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Rheumatology 2001; 40: 750-756
© 2001 British Society for Rheumatology

Original Papers

Monoclonal antibodies against human ribosomal P proteins penetrate into living cells and cause apoptosis of Jurkat T cells in culture

K.-H. Sun, S.-J. Tang1, M.-L. Lin, Y.-S. Wang, G.-H. Sun2 and W.-T. Liu

Faculty of Medical Technology and Institute of Biotechnology in Medicine, National Yang-Ming University,
1 Institute of Marine Biotechnology, National Taiwan Ocean University and
2 Division of Urology, Department of Surgery, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. This study was designed to determine the role of autoantibodies to the ribosomal P protein (anti-P Abs) in the pathogenesis of systemic lupus erythematosus (SLE) using monoclonal anti-P antibodies (anti-P mAbs).

Methods. Anti-P mAbs were prepared by a standard hybridoma procedure using recombinant human P1 and P2 proteins as immunogens. We studied the reactivities of these mAbs to P proteins, their binding and penetration capabilities in different cell lines and their apoptotic effects on Jurkat T cells.

Results. In addition to recognizing human P0, P1 and P2 proteins, the anti-P mAb 9B6-4 bound to 20–40% and penetrated 50–90% of astrocytes, Jurkat T cells and lung cancer cells via the P0 surface protein. Treatment with the mAb 9B6-4 also caused increases in the percentages of Jurkat T cells in the sub-G1 phase of the cell cycle (14.8%) and undergoing apoptosis (21.3%).

Conclusion. Anti-P autoantibodies may play a role in the pathogenesis of lymphopenia or lymphocyte dysfunction in SLE.

KEY WORDS: Anti-P autoantibody, T lymphocytes, Apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Autoantibodies against acidic ribosomal phosphoproteins (anti-P Abs) have been demonstrated in 13–20% of patients with systemic lupus erythematosus (SLE) [1]. These autoantibodies have been reported to have significant associations with lupus psychosis [1–3], nephritis [4] and hepatitis [5, 6]. Moreover, they may also take part in the general disease activity [6–8] and skin manifestations [7, 8] of SLE. These epidemiological findings suggest that anti-P Abs may play an important role in the pathogenesis of SLE.

The P proteins (P0, 38 kDa; P1, 19 kDa; P2, 17 kDa) are generally considered to be associated with the 60 S ribosomal subunit in eukaryotic cells [9]. However, ribosome-free P proteins may exist in the cytoplasm [8] and P0 has been demonstrated on the surface of human hepatoma and neuroblastoma cells as well as fibroblasts [10]. Moreover, affinity-purified anti-P Abs may penetrate into living HepG2 cells and affect the synthesis of apolipoprotein B [11]. Reactions of anti-P Abs with their antigenic targets may have significant effects on the functions and viability of cells.

It has been reported that anti-ribosomal and P-peptide-specific autoantibodies from the plasma of SLE patients may bind to T lymphocytes [12]. These autoantibodies have been considered as a new subtype of antilymphocyte antibody [13]. However, the effect of anti-P Abs on the functions of T cells remains unclear. In this study, we succeeded in preparing monoclonal anti-P Abs (anti-P mAbs) using human recombinant P1 and P2 proteins as immunogens, and we studied the interactions of these antibodies in different cell lines. The results indicate that anti-P mAbs may bind to and penetrate different types of living cells. Moreover, they may also induce apoptosis in T cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell lines
Cell lines of human acute T-cell leukaemia (Jurkat), myeloma (NS-1) and human lung cancer (H1299) were obtained from the American Type Culture Collection. The normal rat brain astrocyte line (RBA-1) was obtained from the cell bank at the Veterans General Hospital, Taipei, Taiwan.

Preparation of recombinant human acidic ribosomal phosphoproteins
The cDNAs of human ribosomal phosphoproteins P0, P1 and P2 were cloned from phytohaemagglutinin-stimulated human mononuclear cells by reverse transcription–polymerase chain reaction [14]. Forward and reverse primers for the P0 and P1 genes were prepared as described in our previous paper [14]. Those for the P2 gene were 5'-GAGGATCCATGCGCTACGTCGCCTCCTACCT-3' and 5'-GAGTCGACAGGGGAGCAGGAATTTAATCAAA-3' respectively. These genes were then inserted into the vector pET-28a (Novagen, Madison, WI, USA) and expressed in BL21DE3 cells (Novagen). Bacterial proteins were solubilized with urea (6 M) and purified by histidine-binding metal chelation resin chromatography (His-bind, Novagen). Because denatured 38-kDa P0 protein could not be renatured in phosphate-buffered saline (PBS), only P1 and P2 proteins were dialysed against PBS (pH 7.2) and employed as antigens in enzyme-linked immunosorbent assays (ELISA). Protein concentrations were determined using a protein assay kit from Bio-Rad (Hercules, CA, USA).

Preparation of anti-P mAbs
The affinity-purified recombinant P1 and P2 proteins were further purified by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The denatured forms of these antigens (10 µg) in CFA (complete Freund's adjuvant) and IFA (incomplete Freund's adjuvant; Sigma, MO, USA) were injected subcutaneously into female BALB/c mice (8–10 weeks old) on days 1 and 35 respectively. On days 21 and 28, native forms of these antigens (10 µg) in CFA and IFA respectively were injected into the footpad of the immunized mice. Anti-P mAbs were prepared by fusion of NS-1 myeloma cells with spleen cells from the immunized mice [15].

Characterization of anti-P mAbs
The 3H10-33 and 9B6-4 hybridoma cells were selected by their production of monoclonal antibodies reactive with the recombinant P1 protein using ELISA [16]. Polystyrene microtitre plates (Corning, New York, NY, USA) were precoated with 100 µl of recombinant P1 or P2 protein (5 µg/ml in 50 mM sodium carbonate buffer, pH 9.6) at 4°C for 18 h. After the plates had been washed with PBS (pH 7.2) containing 0.05% Tween 20 (washing buffer), 200 µl of 1% bovine serum albumin in PBS was added and the plates were incubated for 1 h. The plates were then washed with washing buffer and the samples (100 µl) were added. After incubation for 1 h and washing with the washing buffer, diluted horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson Immunoresearch, West Grove, PA, USA) (100 µl) was added to the plates before incubation at room temperature for 1 h. The plates were washed again and 100 µl freshly prepared substrate solution (1.37 mg/ml of 2,2'-azinodi-13-ethylbenzthiazoline sulphonic acid in 100 mM phosphate buffer, pH 4.2, containing 0.6 µl/ml 30% H2O2) was added to each well. After 15 min incubation, the signal was measured at 410 nm in a Dynatech ELISA reader (Dynex Technologies, Virginia, USA).

To determine the reactivities of the mAbs with the P proteins of cells, non-synchronized, prefixed HEp-2 cells on glass slides (Scimedx, Denville, NJ, USA) were stained by indirect immunofluorescence with the anti-P mAbs. Hybridoma and NS-1 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (10% FBS-RPMI). The concentrations of the anti-P mAbs were determined with an ELISA kit (Boehringer Mannheim Biochemicals, Mannheim, Germany), and a preparation of normal mouse IgG (Sigma) was used as a non-specific isotype antibody control. The endotoxin contents of antibody preparations and culture media were measured with the Limulus amoebocyte lysate assay (Sigma), with a minimal detectable concentration of 0.05 EU/ml (endotoxin units per ml).

Western blotting
The cell lysate (5x105) and affinity-purified P0, P1 and P2 proteins (2 µg/well) were separated by 13% SDS–PAGE. These proteins were electrotransferred and immunoblotted by the use of anti-P mAbs (20 µg/ml). The antigen–antibody complexes on the nitrocellulose paper were detected by peroxidase-coupled anti-mouse IgG and enhanced chemiluminescence detection kits (ECL; Amersham International, Little Chalfont, UK).

Binding and penetration of anti-P mAb into cells
The binding of anti-P mAb to the surface of Jurkat T, H1299 and RBA-1 cells was studied. The cells (3x106) were incubated with the culture supernatant of anti-P hybridoma (10 µg/ml) or mouse immunoglobulin G (IgG; 20 µg/ml) in an ice-bath for 1 h. The cells were washed with ice-cooled PBS in triplicate, then incubated with 200xdiluted fluorescein isothiocyanate (FITC)-labelled goat anti-mouse IgG (Jackson Immunoresearch) in an ice-bath for 40 min. The percentage and mean fluorescence intensity of the positive cells were determined with a flow cytometer (Becton Dickinson, San Jose, CA, USA) after washing the cells with ice-cooled PBS in triplicate.

Penetration of anti-P mAb into the cells was determined according to Avrameas et al. [17]. The cells (3x105/ml) were incubated with the culture supernatants of anti-P hybridomas (10 µg/ml) or mouse IgG (20 µg/ml) in a 5% CO2/95% air incubator at 37°C for 18 h. The cells were washed with PBS in triplicate then fixed with 2% p-formaldehyde in PBS (pH 7.4) for 25 min, washed with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The fixed cells were incubated with 200xdiluted FITC-labelled goat anti-mouse IgG antibodies at room temperature for 40 min. After washing with PBS in triplicate, intracellular mAbs were observed under a confocal scanning microscope (Leica, Heidelberg, Germany). The percentage and mean fluorescence intensity of the positive cells were determined by flow cytometry.

On-dish membrane biotinylation of cells
Membrane labelling of bottom-attached RBA-1 cells with biotin analogue was performed as described by Meier et al. [18]. The cells were biotinylated with 100 µg/ml biotin-7-NHS (Boehringer Mannheim) at 4°C for 30 min. After biotinylation, cells were scraped and washed with PBS. Total cell proteins were extracted from the cell pellets by sonication in an extraction buffer [50 mM borate, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2.5 mM PMSF (phenylmethylsulphonyl fluoride), pH 8.0] and then incubated in an ice-bath for 30 min. The homogenates were cleared by centrifugation at 12 000 r.p.m. for 20 min. The biotin-labelled membrane proteins were immunoprecipitated with streptavidin-conjugated agarose beads (Pierce, Rockford, IL, USA) at 4°C for 18 h. The beads were then washed twice with PBS. After adding 40 µl SDS–PAGE sample buffer, the washing solution was carefully removed from the bead pellet. These samples were analysed by the use of 13% SDS–PAGE. The proteins were electrotransferred and immunoblotted by anti-P mAbs (20 µg/ml).

Cell cycle analysis
Jurkat T cells (3x105/ml) were cultured with 10 µg/ml preparations of 9B6-4, 3H10-33, mouse IgG or 0.01 µM or 0.05 µM preparations of actinomycin D (Clontech, Palo Alto, CA, USA) in a 5% CO2/95% air incubator at 37°C for 18 h. After fixation with 70% alcohol at -20°C for 30 min, these cells were stained with RNase A (1 mg/ml) and propidium iodide (PI) (400 µg/ml) at 37°C for 30 min. The cell cycle was analysed by flow cytometry.

Detection of apoptosis
The ApoAlert Annexin V-FITC apoptosis kit (Clontech) was used to detect apoptosis. Jurkat T cells (3x105/ml) were cultured with 10 µg/ml preparations of anti-P mAb 9B6-4, mouse IgG, NS-1 supernatants or 0.05 µM preparations of actinomycin D at 37°C in a 5%CO2/95% air incubator for 18 h. After rinsing with a binding buffer, the cells were stained with 5 µl Annexin V-FITC (20 µg/ml) and 10 µl PI (50 µg/ml) at room temperature for 5–15 min before analysis by flow cytometry.

Statistical analysis
Data are expressed as mean±S.D. and statistical significance was assessed with Student's t-test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Immunological properties
Preparations of anti-P mAbs 9B6-4 and 3H10-33 contained only IgG1 antibodies and were reactive with P proteins. The 9B6-4 preparation was found to be reactive with proteins P0, P1 and P2 by ELISA, Western blotting (Fig. 1Go) and immunofluorescence staining of HEp-2 cells. The 3H10-33 preparation was only reactive with the recombinant P1 protein and showed weak immunofluorescence staining of HEp-2 cells (Table 1Go). The endotoxin contents of these anti-P mAbs were below the detectable limit (0.05 EU/ml).



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  		FIG. 1. Western blot analysis of whole-cell lysate of RBA-1 cells and recombinant human ribosomal phosphoproteins P0, P1 and P2 with anti-P mAb 9B6-4. Lane 1, recombinant P0 protein; lane 2, recombinant P1 protein; lane 3, recombinant P2 protein; lane 4, whole-cell lysate of RBA-1 cells. The molecular weight of the recombinant proteins was 4 kDa greater than that of the natural proteins as there are an additional 32 amino acids upstream of the multiple cloning sites in the pET-28a expression vector.

 

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  		TABLE 1. Immunological properties of monoclonal anti-P antibodies 3H10-33 and 9B6-4 (20 µg/ml)

 

Binding and internalization
The percentages of positive binding (21.0–39.3%) and penetration (56.6–90.5%) of anti-P mAb 9B6-4 for Jurkat T, H1299 and RBA-1 cells were significantly higher than those of mouse IgG (5.0–5.8%) (Table 2Go). However, the anti-P mAb 3H10-33 only bound to 5.4–7.3% and penetrated 10.2–13.7% of the cells. Although negative results were obtained with mouse IgG (Fig. 2AGo), anti-P mAb 9B6-4 penetrated RBA-1 cells bound to the P proteins in the cytoplasm, as determined by confocal scanning microscopy (Fig. 2BGo).


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  		TABLE 2. Percentages of human acute T-cell leukaemia cells (Jurkat cells), normal rat brain astrocytes (RBA-1 cells) and human lung cancer cells (H1299 cells) bound (4°C, 1 h) and penetrated (37°C, 18 h) by monoclonal anti-P antibodies 9B6-4 and 3H10-33 and mouse IgG (10 µg/ml)

 


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  		FIG. 2. Analysis of penetration of anti-P antibodies (anti-P mAbs) 9B6-4 into rat brain astrocytes (RBA-1) by confocal scanning microscopy and determination of the reactivity of anti-P mAbs 9B6-4 with the membrane proteins on cultured RBA-1 cells. (A and B) Cells penetrated by mouse IgG and anti-P mAbs 9B6-4 respectively. (C) Anti-P mAbs 9B6-4 on the plasma membrane of on-dish biotinylated RBA-1 cells. A 38-kDa membrane protein (lane 1), similar to human acidic phosphoprotein P0 from the whole-cell lysate of RBA-1 cells (lane 2), was reactive with anti-P mAb.

 

Reaction with P0 on cell membrane
The targets of anti-P mAb on the cell surface were determined by precipitating biotinylated membrane proteins of RBA-1 cells with streptavidin–agarose beads (Fig. 2CGo). A protein with the same molecular weight as P0 (38 kDa) on the cell membrane was reactive with anti-P mAb (Fig. 2CGo, lane 1). This antigenic target on the cell surface was similar to human acidic ribosomal phosphoprotein P0 from the whole-cell lysate of RBA-1 cells (Fig. 2CGo, lane 2).

Increase in sub-G1 T cells
Cells with apoptotic DNA fragments (sub-G1 cells) were counted by cell cycle analysis. The percentage of sub-G1 cells (15.8%, P<0.05) increased in the co-culture of Jurkat T cells with anti-P mAb 9B6-4 (10 µg/ml), whereas mAb 3H10-33 only had a small effect on these cells (9.4%) (Fig. 3Go). However, no apparent changes in the growth patterns of RBA-1 and H1299 cells were observed after treatment with anti-P mAbs.



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  		FIG. 3. Effect of anti-P mAb 9B6-4 on the sub-G1 phase of Jurkat T cells. Jurkat T cells (3x105/ml) were cultured with 10 µg/ml preparations of anti-P mAb 9B6-4, anti-P mAb 3H10-33 or mouse IgG (mIgG), or 0.01 µM preparations of actinomycin D (Act D) for 18 h. After fixation, the cells were stained with PI. Jurkat T cells in sub-G1 phase were detected by flow cytometry. The data are mean±S.D. for three separate experiments; 10 000 cells were analysed under each condition. *P<0.05.

 
Actinomycin D was used to determine whether the differences in the sensitivity of the cells to anti-P mAb were common to other inhibitors of RNA or protein synthesis. In the presence of actinomycin D, the increase in sub-G1 Jurkat T cells was dose-dependent (48.5 and 15.2% sub-G1 cells for 0.05 and 0.01 µM respectively) (Fig. 4Go). However, actinomycin D increased the percentage of sub-G1 RBA-1 cells with different sensitivity (38.3% for 0.5 µM and 3.7% for 0.05 µM) (Fig. 4Go).



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  		FIG. 4. Effect of actinomycin D (Act D) on the sub-G1 phase of Jurkat T and RBA-1 cells. Jurkat T and RBA-1 cells (3x105/ml) were cultured with 0.01, 0.05 or 0.5 µM preparations of actinomycin D for 18 h. After fixation, the cells were stained with PI. Jurkat T and RBA-1 cells in sub-G1 phase were detected by flow cytometry.

 

Induction of apoptosis in Jurkat T cells
Externalization of phosphatidylserine on the plasma membrane of viable cells is an early event in apoptosis [19]. In this study, phosphatidylserine-binding protein (annexin V) was used as a probe to determine the redistribution of this phospholipid and the PI uptake of cells in order to detect cell death. Early apoptotic cells will be positive for annexin V-FITC and negative for PI. Although 35.2% of the actinomycin D (0.05 µM)-treated viable Jurkat T cells expressed phosphatidylserine on the membrane (Fig. 5Go), the anti-P mAb 9B6-4 induced externalization of phosphatidylserine on 21.3% of the viable cells in 18-h co-cultures (P<0.05). These changes were not found in the cultures of Jurkat T cells treated with mouse IgG.



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  		FIG. 5. Increase in phosphatidylserine externalization during induction of apoptosis in Jurkat T cells by anti-P mAb 9B6-4. The cells (3x105/ml) were cultured with 10 µg/ml preparations of anti-P mAb 9B6-4 or mouse IgG (mIgG), or a 0.05 µM preparation of actinomycin D (Act D) for 18 h. After rinsing, the cells were stained with annexin V-FITC and PI. Phosphatidylserine-binding protein (annexin V) was used as a probe to detect the redistribution of this phospholipid and PI uptake of cells in order to detect cell death. The percentages of the annexin V-FITC-positive and PI-negative cells undergoing apoptosis were determined by flow cytometry. The data are mean±S.D. of two separate experiments; 10 000 cells were analysed under each condition. *P<0.05.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
It has been reported that anti-P Abs prepared from mice immunized with P1 and P2 of Artemia salina differ from the spontaneously occurring anti-P autoantibodies in their predominant binding to rat P0 and P2 on immunoblots [20, 21]. In this study, we employed human recombinant P1 and P2 proteins as the immunogen in preparing anti-P mAbs. Although the 9B6-4 preparation was reactive to P0, P1 or P2, a significant reaction was observed mainly for the 3H10-33 preparation and P1. Moreover, anti-P mAb 9B6-4 may bind to a membrane protein with the same molecular weight as P0. It has been reported that P0 exists on the surface of human hepatoma and neuroblastoma cells and fibroblasts [10]. P0 may be an important antigenic target on the cell membrane. This antigenic target may also act as a receptor for anti-P Abs penetration. The differences in the binding and penetration capabilities between the two mAb preparations imply that variation in the SLE syndrome among lupus patients may be attributed to heterogeneity in anti-P antibodies. In addition, anti-P mAb 9B6-4 may bind to more than 20% of Jurkat T cells. In this characteristic, the anti-P mAb 9B6-4 is very similar to the anti-P autoantibodies obtained from SLE patients [12, 22]. Therefore, the anti-P mAb 9B6-4 preparation may be helpful in the study of anti-P Abs in the pathogenesis of SLE, especially with respect to the mechanisms of lymphopenia and lymphocyte dysfunction.

Anti-P Abs may bind to and penetrate human hepatoma cells, neuroblastoma cells, fibroblasts, T cells and umbilical vein endothelial cells [10, 12, 23]. Similar findings were obtained in this study in human lung cancer cells and normal mouse brain astrocytes. However, cell types vary in their sensitivity to anti-P mAbs. These results were consistent with the finding that there may be variation in the concentration and expression of P antigens as well as in their reactivity and sensitivity in different tissues [24], and provide a possible explanation for the fact that anti-P Abs have been found to be associated mainly with lupus psychosis [1–3], nephritis [4] and hepatitis [5, 6]. Although anti-P mAbs may penetrate more than 90% of Jurkat T cells, only 20% of the cells become apoptotic. This suggests that sensitivity to anti-P Abs may vary with the stage of the cell cycle.

Apoptosis is an important anti-autoimmune mechanism. In autoimmune diseases, the activation of apoptosis by effector mechanisms, however, is similar to the normal immune response and leads to augmented destruction of the targeted tissues [25]. Caspases are a family of cysteine proteases specific for aspartic acid residues. These proteases are activated during apoptosis. The activated caspases cleave a variety of target proteins and genomic DNA into nucleosomal fragments [26], which is a hallmark of apoptosis. In our preliminary study, activation of caspases (caspases 1 and 3) was not detected in an 18-h co-culture of 9B6-4 mAb with Jurkat T cells (unpublished data), although activation of other caspases may occur in anti-P Abs-induced apoptosis. In addition, anti-P Abs may inhibit the elongation step of protein synthesis [21] and has a profound inhibitory effect on global protein synthesis [11]. Therefore, inhibition of protein synthesis is a possible mechanism for the anti-P Abs-induced apoptosis of Jurkat T cells. These findings are similar to the induction of apoptosis by cycloheximide or actinomycin D [27].

In this study, we prepared the anti-P mAb 9B6-4 for the study of anti-P Abs in the pathogenesis of SLE. These anti-P mAbs may bind to and penetrate various cell types with different sensitivities. The P0 protein on the cell membrane may be the principal antigenic target, and may also be a receptor for the penetration of anti-P Abs into the cells. Although the penetration capabilities of the mAbs may be correlated with their affinities, the variation in the numbers of the P0 receptors on different cell types may also be important and requires further study. In our preliminary study, we observed that application of a single large pulse of anti-P mAb (100 µg/ml) did not increase the percentage of Jurkat T cells in apoptosis (data not shown). This finding may be explained by the observation that cells may metabolize ingested IgG, leading to the recovery of protein synthesis [28]. However, repeated pulses of anti-P Abs may disturb protein synthesis in living cells and lead to cell death [28]. Although Jurkat T cells are leukaemia cells, they are similar to normal cells in some of their characteristics. They have been used as a model in the study of apoptosis induced by Fas ligand [29]. In addition, monoclonal antibodies induced in mice by immunization with ribosomal proteins may have specificity similar to that of lupus antibodies [30]. Therefore, the induction of apoptosis in Jurkat T cells indicates that apoptosis of T cells may also occur in SLE patients. The effects of anti-P mAbs on normal and SLE T cells are now under investigation.


   Acknowledgments
 
This work was supported in part by grants from the National Science Council (NSC 89-2320-B-010-079, 89-2314-B-010-054), the Yen Tjing Ling Medical Research Foundation (CI-89-4-2), National Health Research Institute (NHRI-GT-EX89S508P) and Veterans General Hospital, National Yang-Ming University (VTY89-G5-05), Taiwan. This work was also supported by an award from the Medical Research and Advancement Foundation in memory of Dr Chi-Shuen Tsou.


   Notes
 
Correspondence to: K.-H. Sun, Faculty of Medical Technology, National Yang-Ming University, 155 Section 2, Lie-Nong Street, Taipei, Taiwan 112. Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 9 October 2000; Accepted 19 January 2001


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