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Serum antiguanosine antibodies as a marker for SLE disease activity and pathogen potential
Clinica Chimica Acta 370 (2006) 9 – 16 www.elsevier.com/locate/clinchim
Invited critical review
Serum antiguanosine antibodies as a marker for SLE disease activity and pathogen potential Keith K. Colburn a,b,⁎, Lora M. Green a,b,c,d,1 a
JL Pettis Memorial Veterans Medical Center, Research Service-151, K.K. Colburn M.D. Chief of Rheumatology, L.M. Green Ph.D. Molecular Immunologist, 11201 Benton Street Loma Linda, CA 92357, United States b Loma Linda University Medical School, Department of Rheumatology, K.K. Colburn Professor and Chief of Rheumatology, L.M. Green Professor, Loma Linda, CA 92354, United States c Loma Linda University Graduate School, L.M. Green, Professor-Microbiology, and Physiology, Loma Linda, CA 92354, United States d Radiobiology Program Loma Linda University, L.M. Green Professor Radiation Medicine, Chan Shun Pavilion Room A-1010, 11175 Campus Street Loma Linda, CA 92354, United States Received 15 November 2005; received in revised form 3 February 2006; accepted 7 February 2006 Available online 6 March 2006
Abstract Background: This article reviews research conducted on the immunogenicity of the nucleosides of DNA, especially guanosine, the most immunologically active nucleoside. Discussed is the relationship between circulating antibodies to guanosine, their potential role in SLE disease activity, the binding properties of monoclonal antiguanosine antibody (4H2) compared to polyclonal antiguanosine antibodies in humans with SLE, cell membrane penetration by these antibodies and their interference with signal transduction possibly related to their binding to mitochondria and their apparent GTPase activity. Methods: Enzyme-linked immunosorbent assay methodology was used to show clinical relationships between antiguanosine antibody levels and disease activity in SLE. These results are discussed along with methods of detecting cell penetration by this antibody using special staining techniques, laser-scanning microscope detection of mitochondrial localization, and interference of cAMP and pKA production/activation. Additionally, there is some discussion regarding the assay used to detect enzymatic activity of antiguanosine antibodies. Results: Enhanced circulating levels of antiguanosine antibodies in patients with SLE correlate closely with SLE disease activity. Other factors are discussed that support the pathogenic potential of these antibodies, including their ability to penetrate lymphocytes, bind to mitochondria, inactivate mitochondrial function, interfere with signal transduction, and their potential enzymatically activity. Conclusions: Antiguanosine antibodies correlate with SLE disease activity and may be pathogenically important in SLE by interfering with signal transduction, inactivating mitochondrial and cell function in patients with SLE. © 2006 Elsevier B.V. All rights reserved. Keywords: Antinuclear antibodies; Antiguanosine; Systemic lupus erythematosus
Contents 1. 2. 3. 4.
Introduction . . . . . . . . . . . . . . . . Background on antinucleoside antibodies. Immunodominance of antiguanosine . . . Murine based studies in lupus prone mice 4.1. Antigenic domains. . . . . . . . .
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⁎ Corresponding author. Section of Rheumatology, 111R, Jerry L. Pettis Memorial Veterans Hospital, 11201 Benton Street, Loma Linda, CA 92350, United States. Tel.: +1 909 825 7084x2701/1995; fax: +1 909 796 4508. E-mail addresses: [email protected] (K.K. Colburn), [email protected] (L.M. Green). 1 Tel.: +1 909 558 8371; fax: +1 909 558 0825. 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.02.015
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4.2. Fine specificity studies . . . . . . . . . . . . . . . . . . . . . . . . . . . Human studies of serum antiguanosine antibodies in autoimmunity . . . . . . . 5.1. The correlation of specific features of SLE with antiguanosine antibodies. 5.2. Longitudinal association of antiguanosine antibodies and disease activity . 6. Pathogenic potential of antiguanosine antibodies in SLE . . . . . . . . . . . . . 6.1. Interference of cell signal transduction . . . . . . . . . . . . . . . . . . . 6.2. Cell membrane permeability of antiguanosine antibodies . . . . . . . . . 6.3. Mitochondrial localization of antiguanosine antibodies . . . . . . . . . . 6.4. Interference of mitochondrial function by antiguanosine localization . . . 7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.
1. Introduction Antibodies to DNA are the hallmark of systemic lupus erythematosus (SLE) and include antibodies to native DNA, polynucleotides, and nucleosides [1–3]. The origin of antibodies to native DNA has received much attention because of their specificity and their possible pathogenic role in SLE [4–7]. Numerous studies indicate that anti-DNA autoantibodies in SLE are the result of an antigen-driven, T-cell dependent response. The nature of the stimulus for their production remains controversial. Data from a variety of studies support multiple mechanisms, including stimulation by nucleosomes from apoptotic cells and immunogenic bacterial DNA [8]. Some studies suggest that anti-DNA antibodies bind various extracellular matrix proteins, and their binding to DNA may be a fortuitous cross-reaction [9–11]. Antibodies to certain polynucleotides (poly dT) and nucleosides (guanosine) show remarkable specificity for SLE and correlate with clinical manifestations of the disease such as nephritis [12–19]. This short review is not all inclusive but does provide an overview of the topic of antinuclear antibodies and our work with antiguanosine.
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genetics [25]. These early experiments' technology has enabled investigators to conduct studies with isolated nucleosides to localize cellular components including organelles and modification of specific components in biological systems especially those involving polysaccharides and proteins [25]. Borel, Stollar et al., in 1973, used nucleoside conjugates to induce tolerance in lupus prone mice which prevented the development of glomerulonephritis that naturally occurred in the NZB/NZW/ F2 strain of mice [27,28]. Humayun and Jacob showed that the sugar moiety of the nucleotide played an important role in recognition by antinucleoside antibodies [29]. Eshhar, Benacerraf and Katz in 1975 induced immune tolerance to primary or secondary antibody responses directed against nucleosides in SLE and Balb/c mice by administering nucleosides coupled covalently to copolymers of D-glutamic acid and D-lysine or L-lysine [30]. In vitro studies in 1983 utilizing nucleoside–ricin A conjugates incubated with nucleoside specific B cells isolated from patients with SLE were shown to inhibit specific antibody responses to adenosine (A), guanosine (G), thymidine (T) and cytidine (C) by binding directly to the B cell antigen receptors [31]. The authors concluded that selective elimination of nucleoside specific B cells might be therapeutically beneficial in the treatment of SLE.
2. Background on antinucleoside antibodies 3. Immunodominance of antiguanosine The presence of antibodies to native (n) or double stranded (ds) DNA in patients with SLE has suggested potential pathogenicity. The idea that antinuclear antibodies (ANA) have pathogenic roles has been suggested in many reports including those by Tan, Schur, Kunkel, Ziff and others [20–24]. Studies investigating the antigenicity of the individual nucleosides of DNA and RNA were made possible by the work of Erlanger and Beiser in their 1964 article in the Proceedings of the National Academy of Science [25]. Their report described a process of conjugating nucleosides to the carrier protein bovine serum albumin (BSA). These albumin–nucleoside conjugates were initially used to study the molecular structure of DNA and RNA. The conjugates were found to cross-react with ssDNA and adenosine in RNA preparations [25,26]. They were also used to inhibit the priming ability of DNA in DNA-dependent polymerase systems, thereby, detecting small areas of singlestrandedness in otherwise native DNA molecules. This technology contributed to the study of molecular biology and
In a study of isologous IgG conjugates with individual nucleosides Stollar and Borel [32] showed that guanosine was the most dominant nucleoside for both immunization and induction of tolerance in Balb/c mice [32]. They demonstrated that the nucleosides, especially guanosine, were each highly specific and acted independently as haptens for both immunization and tolerance induction. Stollar reviewed the topic of “Experimental Induction of Antibodies to Nucleic Acids” in Methods in Enzymology in 1980 [33]. In addition, he noted that once antibodies to the nucleic acids were induced with the protein–nucleic acid complex, they react with the nucleic acid in the absence of the carrier protein. Antibodies induced to nucleosides and nucleotides show a marked specificity for the purine or pyrimidine base component [33]. He noted that antibodies to nucleosides react about the same to the nucleotides as to the nucleosides. They also react well with polymers in which the bases were
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accessible, such as denatured DNA. Conversely, antibodies to nucleotides recognize the base, sugar and the phosphate group with a marked specificity for the particular base, and react much better to the nucleotide than the corresponding nucleoside [33]. Antibodies to mono, di and trinucleotides cross-react to denatured DNA, but not to native DNA because the bases are inaccessible. Highly specific antibodies are/can be made to sequences of nucleosides and nucleotides and to their helical conformation [33]. 4. Murine based studies in lupus prone mice and antiguanosine antibodies in SLE In 1980, Borel and Young were able to induce suppression of the response to guanosine and the other nucleosides by injecting specific nucleoside–spleen cell conjugates into C57BL/6 mice [34]. Five years later Naikl, Imai and Osawa showed that this suppression was the result of specifically induced suppressor T cells that blocked the response to guanosine and the other nucleosides [35]. This explanation of the induction of immune tolerance to the nucleosides represented a possible way to treat autoimmune diseases such as SLE. 4.1. Antigenic domains Morimoto, Steinberg, Schlossman and Borel, in 1983, found that in contrast to healthy individuals, lymphocytes in SLE patients spontaneously produced antibodies to adenosine, guanosine, cytidine and thymidine [2]. Normal lymphocytes were, however, capable of producing antinucleoside antibodies only when stimulated with pokeweed mitogen (PWM). A year later Borel et al., coupled nucleosides to spleen cells from female NZB×NZW F1 mice and re-infused them back into mice that normally spontaneously develop fatal lupus nephritis [36]. Without corticosteroids (unlike the results they previously reported when corticosteroid were added to the treatment regimen) there was no change in the course of the lupus nephritis. First it should be pointed out that finding with rodents is not directly applicable to humans, they do however, shed light on possible pathways and mechanisms that may be acting in parallel in humans. Additionally, mice strains differ in their genetics and therefore, will also differ in their immunological responses to challenge and this in it self may explain the differences between the study with Balb/c mice and the subsequent study with NZB crossed with NZW F1 mice. Given this, and what we know about the physical properties of antiguanosine, the lack of a positive response from infused nucleoside coupled spleen cells can be attributed to any number of possible scenarios. Including simply that the exposure to nucleosides could at best physically complex with a fraction of the circulating antinucleoside antibodies ameliorating little of their total immunogenic effects. At worst the presentation of conjugated nucleosides could enhance immunoreactivity leading ultimately to increase disease activity even if there was a concomitant induction of a suppressor subpopulation. At about the same time Weisbart et al. showed that antinucleoside anti-
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bodies to cytidine and guanosine in serum samples from a number of patients with autoimmune diseases were highly specific for SLE [37]. They suggested a possible pathogenic role for these antibodies in SLE. 4.2. Fine specificity studies Munns, Liszewski and Hahn studied the antigenic domains of the nucleosides with ELISA technology in 1984 [38,39]. They found that IgG antiguanosine (anti-G) antibody binding to the guanosine molecule was reduced when the C-6 and N-7 positions on guanosine were methylated, but not with methylation of the C-2, NH2 position. This information indicated that the major epitope for anti-G antibodies on guanosine encompassed the N-7, C-6 and O-6 atoms in the double ringed molecule. Weisbart et al. generated a mouse monoclonal antibody (mAb), reported in 1984, which was highly specific for guanosine and that cross-reacted to polyclonal serum anti-G antibodies from seven patients with SLE [40]. They also made a human mAb to guanosine which was polyspecific in its binding to the nucleosides, whereas the mouse mAb was specific for guanosine. This group also showed that polyclonal anti-G antibodies in serum of SLE patients were highly specific for guanosine when compared with the mouse mAb to guanosine. They postulated that anti-G antibodies might be a subpopulation of pathogenic autoantibodies found in patients with SLE. Weisbart and Colburn reported in 1984 that corticosteroid treatment in patients with SLE markedly reduced their levels of autoantibodies including antiguanosine antibodies [41]. Weisbart et al. found in 1986 that antiguanosine antibodies were present in patients with active drug-induced lupus and suggested that antiguanosine antibodies might be a marker for drug-induced SLE [42]. This idea was challenged by Totoritis, Tan, Rubin, et al., who reported the close association of antibodies to histone complex H2A–H2B in patients with druginduced lupus [43]. 5. Human studies of serum antiguanosine antibodies in autoimmunity Our laboratory took a more in-depth look at antibodies to guanosine in SLE. We reported in 1990 the reduction of antibodies to guanosine and dsDNA by plasmapheresis in patients with severe SLE, and suggested that antiguanosine antibodies might along with anti-dsDNA antibodies participate in pathogenesis of SLE [44]. We also reported in 1990 that B-adrenergic stimulation with isoproterinol increased production of anti-dsDNA, and antiguanosine antibodies in the lupus prone MRL/lpr mice [45]. That same year Weisbart reported on a group of monoclonal anti-DNA antibodies he made from MRL/lpr mice, one of which was 4H2, a monoclonal antiguanosine antibody [46]. He allowed us to use this antibody and collaborated with us in several studies using 4H2. We reported that patients with SLE treated with plasmapheresis had a temporary reduction of serum antiguanosine and antidsDNA antibodies [44]. Patients treated with plasmapheresis
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generally improved temporarily. They usually continued to improve if treated with an immunomodulatory drug. Without the immunosuppressive drugs the antibodies to DNA and guanosine increase within weeks and the symptoms of SLE recurred, thereby suggesting a correlation between the levels of these antibodies and disease activity. 5.1. The correlation of specific features of SLE with antiguanosine antibodies We then studied 86 patients with American College of Rheumatology (ACR) criteria for SLE [47] to determine whether there was relationship between the individual symptoms and laboratory features of SLE with the serum levels of antibodies to dsDNA, ssDNA and each of the nucleosides [48]. We divided patients by whether they had criteria for active SLE or inactive disease. There were 59 patients with active SLE and 27 that were inactive as determined by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) [49]. Active disease symptoms in the SLEDAI include arthritis, skin rashes, central nervous system (CNS) symptoms, nephritis, pleurisy, pericarditis, and typical hematological abnormalities of SLE. The average SLEDAI score of the active patients was 10 times higher than the inactive patients (10.5 vs. 1.1). The serum levels of antiguanosine (p b 0.0001), dsDNA (p b 0.003) and ssDNA (p b 0.005) antibodies, in patients with active SLE were significantly higher than those with inactive disease as measured by enzyme-linked immunosorbent assay (ELISA). The serum levels of antiguanosine antibodies in these patients were more than 2 times higher than levels of antiadenosine antibodies, 3 times those of antithymidine antibodies and 6 times those of anticytidine antibodies (p b 0.0001) [48]. When compared with antiguanosine antibodies the other 3 antinucleoside antibodies tested did not correlate well with the activity of disease in the SLE patients [48]. When we matched the individual organ system involvement of patients with active SLE with the inactive group it became apparent that serum antiguanosine antibody levels compared at least as well if not better than antibodies to dsDNA in differentiating the active from the inactive groups (Table 1). Antiguanosine antibodies in the serum of patients with SLE, especially those with symptoms and signs of active nephritis, polyserositis, arthritis, hematologic manifestations of lupus and CNS lupus, were closely associated to the activity of SLE. This association seemed to be true to a lesser extent for rashes associated with SLE. 5.2. Longitudinal association of antiguanosine antibodies and disease activity If antibodies to guanosine expressed a pathogenic potential similar to anti-dsDNA antibodies then there should be a longitudinal correlation between the serum levels and disease activity in these patients. We followed 12 patients 3 to 7 years and compared the serum levels of antiguanosine antibodies and antibodies to dsDNA and ssDNA in periods of active un-
Table 1 Association of anti-nuclear antibodies with disease manifestations of SLE Abs ssDNA dsDNA anti-G Low C3 ssDNA dsDNA anti-G ssDNA dsDNA anti-G ssDNA dsDNA anti-G ssDNA dsDNA anti-G ssDNA dsDNA anti-G ssDNA dsDNA anti-G a b
SLE feature a Active SLE (n = 59) 0.734 ± 0.144 0.431 ± 0.162 0.357 ± 0.164 51% Nephritis (n = 18) 0.956 ± 0.296 0.802 ± 0.401 0.710 ± 0.422 Polyserositis (n = 10) 1.245 ± 0.025 1.063 ± 0.593 0.745 ± 0.630 Arthritis (n = 8) 1.186 ± 0.439 1.853 ± 0.761 0.415 ± 0.521 CNS (n = 9) 0.744 ± 0.472 0.685 ± 0.718 0.344 ± 0.566 Rash (n = 29) 0.623 ± 0.213 0.309 ± 0.217 0.202 ± 0.141 Hematologic (n = 29) 0.689 ± 0.185 0.359 ± 0.193 0.245 ± 0.160
p values b Inactive SLE (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011 10% (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011 (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011 (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011 (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011 (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011 (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011
0.005 0.003 0.0001
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.008 0.006 0.067 0.024 0.005 0.131 0.104 0.051 0.012 0.010 0.002
Optical density ± standard error of the mean. Mann–Whitney U test.
controlled SLE, active controlled and inactive SLE (Table 2). The levels of antiguanosine antibodies followed the disease activity at a level that was statistically significant. In these studies anti-ssDNA and anti-dsDNA antibodies did not correlate as well as antiguanosine [48]. 6. Pathogenic potential of antiguanosine antibodies in SLE Finding a close correlation of serum levels of antiguanosine antibodies to disease activity in SLE encouraged us to pursue the study of possible pathogenic features of this antibody. We started by attempting to identify the binding sites or epitopes of guanosine and antiguanosine antibodies. Separate preparations of guanosine molecules methylated at the 1, 2, 3 or 7 positions on the guanine ring were used to test their binding affinity using antiguanosine antibodies contained in sera of patients with SLE and monoclonal antiguanosine antibodies generated from MRL/ lpr mice. The methylated groups at 1 and 7 positions blocked the binding of the antiguanosine antibody, whereas the 2 and 3 positions did not. This indicated that the binding site on guanosine was across the 1 and 7 positions. This confirmed the work of Munns et al. [38]. We found that an oxygen molecule attached to the carbon at position 6, between the 1 and 7 positions, was essential to antibody binding. We tested analogs of guanosine with and without oxygen bound at position 6, and found that there was significantly reduced binding if there was
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Table 2 The longitudinal association of antinuclear abs with the phases of SLE disease activity Group I (n = 6)
Mean response a
Abs ssDNA (ELISA) dsDNA (ELISA) dsDNA (RIA) Guanosine (ELISA) Group II (n = 6) Abs ssDNA (ELISA) dsDNA (ELISA) dsDNA (RIA) Guanosine (ELISA)
Active SLE (10 sera) 1.479 ± 0.412 0.684 ± 0.320 50.7 ± 29.3 0.217 ± 0.047
Inactive (12 sera) 1.223 ± 0.341 0.065 ± 0.017 23.8 ± 8.9 0.105 ± 0.017
p value 0.13 0.09 0.09 b0.05
Active (15 sera) 2.711 ± 0.195 2.035 ± 0.291 100.3 ± 20.3 0.745 ± 0.165
Active-improved (15 Sera) 1.346 ± 0.273 0.955 ± 0.266 38.1 ± 17.6 0.243 ± 0.090
0.10 0.16 0.09 b0.02
a b
Statistical analysis b
Mean optical density (OD) for ELISA ± standard error of the mean (S.E.M.); mean units per ml for RIA ± S.E.M. Paired t test.
no oxygen at that position. Adenosine has a similar 9 member double ring structure as guanosine, but has an amine group (NH2) at position 6 whereas guanosine has a hydroxyl group (OH). Antiguanosine antibodies do not bind to adenosine. However, antiguanosine antibodies did bind to hypoxanthine, cGMP and acyclovir, all of which have an oxygen molecule at position 6. We were able to show that the binding properties of the mouse monoclonal antiguanosine 4H2 antibody were conserved exactly like polyclonal antiguanosine antibodies from sera of patients with SLE when binding to guanosine and guanosine analogs [50]. 6.1. Interference of cell signal transduction G-binding proteins active in cell signal transduction exert their function by cycling between inactive GDP and an active GTP-containing conformation described by Pai et al. to be structurally the same as the Ha-ras oncogene product p21 [51]. G-binding proteins are activated by the GDP to GTP reaction that in turn activates adenyl cyclase that then produces cAMP. We found that antiguanosine antibodies bind GDP and GTP at the same epitopes as G-binding proteins, indicating that antiguanosine antibodies have the same internal image as G-binding proteins [50]. These findings led us to consider that antiguanosine antibodies might interfere with cell signal transduction involving G-proteins. Highly specific antiguanosine antibodies provide a marker for SLE disease activity as described above. With the monoclonal antibody, 4H2, we found a tool that could be used to help elucidate the possible pathogenic role of antiguanosine antibodies in SLE. As an initial test we performed RIAs to determine if antiguanosine antibodies interfere with cAMP production by culturing normal human lymphocytes with plasma from patients with high levels of antiguanosine antibodies and with 4H2 monoclonal antiguanosine antibodies. Controls consisted of other monoclonal anti-DNA antibodies, an unrelated monoclonal antibody, sera of patients with Gillian–Barre' syndrome, thrombotic thrombocytopenic purpura, Wegener's granulomatosis, myasthenia gravis, amyotrophic lateral sclerosis and plasma from healthy donors. The plasma from patients with SLE, and 4H2 monoclonal antibody,
qualitatively reduced the cAMP levels (Colburn and Green, unpublished observations). Additionally, using more sophisticated methods (laser scanning cytometry) we were able to confirm that 4H2 reduced cAMP levels in human lymphocytes as well as a number of different cell types. The results of antiguanosine' ability to reduce cAMP levels in epithelial cells are shown in Fig. 1 [52]. 6.2. Cell membrane permeability of antiguanosine antibodies The fact that antiguanosine antibodies interfere with cAMP levels suggested that these antibodies must be internalized into cells to exert their affect on signal transduction. There is a growing body of evidence that some antibodies penetrate cell membranes [18,19]. This topic was the focus of a meeting in
Fig. 1. Quantitative immunocytochemical levels of cAMP in rat epithelial cells treated with antinucleoside antibodies. Rat thyroid epithelial cells were established in 6-well plates containing glass coverslips. The established cells were treated, in duplicate, with: nothing (basal level), 4H2 at 1:8 and 1:16 and 3E10 (an anti-DNA antibody [37]) at 1:8 and 1:16. After a 3 h incubation the cells were fixed and immunofluorescently labeled with anti-cAMP antibodies. The immunolabeled cells were scanned on the laser scanning cytometer and the values compiled and graphed as shown. The estimated concentration of the antibody preparations was 10 μg/ml at the 1:8 dilution. The results indicate that the 4H2 antiguanosine antibody reduced the levels of cAMP nearly 10-fold compared to the untreated levels. The anti-DNA antibody 3E10 only slightly reduced the levels of cAMP.
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Cancun, Mexico in 1998. The presentations from this meeting were published in a volume of the Journal of Autoimmunity. These articles varied in their scope, including topics that revealed that portions of the immunoglobulins were being used as targeting molecules [19], discussions of immunochemical, structural and translocating properties of anti-dsDNA antibodies [53] and cellular dysfunction [54]. This information indicates the previous dogma regarding the functional consequences of antibody binding (complement, opsonization, etc.) are insufficient to account for the dynamic potential and range of mechanisms that are currently emerging. 6.3. Mitochondrial localization of antiguanosine antibodies To show that antiguanosine antibodies penetrated cell membranes we labeled purified 4H2 by direct conjugation to Alexa fluor-594 (a red fluorescent molecule). By fluorescent microscopy we found that labeled 4H2 was incorporated into the cytoplasm of normal human lymphocytes and in live and fixed cells from various sources. In lymphocytes the cytoplasm is difficult to visualize so we used human thyroid epithelial cells (Htori-3-LLU, [55]) to confirm that the binding occurred. A pattern was revealed that suggested that the antibody was localized to the mitochondria. To confirm this we used mitotracker conjugated to FITC (a green fluorescent molecule) and incubated it with live Htori-3-LLU cells. Nuclei were stained with the DNA specific blue dye, DAPI. When the images from the multi-labeled cells were overlaid the red and green signals were in perfect register (Fig. 2). Antiguanosine antibodies were
penetrating the cell membrane and binding to mitochondria. Thus we had found that the antiguanosine antibodies were produced at high levels in patients with active SLE, in vitro these antibodies were found to be capable of altering signal transduction (reduced cAMP levels, Fig. 1) which may be related to their localization to the mitochondria (Colburn and Green, unpublished observation). 6.4. Interference of mitochondrial function by antiguanosine localization The intense localization of antiguanosine to the mitochondria suggested that it might interfere with mitochondrial function. Therefore, the next experiments were to test mitochondrial function using the MTT assay, which measures cell viability and indirectly mitochondrial activity, because the tetrazolium salt is converted to formazan by mitochondrial dehydrogenase activity [56]. The cells with incorporated 4H2 monoclonal antiguanosine antibody had decreased MTT dye reduction, first suggested that the added 4H2 was cytotoxic. However, when the cells were counted, the 4H2 treated cultures were essentially unchanged from the number of cells originally plated, whereas, the non-treated controls cultures had increased in number. Thus, 4H2 was cytostatic; presumably the binding interferes with mitochondrial function. Collectively these results lead us to believe that antiguanosine antibodies in SLE are pathogenic. They penetrate lymphocytic membranes, interfere with signal transduction, bind to mitochondria and altered its function [57].
Fig. 2. Internalization of fluorescently labeled antiguanosine antibody (4H2) in live Htori-3-LLU thyroid epithelial cells. Concentrated hybridoma supernatant containing antibodies to 4H2 (antiguanosine) was concentrated and purified by protein A sepharose and conjugated to Alexa fluor-594 (Molecular Probes, Eugene OR). The four panel composite shows representative images of Htori-3-LLU cells incubated with: a) 4H2—Alexa fluor-594; b) FITC—Mitotracker; c) DAPI nuclear stain; and d) merged image of panels a–c. The overlay of labeled 4H2 (red) with mitotracker (green) resulting in a yellow color indicates that they are binding to the same location, the mitochondria. The magnification of the image was taken at 100× oil. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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We then went in search of a mechanistic explanation for our findings. There is a growing body of evidence that antibodies can have enzymatic activity. From our previous studies, the most logical activity for antiguanosine antibodies would be that it behaved like a GTPase. We designed a method to test the GTPase activity of 4H2 (a murine antiguanosine antibody). GTPase-like properties would explain how antiguanosine could be involved in cell-signal transduction by converting GTP to GDP and thereby indirectly reducing cAMP levels. The results of our preliminary investigation suggest that antiguanosine cleaved the gamma-phosphate bond from a radio-labeled substrate (GTP), which in vivo would disrupt the activation of G-binding proteins and block signal initiation [58]. 7. Summary Our work has demonstrated that circulating antibodies to guanosine are potentially important in SLE. Their serum levels are specific for some features of SLE including nephritis, polyserositis, arthritis, CNS lupus and the hematologic features of SLE. These antibodies closely correlate with SLE disease activity as measured by the SLEDAI scale both at a point in time and in longitudinal studies. Antiguanosine antibodies are highly specific for the 1 through 7 locations on the guanine ring and require oxygen for binding, the same as site of the binding of guanine to the HA-ras P21. HA-ras P21 is an oncogene that has binding properties similar to G-proteins that participate in signal transduction. Antiguanosine antibodies have the internal image of G-binding proteins. Antiguanosine antibodies penetrate cell membranes, bind to and inactivate mitochondria dehydrogenase activity and interfere with signal transduction possibly by their innate GTPase activity. Antiguanosine antibodies are a marker for SLE disease activity, at least as good as, and possibly better than anti-dsDNA antibodies. Furthermore, like anti-dsDNA antibodies, antiguanosine antibodies may be pathogenic for certain features of SLE. Further studies are currently underway to substantiate these findings and contentions. The interesting findings reported here for antiguanosine autoantibodies are only one type of antibody found in SLE patient sera. A relatively recent report details the more than 100 different antibodies found in the sera of patient with SLE [59]. Clearly, there are numerous possible antibodies that are produced in SLE that might, like antiguanosine, help us to better elucidate the complexities of this potentially debilitating disease. References [1] Alarcon-Segovia D, Fishbein E, Estrada-Parra S. The heterogeneity of anti-DNA antibodies in systemic lupus erythematosus and other diseases. J Rheumatol 1975;2:172–7. [2] Morimoto C, Steinberg AD, Schlossman SF, Borel Y. In vitro nucleoside specific immune response by lymphocytes from systemic lupus erythematosus. J Clin Invest 1983;71:1402–9. [3] Weisbart RH, Garrett RA, Liebling MR, Barnett EV, Paulus HE, Katz DH. Specificity of anti-nucleoside antibodies in systemic lupus erythematosus. Clin Immunol Immunopathol 1983;27:403–11. [4] Jacob L, Kerjaschki D, Viard JP, et al. Potential pathogenic role of cellsurface polypeptides, cross-reactive with DNA, in systemic lupus erythematosus. Nephrologie 1989;10(3):139–40.
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[5] Roitt IM, Hutchings PR, Dawe KI, Sumar N, Bodman KB, Cooke A. The forces driving autoimmune disease. J Autoimmun 1992:11–26 Suppl A. [6] George J, Gilburd B, Shoenfeld Y. The emerging concept of pathogenic natural autoantibodies. Hum Antibodies 1997;8(2):70–5. [7] Jang YJ, Stollar BD. Anti-DNA antibodies: aspects of structure and pathogenicity. Cell Mol Life Sci 2003;60(2):309–20. [8] O'Brien TF, Mayer KH, Hopkins JD, Chao L. New concepts in medical bacteriology. J Med 1983;14(4):271–80. [9] Sela U, Hershkoviz R, Cahalon L, Lider O, Mozes E. Down-regulation of stromal cell-derived factor-1alpha-induced T cell chemotaxis by a peptide based on the complementarity-determining region 1 of an anti-DNA autoantibody via up-regulation of TGF-beta secretion. J Immunol 2005;174(1):302–9. [10] Qureshi F, Yang Y, Jaques SM, et al. Anti-DNA antibodies cross-reacting with laminin inhibit trophoblast attachment and migration: implications for recurrent pregnancy loss in SLE patients. Am J Reprod Immunol 2000;44 (3):136–42. [11] Mageed RA, Zack DJ. Cross-reactivity and pathogenicity of anti-DNA autoantibodies in systemic lupus erythematosus. Lupus 2002;11(12):783–6. [12] Ohnishi K, Ebling FM, Mitchell B, Singh RR, Hahn BN, Tsao BP. Comparison of pathogenic and non-pathogenic murine antibodies to DNA: antigen binding and structural characteristics. Int Immunol 1994;6 (6):817–30. [13] Bernstein KA, Kahl LE, Balow JE, Lefkowith JB. Serologic markers of lupus nephritis in patients: use of a tissue-based ELISA and evidence for immunopathogenic heterogeneity. Clin Exp Immunol 1994;98:60–5. [14] Foster MH, Cizman B, Madaio MP. Biology of disease. Nephritogenic autoantibodies in systemic lupus erythematosus: immunochemical properties, mechanisms of immune deposition, and genetic origins. Lab Invest 1993;69:494–507. [15] Krishnan C, Kaplan MH. Immunopathologic studies of systemic lupus erythematosus. II. Antinuclear reaction of γ-globulin from homogenates and isolated glomeruli of kidneys from patients with lupus nephritis. J Clin Invest 1967;46:569–79. [16] Weisbart RH, Noritake DT, Wong AL, Chan G, Kacena A, Colburn KK. A conserved anti-DNA antibody idiotype associated with nephritis in murine and human systemic lupus erythematosus. J Immunol 1990;144(7):2653–8. [17] Schur PH, Sandson J. Immunologic factors and clinical activity in lupus erythematosus. New Engl J Med 1968;278:533–8. [18] Zack DJ, Stempniak M, Wong, Taylor C, Weisbart RH. Mechanisms of cellular penetration and nuclear localization of an anti-double strand DNA autoantibody. J Immunol 1996;157:1082–8. [19] Weisbart RH, Stempniak M, Harris S, Zack DJ, Ferreri K. An autoantibody is modified for use as a delivery system to target the cell nucleus: therapeutic implications. J Autoimmun 1998;11:539–46. [20] Tan EM, Schur PH, Carr RI, Kunkel HG. Deoxyribonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus. J Clin Invest 1966;45:1732–42. [21] Pincus T, Schur PH, Rose JA, Decker JL, Talal N. Measurement of serum DNA-binding activity in systemic lupus erythematosus. N Engl J Med 1969;281:701–5. [22] Stollar D. The specificity and applications of antibodies to helical nucleic acids. Crit Rev Biochem 1975;3:45–69. [23] Hughs GRV, Cohen SA, Christian CL. Anti-DNA activity in systemic lupus erythematosus. Ann Rheum Dis 1971;30:259–64. [24] Linnik MD, Hu JZ, Heilbrunn KR, Strand V, Hurley FL, Joh T, and the LJP 394 Investigator Consortium. Relationship between anti-double stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus. Arthritis Rheum 2005;52(4):1129–37. [25] Erlanger BF, Beiser SM. Antibodies specific for ribonucleosides and ribonucleotides and their reaction with DNA. Proc Natl Acad Sci 1964;52:68–74. [26] Erlanger BF, Senitzer D, Miller OJ, Beiser SM. Nucleic acid-reactive antibodies specific for nucleosides and nucleotides. Karolinska symposia of research methods in reproductive endocrinology, 5th symposium gene transcription in reproductive tissue, May 29–31; 1972. p. 206–21.
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[27] Borel Y, Lewis RM, Andre-Schwartz J, Stollar BD. Treatment of lupus nephritis in adult (NZB+NZW)F1 mice by cortisone-facilitated tolerance to nucleic acid antigens. J Clin Invest 1978;61:276–86. [28] Borel Y, Lewis RM, Stollar BD. Prevention of murine lupus nephritis by carrier dependent induction of immunologic tolerance to denatured DNA. Science 1973;182:76–8. [29] Humayun MZ, Jacob TM. Specificity of anti-nucleoside antibodies. Biochem J 1974;141:313–5. [30] Eshhar Z, Benacerraf B, Katz DH. Induction of tolerance to nucleic acid determinants by administration of a complex of nucleoside D-glutamic acid and D-lysine (D-LG). J Immunol 1975;114(2):872–6. [31] Morimoto C, Masuho Y, Borel Y, Steinberg AD, Schlossman SF. Selective inhibition of anti-nucleoside-specific antibody production by nucleoside– ricin A conjugate. J Immunol 1983;131(4):1762–4. [32] Stollar BD, Borel Y. Nucleoside specificity in the carrier IgG-dependent induction of tolerance. J Immunol 1976;117(4):1308–13. [33] Stollar BD. The experimental induction of antibodies to nucleic acids. Methods Enzymol 1980;70:70–85. [34] Borel Y, Young MC. Nucleic acid-specific suppressor T cells. Proc Natl Acad Sci U S A 1980;79:1593. [35] Naikl M, Imai Y, Osawa T. Establishment of a nucleoside-specific suppressor T cell line from SLE prone (NZB/NZW)F1 mice. J Immunol 1985;135(2):1080–5. [36] Borel Y, Borel H, Schneeberger E, Lewis RM. Influence of intravenous administration of nucleoside-coupled spleen cells on murine lupus nephritis in female (NZB×NZW)F1 mice. Clin Immunol Immunolopathol 1984;30:451–60. [37] Weisbart RH, Garrett RA, Liebling MR, Barnett EV, Paulus HE, Katz DH. Specificity of anti-nucleoside antibodies in systemic lupus erythematosus. Clin Immunol Immunolopathol 1983;27:403–11. [38] Munns TW, Liszewiski MK, Hahn BH. Antibody–nucleic acid complexes. Antigenic domains within nucleosides as defined by solid-phase immunoassay. Biochemistry 1984;23:2958–64. [39] Munns TW, Liszewiski MK. Antibodies specific for modified nucleosides: an immunochemical approach for the isolation and characterization of nucleic acids. Prog Nucl Acid Res Mol Biol 1980;24:109–65. [40] Weisbart RH, Chan G, Kacena A, Saxton RE. Characterization of mouse and human monoclonal antibodies cross-reactive with SLE serum antibodies to guanosine. J Immunol 1984;132(6):2909–12. [41] Weisbart RH, Colburn KK. Effect of corticosteroids on serum autoantibodies in man. J Immunol Pharmacol 1984;8:97–101. [42] Weisbart RH, Yee WS, Colburn KK, Whang SH, Heng MK, Boucek RJ. Antiguanosine antibodies: a new marker for procainamide-induced systemic lupus erythematosus. Ann Intern Med 1986;104:310–3. [43] Totoritis MC, Tan EM, McNally MD, Rubin RL. Association of antibody to histone complex H2A–H2B with symptomatic procainamide-induced lupus. New Engl J Med 1988;318:1431–6. [44] Colburn KK, Gusewitch GA, Pooprasert S, Weisbart RH. Apheresis enhances the selective removal of anti-DNA antibodies in SLE. Clin Rheumatol 1990;9:475–82.
[45] Colburn KK, Boucek RJ, Gusewitch G, Wong AL. B-adrenergic receptor stimulation increases anti-DNA antibody production in MRL/lpr mice. J Rheumatol 1990;17:138–41. [46] Weisbart RH, Noritake DT, Wong AL, Chan G, Kacena A, Colburn KK. A conserved anti-DNA antibody ideotype associated with nephritis in murine and human systemic lupus erythematosus. J Immunol 1990;144:2653–8. [47] Tan EM, Cohen AS, Fries JF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271–7. [48] Colburn KK, Green LM, Wong AL. Circulating antibodies to guanosine in SLE: correlation with nephritis and polyserositis by acute and longitudinal analysis. Lupus 2001;10:411–7. [49] Bombardier C, Gladman DD, Urowitz MB, Carron D, Chang CH, and the Committee on Prognosis Studies in SLE. Derivation of the SLEDAI. A disease activity index for lupus patients. Arthritis Rheum 1992;35:630–40. [50] Colburn KK, Wong AL, Weisbart RH, Green LM. Antiguanosine antibodies in murine and human lupus have the internal image of G-binding proteins. J Rheumatol 2002;30(5):993–7. [51] Pai EF, Kasbach W, Krezel U, Holmes KC, John J, Wittinghofer A. Structure of the guanine nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate confirmation. Nature 1989;341:209–14. [52] Katsaros EP, Colburn KK, Casiano C, Lilly M, Weisbart RH, Green LM. Lupus associated antiguanosine antibodies interfered with cAMP and pKA levels. J Invest Med 2001;49:73A (Supp). [53] Ternynck T, Avramaes A, Ragimbeau J, Buttin G, Avrameas S. Immunochemical, structural, and translocating properties of anti-DNA antibodies from (NZB×NZW)F1 mice. J Autoimmun 1998;11:511–21. [54] Reichlin M. Cellular dysfunction induced by penetration of autoantibodies into living cells: cellular damage and dysfunction mediated by antibodies to dsDNA and ribosomal P proteins. J Autoimmun 1998;11:557–61. [55] Lemoine NR, Mayall ES, Jones T, et al. Characterization of human thyroid epithelial cells immortalized in vitro by simian virus 40 DNA transfection. Br J Cancer 1989;60(6):897–903. [56] Green LM, Reade JL, Ware CF. Rapid calorimetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines. J Immunol Methods 1984;70:257–68. [57] Katsaros EP, Casiano C, Colburn KK, et al. Lupus associated antiguanosine antibodies: potential pathogenic effects. Arthritis Rheum 2001;45:9:S281 (Supp). [58] Taylor MB, Colburn KK, Green LM. Lupus associated antiguanosine antibodies fine specificity and nucleoside binding properties. J Invest Med 2003;51:S143–4 (Supp). [59] Sherer Y, Gorstein A, Fritzler MJ, Shoenfeld Y. Autoantibody explosion in systemic lupus erythematosus: more than 100 different antibodies found in SLE patients. Semin Arthritis Rheum 2004;34(2):501–37.
Invited critical review
Serum antiguanosine antibodies as a marker for SLE disease activity and pathogen potential Keith K. Colburn a,b,⁎, Lora M. Green a,b,c,d,1 a
JL Pettis Memorial Veterans Medical Center, Research Service-151, K.K. Colburn M.D. Chief of Rheumatology, L.M. Green Ph.D. Molecular Immunologist, 11201 Benton Street Loma Linda, CA 92357, United States b Loma Linda University Medical School, Department of Rheumatology, K.K. Colburn Professor and Chief of Rheumatology, L.M. Green Professor, Loma Linda, CA 92354, United States c Loma Linda University Graduate School, L.M. Green, Professor-Microbiology, and Physiology, Loma Linda, CA 92354, United States d Radiobiology Program Loma Linda University, L.M. Green Professor Radiation Medicine, Chan Shun Pavilion Room A-1010, 11175 Campus Street Loma Linda, CA 92354, United States Received 15 November 2005; received in revised form 3 February 2006; accepted 7 February 2006 Available online 6 March 2006
Abstract Background: This article reviews research conducted on the immunogenicity of the nucleosides of DNA, especially guanosine, the most immunologically active nucleoside. Discussed is the relationship between circulating antibodies to guanosine, their potential role in SLE disease activity, the binding properties of monoclonal antiguanosine antibody (4H2) compared to polyclonal antiguanosine antibodies in humans with SLE, cell membrane penetration by these antibodies and their interference with signal transduction possibly related to their binding to mitochondria and their apparent GTPase activity. Methods: Enzyme-linked immunosorbent assay methodology was used to show clinical relationships between antiguanosine antibody levels and disease activity in SLE. These results are discussed along with methods of detecting cell penetration by this antibody using special staining techniques, laser-scanning microscope detection of mitochondrial localization, and interference of cAMP and pKA production/activation. Additionally, there is some discussion regarding the assay used to detect enzymatic activity of antiguanosine antibodies. Results: Enhanced circulating levels of antiguanosine antibodies in patients with SLE correlate closely with SLE disease activity. Other factors are discussed that support the pathogenic potential of these antibodies, including their ability to penetrate lymphocytes, bind to mitochondria, inactivate mitochondrial function, interfere with signal transduction, and their potential enzymatically activity. Conclusions: Antiguanosine antibodies correlate with SLE disease activity and may be pathogenically important in SLE by interfering with signal transduction, inactivating mitochondrial and cell function in patients with SLE. © 2006 Elsevier B.V. All rights reserved. Keywords: Antinuclear antibodies; Antiguanosine; Systemic lupus erythematosus
Contents 1. 2. 3. 4.
Introduction . . . . . . . . . . . . . . . . Background on antinucleoside antibodies. Immunodominance of antiguanosine . . . Murine based studies in lupus prone mice 4.1. Antigenic domains. . . . . . . . .
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⁎ Corresponding author. Section of Rheumatology, 111R, Jerry L. Pettis Memorial Veterans Hospital, 11201 Benton Street, Loma Linda, CA 92350, United States. Tel.: +1 909 825 7084x2701/1995; fax: +1 909 796 4508. E-mail addresses: [email protected] (K.K. Colburn), [email protected] (L.M. Green). 1 Tel.: +1 909 558 8371; fax: +1 909 558 0825. 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.02.015
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4.2. Fine specificity studies . . . . . . . . . . . . . . . . . . . . . . . . . . . Human studies of serum antiguanosine antibodies in autoimmunity . . . . . . . 5.1. The correlation of specific features of SLE with antiguanosine antibodies. 5.2. Longitudinal association of antiguanosine antibodies and disease activity . 6. Pathogenic potential of antiguanosine antibodies in SLE . . . . . . . . . . . . . 6.1. Interference of cell signal transduction . . . . . . . . . . . . . . . . . . . 6.2. Cell membrane permeability of antiguanosine antibodies . . . . . . . . . 6.3. Mitochondrial localization of antiguanosine antibodies . . . . . . . . . . 6.4. Interference of mitochondrial function by antiguanosine localization . . . 7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.
1. Introduction Antibodies to DNA are the hallmark of systemic lupus erythematosus (SLE) and include antibodies to native DNA, polynucleotides, and nucleosides [1–3]. The origin of antibodies to native DNA has received much attention because of their specificity and their possible pathogenic role in SLE [4–7]. Numerous studies indicate that anti-DNA autoantibodies in SLE are the result of an antigen-driven, T-cell dependent response. The nature of the stimulus for their production remains controversial. Data from a variety of studies support multiple mechanisms, including stimulation by nucleosomes from apoptotic cells and immunogenic bacterial DNA [8]. Some studies suggest that anti-DNA antibodies bind various extracellular matrix proteins, and their binding to DNA may be a fortuitous cross-reaction [9–11]. Antibodies to certain polynucleotides (poly dT) and nucleosides (guanosine) show remarkable specificity for SLE and correlate with clinical manifestations of the disease such as nephritis [12–19]. This short review is not all inclusive but does provide an overview of the topic of antinuclear antibodies and our work with antiguanosine.
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genetics [25]. These early experiments' technology has enabled investigators to conduct studies with isolated nucleosides to localize cellular components including organelles and modification of specific components in biological systems especially those involving polysaccharides and proteins [25]. Borel, Stollar et al., in 1973, used nucleoside conjugates to induce tolerance in lupus prone mice which prevented the development of glomerulonephritis that naturally occurred in the NZB/NZW/ F2 strain of mice [27,28]. Humayun and Jacob showed that the sugar moiety of the nucleotide played an important role in recognition by antinucleoside antibodies [29]. Eshhar, Benacerraf and Katz in 1975 induced immune tolerance to primary or secondary antibody responses directed against nucleosides in SLE and Balb/c mice by administering nucleosides coupled covalently to copolymers of D-glutamic acid and D-lysine or L-lysine [30]. In vitro studies in 1983 utilizing nucleoside–ricin A conjugates incubated with nucleoside specific B cells isolated from patients with SLE were shown to inhibit specific antibody responses to adenosine (A), guanosine (G), thymidine (T) and cytidine (C) by binding directly to the B cell antigen receptors [31]. The authors concluded that selective elimination of nucleoside specific B cells might be therapeutically beneficial in the treatment of SLE.
2. Background on antinucleoside antibodies 3. Immunodominance of antiguanosine The presence of antibodies to native (n) or double stranded (ds) DNA in patients with SLE has suggested potential pathogenicity. The idea that antinuclear antibodies (ANA) have pathogenic roles has been suggested in many reports including those by Tan, Schur, Kunkel, Ziff and others [20–24]. Studies investigating the antigenicity of the individual nucleosides of DNA and RNA were made possible by the work of Erlanger and Beiser in their 1964 article in the Proceedings of the National Academy of Science [25]. Their report described a process of conjugating nucleosides to the carrier protein bovine serum albumin (BSA). These albumin–nucleoside conjugates were initially used to study the molecular structure of DNA and RNA. The conjugates were found to cross-react with ssDNA and adenosine in RNA preparations [25,26]. They were also used to inhibit the priming ability of DNA in DNA-dependent polymerase systems, thereby, detecting small areas of singlestrandedness in otherwise native DNA molecules. This technology contributed to the study of molecular biology and
In a study of isologous IgG conjugates with individual nucleosides Stollar and Borel [32] showed that guanosine was the most dominant nucleoside for both immunization and induction of tolerance in Balb/c mice [32]. They demonstrated that the nucleosides, especially guanosine, were each highly specific and acted independently as haptens for both immunization and tolerance induction. Stollar reviewed the topic of “Experimental Induction of Antibodies to Nucleic Acids” in Methods in Enzymology in 1980 [33]. In addition, he noted that once antibodies to the nucleic acids were induced with the protein–nucleic acid complex, they react with the nucleic acid in the absence of the carrier protein. Antibodies induced to nucleosides and nucleotides show a marked specificity for the purine or pyrimidine base component [33]. He noted that antibodies to nucleosides react about the same to the nucleotides as to the nucleosides. They also react well with polymers in which the bases were
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accessible, such as denatured DNA. Conversely, antibodies to nucleotides recognize the base, sugar and the phosphate group with a marked specificity for the particular base, and react much better to the nucleotide than the corresponding nucleoside [33]. Antibodies to mono, di and trinucleotides cross-react to denatured DNA, but not to native DNA because the bases are inaccessible. Highly specific antibodies are/can be made to sequences of nucleosides and nucleotides and to their helical conformation [33]. 4. Murine based studies in lupus prone mice and antiguanosine antibodies in SLE In 1980, Borel and Young were able to induce suppression of the response to guanosine and the other nucleosides by injecting specific nucleoside–spleen cell conjugates into C57BL/6 mice [34]. Five years later Naikl, Imai and Osawa showed that this suppression was the result of specifically induced suppressor T cells that blocked the response to guanosine and the other nucleosides [35]. This explanation of the induction of immune tolerance to the nucleosides represented a possible way to treat autoimmune diseases such as SLE. 4.1. Antigenic domains Morimoto, Steinberg, Schlossman and Borel, in 1983, found that in contrast to healthy individuals, lymphocytes in SLE patients spontaneously produced antibodies to adenosine, guanosine, cytidine and thymidine [2]. Normal lymphocytes were, however, capable of producing antinucleoside antibodies only when stimulated with pokeweed mitogen (PWM). A year later Borel et al., coupled nucleosides to spleen cells from female NZB×NZW F1 mice and re-infused them back into mice that normally spontaneously develop fatal lupus nephritis [36]. Without corticosteroids (unlike the results they previously reported when corticosteroid were added to the treatment regimen) there was no change in the course of the lupus nephritis. First it should be pointed out that finding with rodents is not directly applicable to humans, they do however, shed light on possible pathways and mechanisms that may be acting in parallel in humans. Additionally, mice strains differ in their genetics and therefore, will also differ in their immunological responses to challenge and this in it self may explain the differences between the study with Balb/c mice and the subsequent study with NZB crossed with NZW F1 mice. Given this, and what we know about the physical properties of antiguanosine, the lack of a positive response from infused nucleoside coupled spleen cells can be attributed to any number of possible scenarios. Including simply that the exposure to nucleosides could at best physically complex with a fraction of the circulating antinucleoside antibodies ameliorating little of their total immunogenic effects. At worst the presentation of conjugated nucleosides could enhance immunoreactivity leading ultimately to increase disease activity even if there was a concomitant induction of a suppressor subpopulation. At about the same time Weisbart et al. showed that antinucleoside anti-
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bodies to cytidine and guanosine in serum samples from a number of patients with autoimmune diseases were highly specific for SLE [37]. They suggested a possible pathogenic role for these antibodies in SLE. 4.2. Fine specificity studies Munns, Liszewski and Hahn studied the antigenic domains of the nucleosides with ELISA technology in 1984 [38,39]. They found that IgG antiguanosine (anti-G) antibody binding to the guanosine molecule was reduced when the C-6 and N-7 positions on guanosine were methylated, but not with methylation of the C-2, NH2 position. This information indicated that the major epitope for anti-G antibodies on guanosine encompassed the N-7, C-6 and O-6 atoms in the double ringed molecule. Weisbart et al. generated a mouse monoclonal antibody (mAb), reported in 1984, which was highly specific for guanosine and that cross-reacted to polyclonal serum anti-G antibodies from seven patients with SLE [40]. They also made a human mAb to guanosine which was polyspecific in its binding to the nucleosides, whereas the mouse mAb was specific for guanosine. This group also showed that polyclonal anti-G antibodies in serum of SLE patients were highly specific for guanosine when compared with the mouse mAb to guanosine. They postulated that anti-G antibodies might be a subpopulation of pathogenic autoantibodies found in patients with SLE. Weisbart and Colburn reported in 1984 that corticosteroid treatment in patients with SLE markedly reduced their levels of autoantibodies including antiguanosine antibodies [41]. Weisbart et al. found in 1986 that antiguanosine antibodies were present in patients with active drug-induced lupus and suggested that antiguanosine antibodies might be a marker for drug-induced SLE [42]. This idea was challenged by Totoritis, Tan, Rubin, et al., who reported the close association of antibodies to histone complex H2A–H2B in patients with druginduced lupus [43]. 5. Human studies of serum antiguanosine antibodies in autoimmunity Our laboratory took a more in-depth look at antibodies to guanosine in SLE. We reported in 1990 the reduction of antibodies to guanosine and dsDNA by plasmapheresis in patients with severe SLE, and suggested that antiguanosine antibodies might along with anti-dsDNA antibodies participate in pathogenesis of SLE [44]. We also reported in 1990 that B-adrenergic stimulation with isoproterinol increased production of anti-dsDNA, and antiguanosine antibodies in the lupus prone MRL/lpr mice [45]. That same year Weisbart reported on a group of monoclonal anti-DNA antibodies he made from MRL/lpr mice, one of which was 4H2, a monoclonal antiguanosine antibody [46]. He allowed us to use this antibody and collaborated with us in several studies using 4H2. We reported that patients with SLE treated with plasmapheresis had a temporary reduction of serum antiguanosine and antidsDNA antibodies [44]. Patients treated with plasmapheresis
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generally improved temporarily. They usually continued to improve if treated with an immunomodulatory drug. Without the immunosuppressive drugs the antibodies to DNA and guanosine increase within weeks and the symptoms of SLE recurred, thereby suggesting a correlation between the levels of these antibodies and disease activity. 5.1. The correlation of specific features of SLE with antiguanosine antibodies We then studied 86 patients with American College of Rheumatology (ACR) criteria for SLE [47] to determine whether there was relationship between the individual symptoms and laboratory features of SLE with the serum levels of antibodies to dsDNA, ssDNA and each of the nucleosides [48]. We divided patients by whether they had criteria for active SLE or inactive disease. There were 59 patients with active SLE and 27 that were inactive as determined by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) [49]. Active disease symptoms in the SLEDAI include arthritis, skin rashes, central nervous system (CNS) symptoms, nephritis, pleurisy, pericarditis, and typical hematological abnormalities of SLE. The average SLEDAI score of the active patients was 10 times higher than the inactive patients (10.5 vs. 1.1). The serum levels of antiguanosine (p b 0.0001), dsDNA (p b 0.003) and ssDNA (p b 0.005) antibodies, in patients with active SLE were significantly higher than those with inactive disease as measured by enzyme-linked immunosorbent assay (ELISA). The serum levels of antiguanosine antibodies in these patients were more than 2 times higher than levels of antiadenosine antibodies, 3 times those of antithymidine antibodies and 6 times those of anticytidine antibodies (p b 0.0001) [48]. When compared with antiguanosine antibodies the other 3 antinucleoside antibodies tested did not correlate well with the activity of disease in the SLE patients [48]. When we matched the individual organ system involvement of patients with active SLE with the inactive group it became apparent that serum antiguanosine antibody levels compared at least as well if not better than antibodies to dsDNA in differentiating the active from the inactive groups (Table 1). Antiguanosine antibodies in the serum of patients with SLE, especially those with symptoms and signs of active nephritis, polyserositis, arthritis, hematologic manifestations of lupus and CNS lupus, were closely associated to the activity of SLE. This association seemed to be true to a lesser extent for rashes associated with SLE. 5.2. Longitudinal association of antiguanosine antibodies and disease activity If antibodies to guanosine expressed a pathogenic potential similar to anti-dsDNA antibodies then there should be a longitudinal correlation between the serum levels and disease activity in these patients. We followed 12 patients 3 to 7 years and compared the serum levels of antiguanosine antibodies and antibodies to dsDNA and ssDNA in periods of active un-
Table 1 Association of anti-nuclear antibodies with disease manifestations of SLE Abs ssDNA dsDNA anti-G Low C3 ssDNA dsDNA anti-G ssDNA dsDNA anti-G ssDNA dsDNA anti-G ssDNA dsDNA anti-G ssDNA dsDNA anti-G ssDNA dsDNA anti-G a b
SLE feature a Active SLE (n = 59) 0.734 ± 0.144 0.431 ± 0.162 0.357 ± 0.164 51% Nephritis (n = 18) 0.956 ± 0.296 0.802 ± 0.401 0.710 ± 0.422 Polyserositis (n = 10) 1.245 ± 0.025 1.063 ± 0.593 0.745 ± 0.630 Arthritis (n = 8) 1.186 ± 0.439 1.853 ± 0.761 0.415 ± 0.521 CNS (n = 9) 0.744 ± 0.472 0.685 ± 0.718 0.344 ± 0.566 Rash (n = 29) 0.623 ± 0.213 0.309 ± 0.217 0.202 ± 0.141 Hematologic (n = 29) 0.689 ± 0.185 0.359 ± 0.193 0.245 ± 0.160
p values b Inactive SLE (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011 10% (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011 (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011 (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011 (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011 (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011 (n = 27) 0.379 ± 0.059 0.065 ± 0.017 0.049 ± 0.011
0.005 0.003 0.0001
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.008 0.006 0.067 0.024 0.005 0.131 0.104 0.051 0.012 0.010 0.002
Optical density ± standard error of the mean. Mann–Whitney U test.
controlled SLE, active controlled and inactive SLE (Table 2). The levels of antiguanosine antibodies followed the disease activity at a level that was statistically significant. In these studies anti-ssDNA and anti-dsDNA antibodies did not correlate as well as antiguanosine [48]. 6. Pathogenic potential of antiguanosine antibodies in SLE Finding a close correlation of serum levels of antiguanosine antibodies to disease activity in SLE encouraged us to pursue the study of possible pathogenic features of this antibody. We started by attempting to identify the binding sites or epitopes of guanosine and antiguanosine antibodies. Separate preparations of guanosine molecules methylated at the 1, 2, 3 or 7 positions on the guanine ring were used to test their binding affinity using antiguanosine antibodies contained in sera of patients with SLE and monoclonal antiguanosine antibodies generated from MRL/ lpr mice. The methylated groups at 1 and 7 positions blocked the binding of the antiguanosine antibody, whereas the 2 and 3 positions did not. This indicated that the binding site on guanosine was across the 1 and 7 positions. This confirmed the work of Munns et al. [38]. We found that an oxygen molecule attached to the carbon at position 6, between the 1 and 7 positions, was essential to antibody binding. We tested analogs of guanosine with and without oxygen bound at position 6, and found that there was significantly reduced binding if there was
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Table 2 The longitudinal association of antinuclear abs with the phases of SLE disease activity Group I (n = 6)
Mean response a
Abs ssDNA (ELISA) dsDNA (ELISA) dsDNA (RIA) Guanosine (ELISA) Group II (n = 6) Abs ssDNA (ELISA) dsDNA (ELISA) dsDNA (RIA) Guanosine (ELISA)
Active SLE (10 sera) 1.479 ± 0.412 0.684 ± 0.320 50.7 ± 29.3 0.217 ± 0.047
Inactive (12 sera) 1.223 ± 0.341 0.065 ± 0.017 23.8 ± 8.9 0.105 ± 0.017
p value 0.13 0.09 0.09 b0.05
Active (15 sera) 2.711 ± 0.195 2.035 ± 0.291 100.3 ± 20.3 0.745 ± 0.165
Active-improved (15 Sera) 1.346 ± 0.273 0.955 ± 0.266 38.1 ± 17.6 0.243 ± 0.090
0.10 0.16 0.09 b0.02
a b
Statistical analysis b
Mean optical density (OD) for ELISA ± standard error of the mean (S.E.M.); mean units per ml for RIA ± S.E.M. Paired t test.
no oxygen at that position. Adenosine has a similar 9 member double ring structure as guanosine, but has an amine group (NH2) at position 6 whereas guanosine has a hydroxyl group (OH). Antiguanosine antibodies do not bind to adenosine. However, antiguanosine antibodies did bind to hypoxanthine, cGMP and acyclovir, all of which have an oxygen molecule at position 6. We were able to show that the binding properties of the mouse monoclonal antiguanosine 4H2 antibody were conserved exactly like polyclonal antiguanosine antibodies from sera of patients with SLE when binding to guanosine and guanosine analogs [50]. 6.1. Interference of cell signal transduction G-binding proteins active in cell signal transduction exert their function by cycling between inactive GDP and an active GTP-containing conformation described by Pai et al. to be structurally the same as the Ha-ras oncogene product p21 [51]. G-binding proteins are activated by the GDP to GTP reaction that in turn activates adenyl cyclase that then produces cAMP. We found that antiguanosine antibodies bind GDP and GTP at the same epitopes as G-binding proteins, indicating that antiguanosine antibodies have the same internal image as G-binding proteins [50]. These findings led us to consider that antiguanosine antibodies might interfere with cell signal transduction involving G-proteins. Highly specific antiguanosine antibodies provide a marker for SLE disease activity as described above. With the monoclonal antibody, 4H2, we found a tool that could be used to help elucidate the possible pathogenic role of antiguanosine antibodies in SLE. As an initial test we performed RIAs to determine if antiguanosine antibodies interfere with cAMP production by culturing normal human lymphocytes with plasma from patients with high levels of antiguanosine antibodies and with 4H2 monoclonal antiguanosine antibodies. Controls consisted of other monoclonal anti-DNA antibodies, an unrelated monoclonal antibody, sera of patients with Gillian–Barre' syndrome, thrombotic thrombocytopenic purpura, Wegener's granulomatosis, myasthenia gravis, amyotrophic lateral sclerosis and plasma from healthy donors. The plasma from patients with SLE, and 4H2 monoclonal antibody,
qualitatively reduced the cAMP levels (Colburn and Green, unpublished observations). Additionally, using more sophisticated methods (laser scanning cytometry) we were able to confirm that 4H2 reduced cAMP levels in human lymphocytes as well as a number of different cell types. The results of antiguanosine' ability to reduce cAMP levels in epithelial cells are shown in Fig. 1 [52]. 6.2. Cell membrane permeability of antiguanosine antibodies The fact that antiguanosine antibodies interfere with cAMP levels suggested that these antibodies must be internalized into cells to exert their affect on signal transduction. There is a growing body of evidence that some antibodies penetrate cell membranes [18,19]. This topic was the focus of a meeting in
Fig. 1. Quantitative immunocytochemical levels of cAMP in rat epithelial cells treated with antinucleoside antibodies. Rat thyroid epithelial cells were established in 6-well plates containing glass coverslips. The established cells were treated, in duplicate, with: nothing (basal level), 4H2 at 1:8 and 1:16 and 3E10 (an anti-DNA antibody [37]) at 1:8 and 1:16. After a 3 h incubation the cells were fixed and immunofluorescently labeled with anti-cAMP antibodies. The immunolabeled cells were scanned on the laser scanning cytometer and the values compiled and graphed as shown. The estimated concentration of the antibody preparations was 10 μg/ml at the 1:8 dilution. The results indicate that the 4H2 antiguanosine antibody reduced the levels of cAMP nearly 10-fold compared to the untreated levels. The anti-DNA antibody 3E10 only slightly reduced the levels of cAMP.
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Cancun, Mexico in 1998. The presentations from this meeting were published in a volume of the Journal of Autoimmunity. These articles varied in their scope, including topics that revealed that portions of the immunoglobulins were being used as targeting molecules [19], discussions of immunochemical, structural and translocating properties of anti-dsDNA antibodies [53] and cellular dysfunction [54]. This information indicates the previous dogma regarding the functional consequences of antibody binding (complement, opsonization, etc.) are insufficient to account for the dynamic potential and range of mechanisms that are currently emerging. 6.3. Mitochondrial localization of antiguanosine antibodies To show that antiguanosine antibodies penetrated cell membranes we labeled purified 4H2 by direct conjugation to Alexa fluor-594 (a red fluorescent molecule). By fluorescent microscopy we found that labeled 4H2 was incorporated into the cytoplasm of normal human lymphocytes and in live and fixed cells from various sources. In lymphocytes the cytoplasm is difficult to visualize so we used human thyroid epithelial cells (Htori-3-LLU, [55]) to confirm that the binding occurred. A pattern was revealed that suggested that the antibody was localized to the mitochondria. To confirm this we used mitotracker conjugated to FITC (a green fluorescent molecule) and incubated it with live Htori-3-LLU cells. Nuclei were stained with the DNA specific blue dye, DAPI. When the images from the multi-labeled cells were overlaid the red and green signals were in perfect register (Fig. 2). Antiguanosine antibodies were
penetrating the cell membrane and binding to mitochondria. Thus we had found that the antiguanosine antibodies were produced at high levels in patients with active SLE, in vitro these antibodies were found to be capable of altering signal transduction (reduced cAMP levels, Fig. 1) which may be related to their localization to the mitochondria (Colburn and Green, unpublished observation). 6.4. Interference of mitochondrial function by antiguanosine localization The intense localization of antiguanosine to the mitochondria suggested that it might interfere with mitochondrial function. Therefore, the next experiments were to test mitochondrial function using the MTT assay, which measures cell viability and indirectly mitochondrial activity, because the tetrazolium salt is converted to formazan by mitochondrial dehydrogenase activity [56]. The cells with incorporated 4H2 monoclonal antiguanosine antibody had decreased MTT dye reduction, first suggested that the added 4H2 was cytotoxic. However, when the cells were counted, the 4H2 treated cultures were essentially unchanged from the number of cells originally plated, whereas, the non-treated controls cultures had increased in number. Thus, 4H2 was cytostatic; presumably the binding interferes with mitochondrial function. Collectively these results lead us to believe that antiguanosine antibodies in SLE are pathogenic. They penetrate lymphocytic membranes, interfere with signal transduction, bind to mitochondria and altered its function [57].
Fig. 2. Internalization of fluorescently labeled antiguanosine antibody (4H2) in live Htori-3-LLU thyroid epithelial cells. Concentrated hybridoma supernatant containing antibodies to 4H2 (antiguanosine) was concentrated and purified by protein A sepharose and conjugated to Alexa fluor-594 (Molecular Probes, Eugene OR). The four panel composite shows representative images of Htori-3-LLU cells incubated with: a) 4H2—Alexa fluor-594; b) FITC—Mitotracker; c) DAPI nuclear stain; and d) merged image of panels a–c. The overlay of labeled 4H2 (red) with mitotracker (green) resulting in a yellow color indicates that they are binding to the same location, the mitochondria. The magnification of the image was taken at 100× oil. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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We then went in search of a mechanistic explanation for our findings. There is a growing body of evidence that antibodies can have enzymatic activity. From our previous studies, the most logical activity for antiguanosine antibodies would be that it behaved like a GTPase. We designed a method to test the GTPase activity of 4H2 (a murine antiguanosine antibody). GTPase-like properties would explain how antiguanosine could be involved in cell-signal transduction by converting GTP to GDP and thereby indirectly reducing cAMP levels. The results of our preliminary investigation suggest that antiguanosine cleaved the gamma-phosphate bond from a radio-labeled substrate (GTP), which in vivo would disrupt the activation of G-binding proteins and block signal initiation [58]. 7. Summary Our work has demonstrated that circulating antibodies to guanosine are potentially important in SLE. Their serum levels are specific for some features of SLE including nephritis, polyserositis, arthritis, CNS lupus and the hematologic features of SLE. These antibodies closely correlate with SLE disease activity as measured by the SLEDAI scale both at a point in time and in longitudinal studies. Antiguanosine antibodies are highly specific for the 1 through 7 locations on the guanine ring and require oxygen for binding, the same as site of the binding of guanine to the HA-ras P21. HA-ras P21 is an oncogene that has binding properties similar to G-proteins that participate in signal transduction. Antiguanosine antibodies have the internal image of G-binding proteins. Antiguanosine antibodies penetrate cell membranes, bind to and inactivate mitochondria dehydrogenase activity and interfere with signal transduction possibly by their innate GTPase activity. Antiguanosine antibodies are a marker for SLE disease activity, at least as good as, and possibly better than anti-dsDNA antibodies. Furthermore, like anti-dsDNA antibodies, antiguanosine antibodies may be pathogenic for certain features of SLE. Further studies are currently underway to substantiate these findings and contentions. The interesting findings reported here for antiguanosine autoantibodies are only one type of antibody found in SLE patient sera. A relatively recent report details the more than 100 different antibodies found in the sera of patient with SLE [59]. Clearly, there are numerous possible antibodies that are produced in SLE that might, like antiguanosine, help us to better elucidate the complexities of this potentially debilitating disease. References [1] Alarcon-Segovia D, Fishbein E, Estrada-Parra S. The heterogeneity of anti-DNA antibodies in systemic lupus erythematosus and other diseases. J Rheumatol 1975;2:172–7. [2] Morimoto C, Steinberg AD, Schlossman SF, Borel Y. In vitro nucleoside specific immune response by lymphocytes from systemic lupus erythematosus. J Clin Invest 1983;71:1402–9. [3] Weisbart RH, Garrett RA, Liebling MR, Barnett EV, Paulus HE, Katz DH. Specificity of anti-nucleoside antibodies in systemic lupus erythematosus. Clin Immunol Immunopathol 1983;27:403–11. [4] Jacob L, Kerjaschki D, Viard JP, et al. Potential pathogenic role of cellsurface polypeptides, cross-reactive with DNA, in systemic lupus erythematosus. Nephrologie 1989;10(3):139–40.
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