LDH) leakage, as described by
Immunogold Labeling
The cultures were fixed in different fixatives consisting of 2% or 4% PFA and between 0.05% and 1% GA in 0.15 M sodium cacodylate buffer, pH 7.6, for 20 min at 4C. The cells were washed in PBS and then immersed in a freshly prepared solution of sodium borohydride (0.01% or 0.05%) and 0.1% glycine in PBS, pH 7.4, for 10 min at room temperature (RT). Before and after each immunoincubation, cells were rinsed for at least 1 hr in PBS containing 20 mM NaN3, 0.8% BSA (bovine serum albumin), and 0.1% gelatin. This solution was also used to dilute primary and secondary antibodies. The cells were incubated in 1:100 diluted polyclonal rabbit cathepsin D antibodies overnight at 4C. After rinsing for 5 hr, the cells were incubated in 1:100 diluted goat anti-rabbit IgG or F(ab') tagged with 0.8-nm gold particles overnight at 4C. The cells were then rinsed for 1 hr in PBS containing 20 mM NaN3, 0.8% BSA, and 0.1% gelatin, and for 1 hr in PBS, fixed for 10 min in 2% glutaraldehyde in PBS, and finally washed in PBS at RT. Control cells in which the specific polyclonal antibody had been replaced (with immunoglobulin fraction of nonimmune rabbit serum or PBS containing 20 mM NaN3, 0.8% BSA and 0.1% gelatin) remained unstained.
Silver Enhancement Procedure
The cultures were rinsed three times with 20 mM HEPES buffer containing 280 mM sucrose (pH 6.8) and were then developed for 6 min. The developer solution was prepared by combining 6 parts gum arabic (500 g/liter in deionized water), 1 part HEPES buffer stock (200 mM HEPES; pH adjusted to 6.8 with NaOH), 1.5 parts hydroquinone (0.52 M in deionized water), and 1.5 parts silver lactate (37 mM in deionized water). The silver lactate was protected from light and was added just before use. The cultures were incubated with developer in a dark chamber at 26C, rinsed briefly in deionized water, and processed for EM and LM.
Light Microscopy
The cells, grown on coverslips, were dehydrated in ethanol and xylene and then mounted in Canada balsam (BDH; Poole, UK) and were examined and photographed with a Nikon photomicroscope.
Transmission Electron Microscopy
Cells were dehydrated and immersed in a mixture of absolute ethanol and Epon 812 (1:1) and then embedded in pure Epon 812 in the Petri dishes. Beem capsules containing polymerized Epon were placed upside down at a straight angle to the cell layer and were polymerized for 48 hr at 60C. The Epon layer with the Beem capsules was removed from the Petri dish, leaving the cells on the surface of the Epon layer. The Beem capsules were removed with tweezers and ultrathin sections (60 nm) were cut with a diamond knife (DIATOME; Bienne, Switzerland) on a ReichertJung ultracut (Vienna, Austria) and collected on Formvar-coated Cu 100-mesh grids. The sections were rapidly counterstained with uranyl acetate and lead citrate and examined in a JEOL 2000-EX TEM electron microscope (Tokyo, Japan) at 100 kV.
Quantitation of Silver Precipitates
Cells were randomly selected at a magnification of x8000 and a video recording of the entire cell was made using a Pixie 8 Image Processor (Deben Research; Suffolk, UK). The recording was transferred to a Macintosh computer and the number and localization of silver-enhanced gold particles were determined. Particles found in vacuolar structures surrounded by a visible membrane structure were classified as lysosomal, and disseminated particles not inside structures with visible membranes were referred to as cytoplasmic (cytosolic). Because the cells varied in size, we calculated the number of silver-enhanced gold particles per 2500 µm2 cell area.
Statistical Evaluation
Specimens of rat heart myocytes originated from different animal preparations. All cultures were immunostained, developed, and embedded at the same time. Silver-enhanced gold particles were quantified in entire cells, and significance was analyzed using the Wilcoxon test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As observed in the light microscope, the immunostaining pattern was similar to that noted using immunofluorescence detection of cathepsin D in J-774 cells treated with Triton X-100 during the immuno-incubation (
|
|
|
Silver development at neutral pH (
Cathepsin D was found primarily in lysosomal structures in the rat heart myocytes (Figure 2B), J-774 cells (Figure 3A), and human foreskin fibroblasts (Figure 3B). Using video images, the silver-enhanced gold particles in the myocytes were counted and registered as being either within lysosomal structures or in the cytosol. As shown in Figure 2A and Table 1, control cells that had been enhanced but had not been exposed to the primary antibody exhibited only a small number of particles, and these were evenly distributed in the cells and showed no statistically significant lysosomal predominance.
|
In all three cell types, oxidative stress caused relocalization of cathepsin D from lysosomal structures to the cytosol. This is illustrated in Figure 4, which shows rat heart myocytes exposed to naphthazarin for 30 and 45 min. Lethal cytotoxicity, measured as decreased plasma membrane integrity (leakage of the enzyme LDH from the cytosol to the surrounding medium), was not detected at this stage but was observed later. Viability was 99, 98.7, and 96.4% for cultures treated with naphthazarin for 0, 30, and 45 min, respectively. After 110 min, 50% of the cells were dead and all cells were devitalized after a couple of hours (not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present experiments we applied an immunocytochemical pre-embedding immunogold method to different types of cells as a means of localizing the lysosomal aspartic proteinase cathepsin D. Other investigators have used a postembedding immunogold technique to determine the presence of cathepsins B, H, and L in Epon sections of bronchoalveolar epithelial cells (
To our knowledge, a pre-embedding method for detection of cathepsin D has not yet been described. Pre-embedding immunogold methods have, however, been used for three-dimensional labeling of nuclear matrix proteins in permeabilized cells (
Amounts of silver-enhanced gold particles were analyzed in heart myocytes that had or had not been exposed to the primary antibody before silver enhancement (Table 1). The negligible background labeling that was observed in the antibody-unexposed control cells was evenly distributed in the cell (Figure 2A), whereas the reaction product was mainly found in the lysosomes of cells subjected to the complete procedure.
Oxygen-derived free radicals damage cellular macromolecules and disrupt cellular homeostasis (
We have shown in several studies that lysosomal destabilization is an early event in cellular devitalization (
We conclude that our pre-embedding method, in combination with the use of ultrasmall gold probes and ensuing silver enhancement, is a sensitive method for detection of lysosomal enzymes, and offers fairly good morphological preservation that is enough to determine the localization of the antigen. Moreover, this technique should be a very valuable tool for evaluation of lysosomal membrane stability.
![]() |
Acknowledgments |
---|
Supported by the Swedish Cancer Foundation (grant 2703) and by Östgöta läns landsting.
The technical assistance of Ms Britt-Marie Gustafsson is gratefully acknowledged.
Received for publication March 31, 1997; accepted September 25, 1997.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altona JC, van der Laarse A, Bloys van Treslong CHF (1984) Release of compartment-specific enzymes from neonatal rat heart cell cultures during anoxia and reoxygenation. Cardiovasc Res 18:99-106[Medline]
Brunk UT, Dalen H, Roberg K, Hellquist HB (1997) Photo-oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts. Free Radical Biol Med 23:616-626[Medline]
Brunk UT, Zhang H, Dalen H, Öllinger K (1995a) Exposure of cells to nonlethal concentrations of hydrogen peroxide induces degeneration-repair mechanisms involving lysosomal destabilization. Free Radical Biol Med 19:813-822[Medline]
Brunk UT, Zhang H, Roberg K, Öllinger K (1995b) Lethal hydrogen peroxide toxicity involves lysosomal iron-catalyzed reactions with membrane damage. Redox Rep 1:267-277
Burry RW, Vandré DD, Hayes DM (1992) Silver enhancement of gold antibody probes in pre-embedding electron microscopic immunocytochemistry. J Histochem Cytochem 40:1849-1856
Cohen GM, d'Arcy Doherty M (1987) Free radical mediated cell toxicity by redox cycling chemicals. Br J Cancer 55:46-52
Cremers AFM, de Graaf AJ, van Bergen en Henegouwen PMP, Humbel BM, Verkleij AJ (1990) The use of ultra small gold particles at the light and electron microscopical level. Trans R Microsc Soc 1:609-614
Danscher G, Norgaard JOR (1983) Light microscopic visualization of colloidal gold on resin-embedded tissue. J Histochem Cytochem 31:1394-1398[Abstract]
de Graaf A, Humbel BM, Stuurman N, van Bergen en Henegouwen PMP, Verkleij AJ (1992) Three-dimensional immunogold labeling of nuclear matrix proteins in permeabilized cells. Cell Biol Int Rep 16:827-836[Medline]
de Graaf A, van Bergen en Henegouwen PMP, Meijne AML, van Driel R, Verkleij AJ (1991) Ultrastructural localization of nuclear matrix proteins in HeLa cells using silver-enhanced ultra-small gold probes. J Histochem Cytochem 39:1035-1045[Abstract]
Dickens BF, Mak IT, Werlicki WB (1988) Lysosomal lipolytic enzymes, lipid peroxidation and injury. Mol Cell Biochem 82:119-123[Medline]
Halliwell B (1991) Reactive oxygen species in living systems: source, biochemistry, and role in human disease. Am J Med 91:3C-14C
Horisberger M, Rosset J (1977) Colloidal gold a useful marker for transmission and scanning electron microscopy. J Histochem Cytochem 25:295-305[Abstract]
Ishii Y, Hashizume Y, Watanabe T, Waguri S, Sato N, Yamamoto M, Hasegawa S, Kominami E, Uchiyama Y (1991) Cystein proteinases in bronchoalveolar epithelial cells and lavage fluid of rat lung. J Histochem Cytochem 39:461-468[Abstract]
Lah JJ, Hayes DM, Burry RW (1990) A neutral pH silver development method for the visualization of 1-nanometer gold particles in pre-embedding electron microscopic immunochemistry. J Histochem Cytochem 38:503-508[Abstract]
Mak IT, Misra HP, Weglicki WB (1983) Temporal relationship of free radical-induced lipid peroxidation and loss of latent enzyme activity in highly enriched hepatic lysosomes. J Biol Chem 258:13733-13737
Newman GR, Jasani B, Williams ED (1983) A simple post-embedding system for rapid demonstration of tissue antigens under the electron microscope. Histochem J 15:543-555[Medline]
Öllinger K, Brunk UT (1995) Cellular injury induced by oxidative stress is mediated through lysosomal damage. Free Radical Biol Med 19:565-574[Medline]
Öllinger K, Brunmark A (1991) Effect of hydroxy substituent position on 1,4-naphthoquinone toxicity to rat hepatocytes. J Biol Chem 266:21496-21503
Öllinger K, Brunmark A (1994) Effect of different oxygen pressures and N,N-diphenyl-p-phenylenediamine on adriamycin toxicity to cultured neonatal rat heart myocytes. Biochem Pharmacol 48:1707-1715[Medline]
Raczek S, Yeung CH, Hasilik A, Robenek H, Hertle L, Schulze H, Cooper TG (1995) Immunocytochemical localisation of some lysosomal hydrolases, their presence in luminal fluid and their directional secretion by human epididymal cells in culture. Cell Tissue Res 280:415-425[Medline]
Romano EL, Stolinski C, HughesJones NC (1975) Distribution and mobility of A,D, and c antigens on human red cell membranes: studies with a gold-labelled antiglobulin reagent. Br J Haematol 30:507-516[Medline]
Ryan TP, Aust SD (1992) The role of iron in oxygen-mediated toxicity. Crit Rev Toxicol 22:119-141[Medline]
Sterling JW (1990) Immuno- and affinity probes for electron microscopy: a review of labeling and preparation techniques. J Histochem Cytochem 38:145-157[Abstract]
Van Lookeren Campagne M (1993) Pre-embedding immuno gold labelling of intracellular antigens using ultra small gold probes. Aurion Newsletter
Van Lookeren Campagne M, Dotti CG, Jap Tjoen San A, Verkleij AJ, Gispen WH, Oestreicher AB (1992) B-50/GAP43 localization in polarized hippocampal neurons in vitro: an ultrastructural quantitative study. Neuroscience 50:35-52[Medline]
Vassault A (1985) Lactate dehydrogenase. In Bergmeyer HU, ed. Methods of Enzymatic Analysis. Vol 3. London, Academic Press, 118-126
Yamamoto K (1995) Cathepsin E and cathepsin D: biosynthesis, processing and subcellular location. In Takahashi K, ed. Aspartic Proteinases: Structure, Function, Biology and Biomedical Implications. New York, Plenum Press, 223-229
Zdolsek JM, Svensson I (1993) Effect of reactive oxygen species on lysosomal membrane integrity. Virchows Arch [B] 64:401-406[Medline]
Zdolsek JM, Zhang H, Roberg K, Brunk UT (1993) Effects of H2O2 on lysosomal membrane integrity in cultured cells. Free Radical Res Commun 18:71-85[Medline]
Zhang H, Öllinger K, Brunk U (1995) Insulinoma cells in culture show pronounced sensitivity to alloxan-induced oxidative stress. Diabetologia 38:635-641[Medline]