Mercury-induced anti-nucleolar autoantibodies can transgress the membrane of living cells in vivo and in vitro

Manuchehr Abedi-Valugerdi, Hui Hu and Göran Möller

Department of Immunology, Wenner-Gren Institute, Arrhenius Laboratories for Natural Sciences, Stockholm University, 10691 Stockholm, Sweden

Correspondence to: M. Abedi-Valugerdi


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Treatment with HgCl2 induces a systemic autoimmune disease in certain mice and rats. The major characteristic of this disease in mice with H-2s genotype is the production of anti-nucleolar autoantibodies (ANolA). The exact mechanism(s) for the production and the functional role of mercury-induced ANolA are not known. We have studied the ability of mercury-induced ANolA to enter the living cells in vivo and in vitro. We found that in highly susceptible mice, treatment with mercury induced ANolA capable of localizing in the nucleoli of kidney and liver cells in vivo. No detectable nucleoli localization of ANolA were found in the cells of the heart, stomach, intestine and spleen. Consistent with the in vivo studies, mercury-induced ANolA were also able to enter and translocate in the nucleoli of certain cells in vitro. The highest degree of antibody penetration was found in A-498 cells (a human kidney cell line) followed by 3T3 cells (a mouse fibroblast cell line), whereas the cells of lymphoid origin exhibited a very low degree of antibody penetration. Penetrated ANolA could be recovered from the nucleoli of live 3T3 cells previously treated with ANolA. The in vitro nucleolar translocation by ANolA did not affect the DNA synthesis, but was found to be an active process dependent on time and temperature. Furthermore, pre-treatment of living cells with trypsin markedly inhibited both cell entry and nucleolar accumulation of ANolA. Thus, mercury-induced ANolA have a unique ability to transgress the membrane of certain living cells in vivo and in vitro, and to localize in the nucleoli.

Keywords: anti-nucleolar autoantibodies, fibrillarin, H-2s mice, HgCl2, penetrating autoantibodies


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
It is well established that the heavy metal ion mercury induces a systemic autoimmune-like disease in genetically susceptible mice and rats (reviewed in 1,2). In highly susceptible mouse strains such as SJL (H-2s), A.SW (H-2s) and B10.S (H-2s), the mercury-induced autoimmune disease is characterized by a T cell-dependent polyclonal B cell activation, an increase in the serum levels of IgG1 and IgE antibodies, the formation of renal immune complex deposits, and most strikingly the production of anti-nucleolar autoantibodies (ANolA) mainly of IgG1 isotype (14).

It has been shown that most of the mercury-induced ANolA react with fibrillarin, a 34–36 kDa protein (57) which is associated with the U3, U8, U13, U14, X and Y small nucleolar RNAs in vertebrates (8). Fibrillarin is also a target for autoantibodies in a subset of patients with scleroderma (7,910). Interestingly, it has been found that murine mercury-induced and spontaneous human ANolA share several similarities in binding to fibrillarin (7). This finding led to the suggestion that both experimentally induced and spontaneously developed ANolA interact with similar evolutionary conserved epitopes in fibrillarin (7).

Although the production of autoantibodies against intracellular antigens is a common feature of systemic autoimmune diseases in human and animal models (reviewed in 11,12), in most instances the possible pathogenic role(s) and more important the function(s) of these autoantibodies are unknown. Since it is commonly believed that intracellular components are immunologically `privileged' and are not normally accessible to the circulating autoantibodies (11), the possibility that these autoantibodies can reach their intracellular targets while cells are alive has not been appreciated (11). However, studies have shown that autoantibodies with certain specificity such as anti-ribonucleoprotein (anti-RNP), anti-DNA and anti-ribosomal protein P obtained from patients with systemic autoimmune diseases are able to penetrate the living cells, react with their intracellular antigens and thereby interfere with the cell functions (reviewed in 13). These findings and the fact that the production of ANolA is the major characteristic of mercury-induced systemic autoimmune disease in highly mercury-susceptible mice led us to test the penetrating ability of mercury-induced ANolA into the alive cells both in vivo and in vitro.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female or male A.SW (H-2s) and SJL (H-2s) mice were bred and kept in our animal facilities at the Department of Immunology, Stockholm University. Both mouse strains were 5–8 weeks old at the beginning of each experiment.

Treatment with HgCl2
Mercury treatment was done as described (14). Briefly, Groups of SJL (H-2s) and A.SW (H-2s) mice (three to four mice per group) were injected s.c. with either 0.1 ml of HgCl2 solution (1.6 mg/kg body weight) or 0.1 ml of sterile saline (controls) every third day for 4 weeks.

Organs and blood collection
At the end of each experiment, mercury- and saline-treated mice were first bled, and thereafter were killed by cervical dislocation. Kidney, spleen, liver, stomach and intestine tissues were removed and immediately preserved either in liquid nitrogen or in dry ice and then stored at either –70 or –20°C until used. The blood of each mouse was allowed to clot at room temperature. Serum was separated after centrifugation and stored at –20°C until used.

Separation of IgG from the serum
IgG antibodies were purified from pooled sera collected from the blood of either mercury- and/or saline-treated A.SW (H-2s) mice by using a Protein G column (Pharmacia, Uppsala, Sweden) according to the manufacturer's instruction.

Detection of ANolA
The presence of IgG1 ANolA in the mice sera and the nuclear eluates was determined by indirect immunofluorescence (IIF) using rat liver sections as a substrate and FITC-conjugated anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL) as a detecting reagent according to the method described previously (14). The initial dilution for the sera was 1:30 and for the nuclear eluates was 1:1. The highest serum or nuclear eluate dilution at which nucleolar fluorescence could be detected was defined as the titer of IgG1 ANolA.

Assay for in vivo binding of mercury-induced ANolA to the nucleoli of living cells
The frozen tissues of the kidney, the liver, the heart, the intestine, the stomach and the spleen from the mercury- and saline-injected SJL mice were horizontally cut into two slices and embedded in OCT compound (Miles Scientific, Nunc, Naperville, IL) to make composite blocks. Sections 5 µm thick were cut from the composite blocks in a Jung (Leica, Heidelberg, Germany) chilled (–20°C) cryostat. The localization of the mercury-induced IgG1 ANolA within the cell nucleoli was detected by the direct immunofluorescence (DIF) method. Briefly, the sections were fixed in cold acetone for 5 min and air dried. Alternatively, in some experiments, the kidney tissue sections were first fixed with 4% paraformaldehyde (PFA)/PBS for 10 min at room temperature. Then, the fixation was followed by a 15 min treatment with 0.2% Triton X-100/PBS to permeabilize cell membrane and to allow free access to FITC-conjugated antibody. Thereafter, the fixed sections were incubated with serial dilutions of FITCconjugated goat anti-mouse IgG1 antibody (Southern Biotechnology) for 30 min. The initial dilution for FITC-conjugated antibody was 1:40, diluted either in PBS (acetone fixation) or PBS containing 0.2% Triton X-100 (PFA fixation). After the incubation period, the tissue sections were washed 3 times with PBS and examined with a fluorescent microscope as described previously (14). The highest dilution of antibody at which nucleolar fluorescence could be detected was defined as the titer of penetrated IgG1 ANolA.

Assay for in vitro binding of mercury-induced ANolA to the nucleoli of living cells
The cell lines 3T3 (a mouse embryo fibroblast line), A-498 (a human kidney carcinoma line), A20 (a mouse B cell lymphoma line) and CTLL-2 (a mouse T cell line) were originally obtained from the ATCC (Rockville, MD). 3T3 and A-498 cells were maintained in DMEM medium, supplemented with 10% FCS (Gibco/BRL). A20 cells were maintained in RPMI 1640 medium, supplemented with 10% FCS and 0.05 mM 2-mercaptoethanol. CTLL-2 cells were maintained in RPMI 1640 medium, supplemented with 10% FCS, 15 mM HEPES, 2 mM L-glutamine, 2 mM sodium pyruvate, 10% rat concanavalin A supernatant and recombinant IL-2 (diluted 1/1000). The cell cultures were incubated at 37°C in a humidified atmosphere containing either 5% (A-498, A20 and CTLL-2 cells) or 10% (3T3 cells) CO2. For penetration assay, Cells were grown either in 25 cm2 flasks (Costar, Cambridge, MA) or on glass slides in sterile dishes for 3 days. The cells grown as monolayers in flasks (A-498 and 3T3 cells) were first detached from the flasks either mechanically (by using a policeman rubber) or enzymatically (by incubation with 0.05% trypsin, 0.02% EDTA in PBS for 5 min at room temperature and by gentle shaking). Cell suspensions (1–2x105/ml) were prepared from the detached cells by washing the cells twice with either PBS or the corresponding media. Simultaneously, the media were aspirated from the cells grown as monolayers on glass slides (80% confluency). Thereafter, both preparations (cells as suspensions and cells as monolayers on glass slides) were incubated with a 1:50 dilution (diluted in either sterile PBS or the corresponding medium containing 10% FCS) of a serum obtained either from a mercury- or a saline-treated mouse for 1 h at 37°C. In a kinetics study, 3T3 and A-498 cells were incubated with the test sera for different time intervals and at different temperatures (as indicated in the footnotes for the tables and legends for the figures). After the incubation period, the cells were washed twice with PBS. Cells in suspensions were smeared onto glass slides, and then these cells and the cells grown as monolayers on slides were fixed with cold acetone for 5 min. In some experiments, the smeared cells were first fixed with 4% PFA/PBS for 10 min at room temperature, and then the cells were washed with PBS and treated with 0.2% Triton X-100/PBS for 15 min to permeabilize the cell membrane. To detect the intracellular mouse IgG1 bound to nucleoli, the slides were incubated with fluoresceinated goat anti-mouse IgG1 (Southern Biotechnology), diluted 1:40 with either PBS (acetone fixed cells) or PBS containing 0.2% Triton X-100 (PFA-fixed cells). After the incubation period (30 min at room temperature), the cells were washed 3 times with PBS. The stained cells were examined with a fluorescent microscope as described previously (14).

Elution procedure for penetrated ANolA
3T3 cell monolayers grown in glass Petri dishes were washed twice with sterile PBS and then incubated with a 1:50 dilution (in sterile PBS) of a serum of either a mercury- or a saline-treated SJL mouse for 1 h at 37°C. Thereafter, cells were washed twice with PBS, harvested with a policeman rubber and collected by centrifugation at 400 g. Nuclear extracts from the treated cells were prepared as described by Ochs et al. (15). Briefly, the collected cells were suspended in 10 volumes (of the cell pellet) of reticulocyte standard buffer (RSB-5) and allowed to swell on ice for 30 min. Nonidet P-40 (Boehringer Mannheim, Mannheim, Germany) was added to a final concentration of 0.3%. The mixture was homogenized with a high-speed rotor-stator-type homogenizer (Tissue-Tearor, Bartlesville, OK) using speed range of 4500–8000 r.p.m. for three to four bursts of 20 s. The homogenates were centrifuged at 1000 g for 10 min to sediment crude nuclei. To elute the penetrated IgG ANolA from the nucleoli, the isolated nuclei were suspended in 1 ml glycine–HCl buffer (0.1 M glycine–HCl, pH 2.6) and sonicated on ice for three or four bursts of 30 s. The disrupted nuclei were stirred at 4°C for 15 min and then centrifuged for 10 min at 1000 g. The eluates were immediately neutralized with 2 M NaOH solution and concentrated to 0.2 ml. Thereafter, the nuclear eluates were tested for the presence of IgG1 ANolA using IIF as described for the detection of ANolA.

[3H]Thymidine incorporation assay
3T3 and A-498 cells were cultured in 96-well sterile flat-bottomed microculture plates (Costar, Cambridge, MA) at a concentration of 105 cells/well. A serum from either a mercury- and/or a saline-injected SJL mouse, diluted to a final concentration of 1:50, were added to cultures. All the experimental and control cultures were run in triplicate. The cultures were incubated in a 37°C humidified incubator as previously indicated. After a 24 h incubation period, [3H]thymidine (Amersham International, Aylesbury, UK) at a final concentration of 2 µCi/ml was added to the cultures. After either a 24 h pulse of [3H]thymidine, the cells were harvested and the incorporated radioactivity was analyzed by a liquid scintillation counting in a ß-counter (Wallac Sverige, Upplands Väsby, Sweden).

Statistical analysis
Statistical analysis was performed using Student's two-tailed t-test. P < 0.05 was considered to be statistically significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mercury-induced ANolA bind to the nucleoli of the kidney and liver cells in vivo
Continuous administration of subtoxic doses of HgCl2 to highly mercury-susceptible mouse strains SJL (H-2s) and A.SW (H-2s) induced high serum titers of IgG1 antibody against the nucleoli as tested by IIF staining (data not shown, see also 14). We also found that the mercury-induced high serum levels of IgG1 ANolA corresponded to the high increase of splenic IgG1 antibody-secreting cells in both strains (data not shown, see also 14). To test the possible penetration of mercury-induced ANolA into viable cells in vivo, we first analyzed the tissue sections prepared from different organs of mercury- and saline-treated A.SW and SJL mice for the intracellular presence of autoantibodies, using a DIF staining. As shown in Fig. 1Go(a and b), most of the cells in the kidney and liver sections of mercury-treated mice (unless otherwise stated, the results from SJL mice are shown in this study) exhibited a strong nucleolar staining pattern (anti-IgG1, FITC-conjugate) with high titers (not shown) as compared with those of saline-treated mice (Fig. 1f and gGo). We did not find clear nucleolar staining in the cells of the heart, stomach (not shown), intestine and spleen sections of either mercury- or saline-treated mice (Fig. 1cGo–e and h–j respectively).



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Fig. 1. Cellular penetration and nucleolar localization of mercury-induced ANolA in vivo. Acetone-fixed sections of kidney (a and f), liver (b and g), heart (c and h), intestine (d and i) and spleen (e and j) from a mercury- (left panel) and a saline- (right panel) injected SJL mouse (a representative from several mice) were directly incubated with FITC-conjugated anti-mouse IgG1. Thereafter, the tissue section were examined with a fluorescent microscope for the presence of IgG1 anti-nucleolar antibodies. Note the clumpy nucleolar staining in the majority of kidney and liver cells of mercury-injected mice (arrows in a and b). Titers for FITC-conjugated antibody, kidney, liver and spleen sections 1:160, intestine sections 1:80, heart sections 1:40. Magnification x400 (reduced 1:6 for the illustration). The yellow color background seen in (f) and (g) is due to the longer light-exposure time used in order to visualize the sections.

 
It has been suggested that the in vivo cellular penetration of autoantibodies is a fixation artifact (16), i.e. autoantibodies move from the periphery into the cells during fixation with acetone. To exclude this possibility, in a separate study, the tissue sections prepared from the kidneys of mercury- and saline-injected mice were first fixed with PFA, and then the cells were permeabilized with Triton X-100 and stained with FITC-conjugated anti-IgG1 antibodies. Again, a nucleolar green fluorescence was found in the cells of the kidney sections prepared from the mercury- but not saline-injected mice (not shown). However, the fluorescence intensity and the titers of FITC-conjugated antibodies were lower than those we found in the preparations fixed with acetone (not shown). These results demonstrate for the first time that injection of mercury into the susceptible mouse strains with the H-2s genotype induced autoantibodies which are able to penetrate into the cells of certain organs and react with their corresponding antigens in vivo.

Mercury-induced ANolA bind to the nucleoli of certain living cells in vitro
The presence of high titers of serum IgG1 ANolA and nucleolar localization of these autoantibodies in the liver and kidney cells in the mercury-treated mice led us to determine whether circulating ANolA are also able to penetrate the living cells in vitro. We first used a human epithelial cell line of kidney origin (A-498) and a mouse fibroblast cell line of embryo origin (3T3). As shown in Fig. 2Go(a–d), by applying DIF staining, an intensive nucleolar fluorescent staining pattern was observed in both viable cell types after only 60 min incubation with a serum of a mercury- (Fig. 2a and bGo) but not of a saline- (Fig. 2c and dGo) injected SJL mouse. Interestingly, the degree of ANolA penetration varied in each cell line. Nucleolar fluorescence was most intense in the human kidney cells followed by the mouse fibroblast cells (Table 1Go). Treatment with the mouse sera did not affect the viability of the cells as demonstrated by the exclusion of Trypan blue (not shown).



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Fig. 2. Mercury-induced ANolA penetrate and translocate in the nucleoli of living cells in vitro. Human kidney, A-498 (a and c) and mouse fibroblast 3T3 cells (b and d) were incubated with a serum obtained either from a mercury- (a and b) or a saline- (c and d) treated SJL mouse for 1 h at 37°C. Thereafter, the cells were washed, fixed with acetone and incubated with FITC-conjugated anti-mouse IgG1 for 30 min. Then the cells were examined with a fluorescent microscope for the presence of IgG1 anti-nucleolar antibodies. Note the intensive clumpy nucleolar staining of both cell lines incubated with the serum of mercury-treated mice (arrows in a and b). Magnification x400 (reduced 1:2 for the illustration).

 

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Table 1. Localization of mercury-induced ANolA into the nucleoli of various cell lines in vitroa
 
In order to ascertain the in vitro findings of cellular penetration and nucleolar localization by mercury-induced ANolA, in an additional experiment, human kidney cells (A-498) incubated with a serum of either a mercury- and/or a saline-injected SJL mouse were fixed with either acetone and/or PFA. Then the PFA-fixed cells were permeabilized with Triton X-100 and both acetone- and PFA-fixed cells were stained with FITC-conjugated anti-IgG1 antibodies. As shown in Fig. 3Go(a and c), cells incubated with mercury-injected serum and fixed with either PFA (Fig. 3aGo) or acetone (Fig. 3cGo) exhibited a very similar (if not identical) nucleolar fluorescence staining pattern. As expected, no nucleolar fluorescence was observed in those A-498 cells, which were incubated with the saline-injected serum (Fig. 3b and dGo).



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Fig. 3. In vitro cellular penetration and nucleolar localization by mercury-induced ANolA is not a fixation artifact. A-498 cells (a–d) were incubated with a serum of either a mercury- (a and c) and/or a saline- (b and d) injected SJL mouse for 1 h at 37°C. Thereafter, the cells were washed and fixed with either PFA (a and b) and/or acetone (c and d). The PFA-fixed cells were permeabilized with Triton X-100, and then the acetone- and PFA-fixed cells were incubated with FITC-conjugated anti-IgG1 antibodies. Then the cells were examined with a fluorescent microscope for the presence of IgG1 anti-nucleolar antibodies. Note the intensive clumpy nucleolar staining of the cell lines incubated with the serum of mercury-treated mice (arrows in a and c). Magnification x400 (reduced 1:2 for the illustration). The yellow color background seen in (b) and (d) is due to the longer light-exposure time used in order to visualize the cells.

 
Our in vivo finding that mercury-induced ANolA did not penetrate into the cells of the spleen, led us to investigate whether cellular penetration by mercury-induced ANolA could be observed in the cell lines of lymphoid origin in vitro. A mouse B cell lymphoma cell line (A20) and a mouse cytotoxic T cell line (CTLL-2) were used. Only a small percentage of A20 (1–3%) and CTLL-2 (6–12%) cells exhibited low intensity of nucleolar fluorescence after 60 min (at 37°C) incubation with a serum of a mercury-injected SJL mouse (Table 1Go). Interestingly, the nucleolar fluorescence pattern in A20 and CTLL-2 cells differed from the one seen in A-498 and 3T3 cells. Several distinct fluorescence dots (nucleoli) were seen in the nuclei of A-498 and 3T3 cells after treatment with serum of a mercury-injected mouse, whereas A20 and CTLL-2 cells mainly exhibited one fluorescence dot with the same treatment (not shown)

To confirm that it was actually the mercury-induced ANolA that penetrated into the living cells, in the next series of experiment, the IgG (here mainly of IgG1 isotype) antibodies were purified from the sera of both mercury- and saline-treated A.SW mice, and were examined on 3T3 living cells. As shown by DIF staining (Fig. 4a and bGo), only 60 min incubation with 3T3 cells at 37°C was sufficient for the purified serum IgG1 from mercury- (Fig. 4aGo) but not saline- (Fig. 4bGo) treated mice to enter the cells and to localize in the nucleoli. This result strengthened the finding that mercury-induced ANolA are penetrating autoantibodies.



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Fig. 4. Entrance and nucleolar localization of 3T3 cells by IgG from mercury-injected mice. 3T3 cells were incubated with IgG antibodies purified from the sera of either mercury- (a) or saline- (b) treated A.SW (H-2s) mice for 1 h at 37°C. Following a wash step, the cells were fixed with acetone and then incubated with FITC-conjugated anti-IgG1 antibodies. Then the cells were examined with a fluorescent microscope for the presence of IgG1 anti-nucleolar antibodies. Note the fine clumpy nucleolar staining in the cells incubated with IgG antibodies obtained from the sera of mercury-treated mice (arrows in a). Magnification x400. The slight yellow color background seen in (b) is due to the longer light-exposure time used in order to visualize the cells.

 
Recovery of the nucleoli localized antibodies from the live cells
If mercury-induced ANolA were translocated to the nucleoli of the living cells, they could be recovered from these intracellular organelles. Therefore, the nuclei from 3T3 cells incubated with the serum of a mercury- and/or a saline-treated SJL mouse were isolated, and the nucleoli localized antibodies were eluted in acid conditions. Thereafter, the nuclear eluates were tested for the presence of IgG1 ANolA, using an IIF method. As shown in Fig. 5Go(b and d), only nuclear eluate of the 3T3 cells treated with the serum of a mercury-injected mouse exhibited reactivity against nucleolar antigens (Fig. 5bGo). In fact, the nuclear eluate obtained from the 3T3 cells treated with mercury-injected serum and its corresponding serum had very similar (if not identical) nucleolar patterns of fluorescence staining on the rat liver sections (Fig. 5a and bGo).



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Fig. 5. Recovery of mercury-induced penetrating ANolA. 3T3 cell monolayers grown in glass Petri dishes were incubated with a 1:50 dilution of a serum of either a mercury- or a saline-treated SJL mouse for 1 h at 37°C. Thereafter, the cells were harvested and nuclear extracts from the treated cells were prepared as described in Methods. Nuclear eluates (b and d) and their corresponding sera (a and c) were tested for the presence of IgG1 ANolA by using an IIF method (see Methods for further description). Only nuclear eluate of 3T3 cells treated with the serum of mercury-injected mouse (b) and its corresponding serum (a) exhibited a strong nucleolar staining (arrows). Dilutions for the sera 1:40 and for the eluates 1:1. Magnification x400 (reduced 1:2 for the illustration). The yellow color background seen in (c) and (d) is due to the longer light-exposure time used in order to visualize the sections.

 
Kinetics of mercury-induced penetrating ANolA
To study the kinetics of cellular penetration and nucleolar localization of mercury-induced ANolA in vitro, A-498 and 3T3 cells were incubated with a serum of either a mercury- and/or a saline injected SJL mouse for different time intervals and at different temperatures. Thereafter, a DIF staining was used to detect the penetrated antibodies. As shown in Table 2Go, the percentage of nucleolar DIF-positive cells was directly related to the length of time that the mercury-injected serum was incubated with the cells (both cell types). However, the two cell types showed different binding processes. The nucleolar binding process was more rapid in A-498 (>50% of nucleoli were positive after 15 min incubation with the mercury-injected serum) than in 3T3 cells (only 4–10% of the cells were positive after a 15 min incubation with the mercury-injected serum). After a 24 h incubation with the mercury-injected serum, 78–83% of A-498 cells exhibited an intense nucleolar fluorescence (Table 2Go and Fig. 6eGo), whereas 44–50% of nucleoli of 3T3 cells were positive (Table 2Go). The intensity of nucleolar fluorescence in these cells was lower than those in A-498 cells (Table 2Go). Furthermore, the nucleolar binding process was found to be temperature dependent, because only 3–4% of 3T3 cells and 21–32% of A-498 cells demonstrated a faint nucleolar fluorescence after incubation with the mercury-injected serum for 60 min at 4°C (3T3 cells) or 1°C (A-498 cells), while 13–24% of 3T3 cells and 65–67% of A-984 cells showed intense nucleolar staining when incubated with the same serum at 37°C for the same length of time (Table 2Go and Fig. 6aGo–d).


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Table 2. Penetration of mercury-induced ANolA into the nucleoli is time and temperature dependenta
 


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Fig. 6. Kinetics of cellular uptake and nucleolar translocation of mercury-induced ANolA. A-498 cells were incubated with a 1:50 diluted serum from either a mercury- (left panel) and/or a saline- (right panel) injected SJL mouse for different time intervals, and at different temperatures (a and b, for 1 h at 1°C, c and d for 1 h at 37°C, e and f for 24 h at 37°C). Thereafter, the cells were fixed with acetone and examined for the presence of intranuclear mouse IgG1 antibodies by using a DIF method as described in Methods. Note the intense nucleolar fluorescence in the cells incubated with mercury-injected serum for either 1 or 24 h at 37°C (arrows in c and e respectively). Magnification x400 (reduced 1:3 for the illustration). See also Table 2Go. The yellow color background seen in (a), (b), (d0 and (f) is due to the longer light-exposure time used in order to visualize the cells.

 
Effect of trypsin on cellular penetration and nucleolar localization by mercury-induced ANolA
To determine the possible involvement of a cell surface protein for binding and internalization of the mercury-induced ANolA, 3T3 and A-498 cells were first treated with trypsin, and then incubated with a serum of either a mercury- and/or a saline-injected SJL mouse. As shown in Table 3Go, pre-treatment of both cell lines with trypsin caused a dramatic decrease in the cellular entrance and nucleolar localization of ANolA.


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Table 3. Effect of trypsin treatment on cellular penetration and nucleolar localization of mercury-induced ANolA in vitroa
 
Effect of penetrated ANolA on the DNA synthesis
To determine the effect of prolonged incubation of penetrating mercury-induced ANolA on the cell functions such as replication, DNA synthesis was measured on both A-498 and 3T3 cells after 48 h incubation with a serum from either a mercury- or a saline-injected mouse. As shown in Table 4Go, no significant differences in [3H]thymidine uptake were observed between the cells treated with either a mercury- and/or a saline-injected serum.


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Table 4. Effect of penetrated mercury-induced ANolA on DNA synthesisa
 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the present study, we demonstrated that mercury-induced circulating ANolA can transverse the plasma and nuclear membrane of living cells, and translocate to the nucleoli both in vivo and in vitro.

In vivo, we found that mercury-induced ANolA penetrated into the cells of certain organs (kidney and liver), and in vitro we observed that transformed kidney epithelial cells (A-498) and normal fibroblasts (3T3) were much more vulnerable than lymphoid cells (A20 and CTLL-2) to the cellular penetration and nucleolar localization by mercury-induced ANolA. These findings suggest that cellular penetration by mercury-induced ANolA is relatively selective for certain cells and is limited by the cell origin, i.e. cell lines of epithelial and connective tissue origins are more sensitive than lymphoid cell lines to the antibody penetration. The observation that the percentage of stained cells and the nucleolar fluorescence intensity were higher in transformed A-498 than in normal 3T3 cells also suggests that the state of activation or differentiation of the cell might affect the antibody penetration. The results from other studies also support the above-mentioned suggestions. For instance, Golan et al. (17) have demonstrated that IgG antibodies obtained from a subset of patients with systemic lupus erythematosus (SLE) were able to penetrate into the transformed cell lines of epithelial origin but not of lymphoid origin. This group also found that the same antibodies were not capable of penetrating a normal cell line of epithelial origin (17). In another study, Zack et al. (18) have observed that an anti-double-stranded DNA monoclonal autoantibody was internalized and transported to the nucleus only in renal tubular cells but not in liver, cardiac, muscle and glomerular cells in vivo.

Although the phenomenon of autoantibodies penetrating into living cells and reacting with their intracellular antigens was described 20 years ago (19), the issue is still highly controversial. For example, it has been argued that cellular penetration by autoantibodies is rather a fixation artifact phenomenon (16), i.e. using organic solvents such as acetone as a fixative agent will allow the movement of autoantibodies into the cells during the tissue preparation. Two lines of evidence suggest that penetration of mercury-induced ANolA into living cells is not caused by a fixation artifact. First, ANolA-penetrated cells fixed with PFA (known as a cross-linking reagent, which forms intermolecular bridges and does not remove lipids) also exhibited an intensive nucleolar green fluorescence comparable to those fixed with acetone. Second, if mercury-induced ANolA move into the cells just during the tissue preparation, one would expect to see a similar pattern of fluorescence staining in the cells of most (if not all) organs and cell lines, not only in the kidney and liver cells (in vivo) or A-498 and 3T3 cells (in vitro).

Like human anti-nuclear IgGs (17) and mouse anti-DNA (20) penetrating antibodies, the kinetics of both cellular uptake and nucleolar localization of mercury-induced ANolA was found to be time and temperature dependent, suggesting that binding and penetration processes are active, energy dependent and receptor mediated. Obviously, further experiments are needed to clarify which cell surface molecule(s) is involved for binding and internalization of mercury-induced ANolA. However, the finding that trypsinization of the cells before co-culture with mercury-induced ANolA significantly inhibited both cellular and nucleolar translocation of the Ig suggests that internalization is mediated by a cell surface protein. In fact, various cell membrane proteins such as Fc{gamma} receptors on human T cells (21), brush border myosin (myosin 1) on H35 rat hepatoma cells (22) and a Po 38-kDa ribosomal phosphoprotein on human hepatoma cells (23) were found to mediate the antibody penetration. Studies are in progress to test whether either of these molecules or another hitherto unknown cell surface protein is involved in uptake of mercury-induced ANolA.

The mechanism(s) for the nucleolar translocation of ANolA also remains unknown, but it has been postulated that large nuclear proteins such as transcription factors, steroid hormone receptors and enzymes involved in DNA replication contain a short stretch of positively charged amino acids (nuclear localization sequences), which target them for transport through the nuclear pore to the nucleus (reviewed in 24). Interestingly, the structural analysis of three anti-DNA monoclonal autoantibodies with nuclear localizing property derived from lupus-prone mice has shown that at least two of the three mAb shared a conformational motif in the heavy chain CDR3 region and all three mAb contained multiple positively charged amino acids in their complementarity-determining regions, resembling nuclear localization sequences which direct nuclear import of proteins (25). Therefore, it is likely that mercury-induced ANolA also contain basic amino acid-rich sequences similar to those seen in above mentioned penetrating anti-DNA autoantibodies. Generation and molecular analysis of mercury-induced anti-nucleolar mAb will permit us to test this likelihood.

Several studies have shown that penetrating autoantibodies cause cellular dysfunction after entering the cell and reacting with their intracellular antigens (23,2627). Therefore, it has been suggested that these antibodies might have pathogenic roles (23 and reviewed in 28). It has been found monoclonal anti-DNA antibodies derived from lupus-prone, MRL/lpr/lpr mice penetrated and localized within the nuclei of different organs, after administration to normal mice (27). In the kidney, this was associated with functional abnormalities, including glomerular hypercellularity and proteinuria (27). It has also been shown that anti-ribosomal P protein autoantibodies, which are present in the sera of a subset of patients with SLE, penetrate into live hepatocytes and adversely affect the synthesis of apolipoprotein B resulting in an increase in cellular cholesterol with lipid droplet accumulation as seen in some chronic liver diseases (23). In contrast, others have found that cellular penetration and nuclear localization by anti-nuclear autoantibodies had no adverse effects on either cell viability or DNA synthesis (17,29). Consistent with these observations, we also found that DNA synthesis was not affected by mercury-induced penetrating ANolA. This can be explained by two possibilities. As we mentioned previously, the main target for mercury-induced ANolA is fibrillarin, which is known to be associated with snRNAs (8). In mammals the exact function of fibrillarin is not known, but it has been suggested that this nucleolar protein possibly participates in ribosomal biosynthesis. Based on this suggestion, it is likely that fibrillarin does not have a crucial role in the DNA synthesis and interfering with its function/structure by ANolA would not impair the cell proliferation. Second, since besides fibrillarin, several other nucleolar proteins (nucleolin, Nsr1p, Gar1p, Nop3p, B23, Drs1p, etc.) are also present in the mammalian nucleoli (30), it is likely that if fibrillarin is required for DNA synthesis and if binding of ANolA to fibrillarin impairs it's function, other nucleoproteins will take over fibrillarin's function. Since it has been suggested that fibrillarin is involved in the synthesis of ribosomal RNA, further studies are needed to test if nucleolar localization of ANolA would affect other cell functions such as protein synthesis. It is also of interest to determine whether similar ANolA, which are produced in subsets of scleroderma patients (7,910), are also able to enter the living cells.


    Acknowledgments
 
We thank Mrs. Lena Israelsson for excellent technical assistant. This study was supported by grants from the Swedish Medical Research Council.


    Abbreviations
 
ANolAanti-nucleolar autoantibodies
DIFdirect immunofluorescent
IIFindirect immunofluorescent
PFAparaformaldehyde
RNPribonucleoprotein

    Notes
 
Transmitting editor: C. Martinez-A

Received 27 April 1998, accepted 22 December 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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