Visualization of iron in cultured macrophages: A cytochemical light and electron microscopic study using autometallography

Free RadicalBiology& Medicine, Vol. 15, pp. 1-11, 1993 Printed in the USA. All rights reserved.

0891-5849/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.

Original Contribution VISUALIZATION OF IRON IN CULTURED MACROPHAGES: CYTOCHEMICAL LIGHT AND ELECTRON MICROSCOPIC STUDY USING AUTOMETALLOGRAPHY

A

JOHANN M. ZDOLSEK, KARIN ROBERG, and U L F T . B R U N K Department of Pathology II, Faculty of Health Sciences, University of LinkOping, S-581 85 Linkrping, Sweden

(Received 10 July 1992; Revised 15 October 1992; Re-revised 21 January 1993; Accepted 26 January 1993) A b s t r a c t - - T h e objective of this study was to develop a sensitive cytochemical method for the visualization of iron, both at light microscopical (LM) and at electron microscopical (EM) levels, in glutaraldehyde-fixed cultured cells with reasonable morphological preservation. The method is based on autometallography (also called the sulfide silver method or the Timm technique). Gold, silver, and various metal sulfides have previously been shown to act as catalysts for cellular silver deposition from a physical developer (autometallography). In our modification of this cytochemistry, a high pH is used during the initial sulfidation step to guarantee adequate levels of sulfide ions to generate enough Fe(II or III) sulfide. Since this procedure may cause severe cellular distortion, we initially stabilize the cultured cells by a glutaraldehyde fixation. We have compared our new high pH, high S2- LM and EM variety of autometallography with other modifications of this technique that have previously been used for LM and EM demonstration of easily sulfidated heavy metals, such as zinc. Cultured mouse macrophages were examined for the localization of reactive metals following endocytosis of ferritin or inorganic Fe(III) iron. Ag-precipitates, presumed to indicate the presence of iron, were predominantly found within secondary lysosomes of the acidic vacuolar apparatus. The relation of the Ag-precipitates to iron was proven by the fact that iron-exposed cells showed a much reduced amount of silver precipitates after subsequent exposure to deferoxamine a potent iron chelator. Moreover, control macrophages neither exposed to iron nor to ferritin showed only a low normal lysosomal content--and a few extralysosomal sites--of reactive substances, believed to be iron. Keywords--Macrophages, Cultured cells, Heavy metals, Iron, Lysosomes, Cytochemistry, Autometallography, Oxygen radicals, Free radicals

INTRODUCTION

Many cellular injuries are attributed to the effects of reactive oxygen species (ROS), ~and in most cases the hydroxyl radical (HO") is considered to be the main causative agent. The hydroxyl radical is usually formed only secondarily (apart from radiolysis of water) following interactions between iron and hydrogen peroxide (H202), formed directly or via dismutation of superoxide anion radicals (02"-). Very small quantities of reactive Fe(II)-complexes are enough to catalyse the formation of HO" from H202 (Fenton reaction) with further induction of chain peroxidative reactions. Consequently, the cellu-

Supported by the Swedish Cancer Society, grant no. 2703, the Swedish Medical Research Council, grant no. 448 l, and Link0ping University Hospital research grants. Address correspondence to: Johann M. Zdolsek.

lar distribution of iron in loosely bound, reactive form may determine where HO"-mediated oxidative damage will take place (site specificity),a'3 In most cells iron is by far the dominating heavy metal, although it is virtually always part of stable metallo-organic compounds in which iron is prevented from inducing Fenton chemistry. Reactive iron, however, may exist for short periods of time and particularly so in the lysosomal vacuome due to release of iron from degraded autophagocytosed ferritin and heme-containing proteins or to endocytic uptake of protein-bound iron. This iron, capable of redox cycling, may thereby determine the site specificity for the hydrogen peroxide-induced damage. 1,4-6Oxygen free radical research would thus greatly benefit from EM techniques for the selective cytochemical visualization of iron, but present techniques are insensitive and only demonstrate extreme iron o v e r l o a d . 7 Autometallography8 (initially named the Timm

2

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visualized by these earlier EM modifications (due to low concentrations of HS and S2- during sulfidation). The present work presents an EM and LM method based on autometallography for the detection of heavy metals, including iron and iron complexes, in cultured cells while preserving an acceptable ultrastructure.

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MATERIALS AND METHODS

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1 Fig. 1. In a protective colloid (shaded area) and in the presence ol hydroquinone, a core of metal or metal sulfide/selenide (MeS) catalyses the reduction of silver ions (Ag÷) to silver atoms (Ag), which are deposited on the catalytic core. The deposited silver atoms then in turn function as new catalytic sites and promote the further reduction of silver ions on their surface, finally forming a microscopically detectable Ag precipitate.

technique or the sulfide silver method, later the silver enhancement technique) is a sensitive cytochemical method for the demonstration of heavy metals (for principles see Fig. 1). The method was originally developed by Tirnm 9 and has so far mainly been used at the LM level. It demonstrates extremely low concentrations of a variety of cellular heavy metals, both naturally occurring and of foreign origin, provided the metal is not too firmly bound within metallo-organic complexes. Autometallography (AMG) demonstrates some metals directly (e.g., Au, Ag) ~°'ll and has been used to demonstrate sulfides and/or selenides of some other heavy metals at the LM and EM levels. 8"12-~4 Theoretically less than 10 atoms/molecules of gold, silver, or heavy metal sulfide would initiate the autometallographic process.a'~ 5 Autometallography has recently found a new application as an amplifying technique in gold-immunocytochemistry, making it possible to utilize l nm gold particles for both LM and EM. 16'iv Several previous investigators have modified the autometallographic technique for EM, always using a neutral pH (instead of alkaline conditions) during the initial sulfidation process to preserve ultrastructure. t8-2° Previous cytochemical EM modifications seem to demonstrate (1) a few nonnormally occurring metals such as gold, mercury, and silver (if delivered to and taken up by the cell); or (2) zinc where it occurs in large amounts (e.g., in pancreatic/3-cell granules ~s or in the hippocampus region of the brain), s'~9 It is questionable, however, whether metals other than those with very low solubility of their sulfides may be

Animals and chemicals Mice, Balb/c strain, were from Anticimex (Stockholm, Sweden). Thioglycollate medium was obtained from DIFCO (Detroit, M1). Ham's F-10 medium, fetal calf serum (FCS), and HEPES were from GIBCO (Paisley, UK). Glutamine, penicillin-G, and streptomycin were from Flow (Rickmansworth, UK). Ammonium-sulfide and hydroquinone were from BDH Ltd (Poole, UK). Epon-812 and silver-lactate were from Fluka AG (Buchs, Switzerland). Deferoxamine (Desferal ®) was purchased from Ciba-Geigy AG (Basel, Switzerland). Cationized ferritin was from Serva Feinbiochemica (Heidelberg, Germany). Glutaraldehyde (GA) was from Bio-Rad (Cambridge, MA), and osmium tetroxide was purchased from Johnson Matthey Chemicals (Royston, UK). All other chemicals were of analytical grade and purchased from Merck (Darmstadt, Germany).

Cell cultures Macrophages. Unstimulated mouse peritoneal macrophages and thioglycollate elicited mouse peritoneal macrophages (MPMs) were obtained from adult male mice (weighing 20-30 g) by lavage of the peritoneal cavity with 5 ml Ham's F-10 medium, zu22 The latter were obtained by injecting 1 ml ofthioglycollate medium into the peritoneal cavity of the mice 4 d prior to cell harvesting. The animals were killed by spinal shock. The abdominal skin was carefully removed, without damaging the peritoneum, and cold F-10 (4°C) was injected into the peritoneal cavity. The abdomen was gently massaged for 1 rain, after which a small hole was cut in the peritoneum and the peritoneal fluid was withdrawn. The cell suspension was centrifuged at 200 g for 10 min. The pellet was resuspended in Ham's F-10 medium supplemented with 10% NCS, 10 mM Hepes buffer (pH 7.4), 100 ~g]ml streptomycin, 2 mM L-glutamine, and 100 U/ml penicillin-G. Approximately 6 × 105 cells were then plated into 35-mm polystyrene dishes (Costar ~) and allowed to attach for 45 min in an incubator at 37°C in an atmosphere of 5% CO2 in humid air. Subse-

Visualization of iron

quently the medium was removed and the dishes rinsed with F-10 (37°C) to remove nonadhering cells. Then 2 ml of culture medium were added to each dish. For LM, round glass coverslips (diam. 22 mm) were placed in the culture dishes before cell plating. Erythrocytes were from peripheral blood obtained from one of the authors.

Experimental scheme Macrophages. Following 24 h in culture, cells were prepared for light and electron microscopy. For light microscopy the procedure was as follows: 1. Macrophages were exposed to 25 uM FeC13 in complete culture medium with FCS for 24 h, rinsed with 5 ml warm F-10 medium, and then immediately fixed, sulfidated with ammonium sulfide, or sodium sulfide (see below), and subjected to AMG (see below). Control macrophages were identically treated, although not exposed to iron. 2. Macrophages were exposed to 25 uM FeCI3 in complete culture medium with FCS for 8 h followed by a rinse with 5 ml warm F-10 medium and a further 18-h culture in complete culture medium with or without 100 #M deferoxamine. Control macrophages were cultured in an identical fashion, although they received no iron during the initial 8-h culture period. Thereafter fixation (2, 24, or 48 h), sulfidation with ammonium sulfide, and AMG were carried out (see below). 3. Elicited macrophages were exposed to cationized ferritin ( 1 mg/ml) at 4°C for 30 min, washed three times with cold medium, and changed to ferritinfree culture medium for another 1 h at 37°C, followed by fixation, sulfidation with ammonium sulfide, and AMG (see below).

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for 2 h at 22°C (unless otherwise stated) followed by short rinses (× 5) in glass-distilled water at 22°C. Cells were then sulfidated either in 1% ammonium sulfide in 70% ethanol at 22°C for 15 min (pH ~ 9) or in 0.1 or 1% sodium sulfide in PBS at 22°C for 15 min or 24 h (pH 7.2). Following careful rinsing with running glass-distilled water for 10 min at 22°C, development was performed using a colloid-protected developer according to Danscher l° containing Ag-lactate (0.11 g in 15 ml distilled water), hydroquinone (0.85 g in 15 ml distilled water), 60 ml 25% gum arabic, and 10 ml Na-citrate buffer, pH 3.8. Development (silver amplification) was performed at 26°C in the dark for 10-30 min. Following dehydration in a graded series of ethanol solutions, cells grown on coverslips were mounted with Canada balsam for LM. Cells for transmission electron microscopy (TEM) were dehydrated, directly or after an additional postfixation in 1% OsO4 in 0.15 M Na-cacodylate buffer for 90 min at 22°C, in a graded series of ethanol solutions and embedded in Epon-812 in the plastic Petri dishes as described earlierY Some cells were exposed en bloc to 1% uranyl acetate (UAc) in 50% ethanol overnight during dehydration. Thin sections were cut with a diamond knife and rapidly stained with uranyl-acetate and lead-citrate. For LM cells were examined and photographed in an Olympus AHBS photomicroscope (Tokyo, Japan) and for TEM in a JEOL 2000EX electron microscope (Tokyo, Japan) at 100 kV. RESULTS

Effect of sulfidation with sodium (pH ~ 7.2) or ammonium sulfide (pH ~ 9)

Erythrocytes. Following fixation (2 h in GA fixative) of air-dried erythrocyte smears, sulfidation with ammonium sulfide was performed (see below), followed by AMG for 10, 15, 20, 25, and 30 min.

Macrophages exposed to FeC13 for 24 h and further treated with 0.1% sodium sulfide (15 min) at pH 7.2 displayed only few and very small cytoplasmic granules (Fig. 2A). Prolonged sulfidation (24 h) using 0.1% sodium sulfide at pH 7.2 resulted in a cytoskeletonlike silver impregnation type of staining with only occasional granular cytoplasmic Ag deposits (not shown). Similar results were obtained if cells were treated with 1% sodium sulfide (not shown). In comparison, identically FeC13-exposed macrophages showed dense granular and cytoplasmic precipitations when utilizing ammonium sulfide at pH 9 during the sulfidation step (Fig. 2B).

Fixation, sulfidation, and autometallography

Effect ofFe, deferoxamine, and ferritin exposure

All cells were briefly rinsed in phosphate-buffered saline (PBS) (22°C) prior to fixation with 2% glutaraldehyde (GA) in 0.1 M Na-cacodylate with 0.1 M sucrose (pH 7.2; effective osmotic pressure 300 mOsm)

Following sulfidation with ammonium sulfide at pH ~ 9 and autometallography, there was a clearly visible and predominantly granular reaction product (Ag) in the cytoplasm of 8 h FeCla-exposed cells even

For transmission electron microscopy, control and FeCl3-exposed cells (25 #M, 24 h) were fixed in GA for 2 h followed by sulfidation with ammonium sulfide, AMG, and embedding in Epon (see below).

J. M. ZDOLSEK rt al.

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Fig. 2. Macrophages exposed to FeCl, (25 PM) for 24 h, followed by glutaraldehyde-fixation for 2 h, and sulfidation with (a) 0.1% Na,S at pH 7 for I5 min, and (b) 1% (NH,)$ at pH 9 for I5 min. Autometallography was performed for 15min. In (a) only few and minute silver grains arc visible, whereas in (b) there is a strong granular silver precipitate in the cytoplasm with a lysosomal distribution pattern. Bars = 20 pm.

after such short periods of development as 15 min (Fig. 3A). If the sulfidation or autometallography steps were omitted, no staining of the cells (iron exposed and control) was seen (not shown). If, however, the 8-h iron treatment was followed by an 18-h treat-

ment period with 100 PM deferroxamine, the amount of the cytoplasmic Ag precipitations was dramatically reduced (Fig. 3B). Control cells (no added iron) showed neither granular nor cytoplasmic staining patterns after a 15min development period (Fig. 3C),

Visualization of iron although if development time was increased to 25 min a weak granular and cytoplasmic staining was seen (not shown). Increasing the GA fixation time of FeCl3-exposed macrophages resulted in a slightly weaker "staining" pattern that was most noticeable in cells fixed for 48 h (not shown). When previously ferritin-treated macrophages (30 min, 4°C) were rinsed and exposed to complete culture medium without ferritin (1 h, 37°C) allowing endocytosis, precipitates were found in the vacuolar apparatus and in the form of fine black precipitates on the substratum (Fig. 4A). If the macrophages were fixed, sulfidated, and subjected to AMG immediately after the ferritin exposure, the cell surfaces were covered with fine black precipitates (not shown). Control cells, developed for the same period of time (10 min), neither showed precipitates on the cell surface, in the cytoplasm, nor on the substratum (Fig. 4B).

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detection of iron in cultured cells has not previously been performed at the EM level. The formation of metal sulfides from cellular metallo-organic compounds is crucial for the cytochemical detection of metals (other than Au or Ag) by autometallography and is dependent on (a) the concentration of S2- during the sulfidation process, (b) the solubility product of the formed metal sulfides (Table 1), and (c) the stability constant of the metallo-organic compound of interest. The faint LM reaction pattern in iron-loaded macrophages after development following an initial treatment with 0.1, or even 1%, NazS at pH ~ 7.2, as compared to the strong reaction following the treatment with (NH4)2S at pH ~ 9 (Figs. 2A-C), indicates the need for high [S2-] to achieve sufficient sulfidation of the protein-complexed iron according to reaction (1): Fe-protein +

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F e S -~-

"apo-protein"

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Erythrocytes Air-dried smears of human blood did not show any Ag precipitates in the erythrocytes, irrespective of development time. Leucocytes, however, revealed cytosolic "staining" and dense granules (not shown). TRANSMISSION ELECTRON MICROSCOPY

Effect of Fe exposure Following autometallography for 20 min of 24 h FeC13-exposed macrophages, coarse aggregations of Ag precipitates were seen in lysosomes, while some single precipitates occurred in the cytosol as well (Fig. 5A). In control macrophages cytoplasmic and vacuole-associated precipitates were evident following development for 20 min (Fig. 5B). The amount of precipitate in the lysosomes of control cells was considerably lower than in Fe-exposed cells. If the development time in the physical developer was increased to 30 min for control cells, the size of the individual Ag precipitates increased, not the number (not shown). Aggregated Ag precipitates were not found in nuclei, mitochondria, or in the endoplasmic reticulum of control or Fe-exposed cells. DISCUSSION Autometallography is an extremely sensitive cytochemical technique which at the LM level detects gold and silver as well as the sulfides of a number of heavy metals) The principles are outlined in Fig. 1. Modifications for EM have been described for gold, silver, mercury, and zinc. lo,11,13,~9 The autometallographic

Hydrogen sulfide (H2S) is a weak acid, and a high pH is required to obtain S2- concentrations high enough to drive reaction (l) to the right (Fig. 6). When FeC13 is added to the growth medium of cultured macrophages the iron, probably in the form of protein-iron-hydroxyl-phosphate complexes, 24 is taken up 25 by endocytosis and transferred to the acidic vacuolar apparatus of the cells. 26 The resulting lysosomal accumulation of iron is reflected by heavy silver precipitation following completion of autometallography even after even short periods ( 10-15 min) of development by the physical developer. Our modification of the autometallographic technique for visualization of heavy metals at the EM level is based on (l) an initial GA fixation carefully performed at physiological pH under isotonic effective osmotic conditions, followed by (2) exposure to S2- at high pH (pH -~ 9) and (3) autometallography. The initial glutaraldehyde fixation protects the ultrastructural morphology from the damaging effects of the high pH during the ensuing sulfidation step. GA fixation for prolonged periods of time (24/48 h) may slightly decrease the intensity of the silver precipitation at the light microscopic level, but it leaves the general staining pattern unaltered. The slight reduction in staining intensity observed after prolonged GA fixation may be due to GA-induced cross-linking of the protein components of the Fe°protein complexes, preventing iron displacement and sulfide conversion. In practice, however, this should be of no importance and sulfidation with (NH4)2 S at pH -~ 9 seems to ensure that a high enough fraction of iron in the Fe-

J. M.

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Fig. 3. Macrophages exposed to FeCI, (25 hM) under ordinary culture conditions for 8 h followed by (a) culture in iron-free medium for another 18 h and (b) medium with deferoxamine (100 PM for I8 h). Control cells (c) were cultured in complete culture media without additives for 8 + I8 h. All cultures were fixed in GA, sulfidated in (NH,),S, and physically developed for 15min. Note pronounced reduction in staining following deferoxamine treatment. Bars = 20 +m.

protein complexes are converted (where such conversion is at all possible) to Fe(I1 or III) sulfide in cells stabilized by GA for 1 to 48 h. Since the addition of deferoxamine (which is a very

efficient specific iron chelator) considerably reduced Ag depositions in Fe-exposed macrophages, it is reasonable to assume that our method mainly detects iron in these cells, especially since control cells not

Visualization o f iron

7

Fig. 3. Continued.

exposed to iron show no silver precipitates after 1015 min development (they do so only after 25-30 min. development). Our findings correlate well with those of Esparza and Brock 27who, using radioactively labelled iron, found a marked increase in iron release of iron-loaded cultured macrophages following exposure to deferoxamine. This chelator enters cells through fluid-phase endocytosis28 and probably remains in the vacuolar compartment (mainly secondary lysosomes) where it may, preferably at low pH, bind to iron released from degraded iron-containing biomolecules (e.g., autophagocytosed ferritin or heme groups). 29 The ferrioxamine complexes are then secreted (exocytosed) in the extracellular medium. Control (not iron treated) cells exposed to ammonium sulfide after GA fixation and then developed for 20-30 min also showed intralysosomal silver granules

Table 1. Solubility Products of Some Heavy Metal Sulfides Metal Sulfide FeS ZnS PbS CuS AgES HgS

Solubility Product 6.3 1.6 1.3 6.3 6.3 1.6

× X x × X ×

10 -~s 10-24 10 28 10 -36 10 T M 10 -52

when studied in the electron microscope. This reaction product corresponds to naturally occurring heavy metals with lysosomal locationJ 2 Since iron is by far the most common metal intracellularly, it is presumed that this metal is the cause of the silver precipitation, although additive effects of minute amounts of metals, such as zinc and copper, cannot be excluded. Deferoxamine did not significantly reduce this normal amount of iron, which might be explained by competition from normally occurring intralysosomal iron-chelating substances preventing formation of exocytosable ferrioxamine complexes. Ferritin is a cytosolic storage protein for Fe(III) in the cytoplasm of living cells. It is also a suitable tracer for endocytosis. 3° Using the cationized derivative of ferritin which preferentially binds to anionic membrane glycoproteins and then is endocytosed, we were able to demonstrate that our method detects iron in ferritin since precipitated Ag granules were visible both attached to the plasma membrane and on the microprecipitate 26'31 of the culture substratum as well as intracellularly in endosomes and lysosomes after endocytosis. The finding of Ag precipitates outside the cells, on the substratum, also demonstrates that proteolytic degradation of the ferritin molecules is not necessary to convert the associated iron into sulfide and that a high [S2-] is sufficient to drive reaction (1) to the right.

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Fig. 4. (a) Elicited macrophages exposed at 4°C to cationized ferritin. which binds to the plasma membrane, for 30 min followed by rinsing and further culture in ferritin-free medium for I h at 37°C allowing endocytosis of surface-bound ferritin. Control macrophages (b) show no Ag precipitate. Fixation and sulfidation as described in Fig. 3. Physical development for 10 min. Bars = 20 gum.

The cytochemical reactions of erythrocytes, mitochondria, and endoplasmic reticulum were constantly negative, indicating that the method is unable to detect heme-bound iron within hemoglobin and

cytochromes. The mode of iron binding in these compounds effectively prevents the formation of Fe(I1 or III) sulfide, even at exposure to very high concentrations of S2-.

Visualization of iron

Fig. 5. Electron micrographs ofFeCl,-exposed (Fig. 5a) and control (Fig. 5b) macrophages where the high pH, high S2- technique for metal visualization as described in Fig. 3 was used, with physical development for 20 min. In the former, presumably iron-rich cells, the amount of electron-dense Ag grains per vacuole is much increased. The smaller amount of Ag precipitate within the vacuolar apparatus of control cells with normal contents of iron as compared to iron-exposed ones is obvious. Bars = 1 pm.

J.M. ZDOLSEK et al.

10 100 80" 60" 40 20 i

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pH Fig. 6. Dissociation plots of the H2S, HS , S2- system. A high pH value (>_ 9) is needed to obtain any significant concentration of S2-.

Osmium postfixation followed by sulfidation resulted in the formation of compounds reacting with Ag ions during the development. This resulted in heavy non-specific Ag precipitation (not shown). Osmium postfixation performed after the silver amplification reaction resulted in considerable reduction in the amount of silver precipitates. Moreover, the combination of OsO4 postfixation and UAc staining en bloc resulted in almost complete disappearance of silver granules, indicating that OsO4 treatment causes oxidation of Ag to a product which is dissolved during subsequent procedures. Treatment with UAc for prolonged periods seems to increase this effect, requiring the omission of both OsOa fixation and UAc staining en bloc which, unfortunately, results in suboptimal EM contrast. UAc and Pb-citrate grid staining, however, did not give rise to any obvious dissolving of Ag precipitates and a reasonable contrast was thus obtained, allowing the identification of most cellular details. In conclusion, we present a high pH, high S2- variety of autometallography for the visualization of heavy metals in cells at both LM and EM levels which allows the demonstration of very small amounts of intracellular iron. The method involves careful initial stabilization of the cell structure by glutaraldehyde fixation followed by sulfidation at a high pH ( ~ 9 ) followed by autometallography in a colloid-protected Ag + redox system. This technique may be of importance in the evaluation of recently proposed intralysosomal Fenton reactions which may explain the mechanisms behind lipofuscinogenesis 32 and alteration of lysosomal stability by oxidative stress. 33-35 REFERENCES

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