Eur. J. Immunol. 1992. 22: 3051-3056
Stuart KahnO Maria Kahn* and Harvey Eisen" Department of Pediatricso, School of Medicine, University of Washington, Seattle and Division of Basic Sciences", Fred Hutchinson Cancer Research Center, Seattle
Autoantibodies to negatively charged epitopes following 7: cvuzi infection
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Polyreactive autoantibodies to negatively charged epitopes following Trypanosoma cruzi infection* During the course of many human autoimmune diseases, antibodies which recognize negatively charged epitopes on self antigens are detected. Trypanosoma cruzi, an intracellular protozoan parasite capable of infecting a wide variety of vertebrates, is the cause of Chagas disease in humans. Infection with the parasite frequently results in autoimmune and inflammatory pathology.We report here on an affinity-purified population of antibodies that bind to a broad class of antigens that contain runs of acidic amino acids, including tubulin. Although these antibodies can be isolated from both uninfected and 7: cruzi chronically infected C3H/He mice, the antibodies from the normal mice (the natural autoantibodies) bind t o tubulin poorly at physiological pH, whereas the antibodies isolated from the infected animals bind well at physiological pH. We propose that similar processes may occur in humans following other infections accounting for the detection of antibodies to negatively charged epitopes in a variety of autoimmune diseases.
1 Introduction In many autoimmune diseases, antibodies have been described which bind to negatively charged epitopes on common cellular components e.g. anti-phospholipid antibodies [l] and anti-DNA antibodies [2]. In addition, cellular proteins which contain long runs of charged amino acids are the common autoantigens in human rheumatologic diseases 131. The mechanisms leading to the generation of theye and other autoantibodies detected in various disease states remains unclear. A polyclonal proliferation of B lymphocytes has been implicated in the generation of autoantibodies 141. Furthermore, some autoantibodies appear to undergo selection for increased affinity in response to specific self antigens [S-7]. Since low titers of polyreactive autoantibodies (natural autoantibodies) can be detected in normal individuals, it is possible that the deregulation of these natural autoantibody-producing lymphocytes may generate the autoantibodies found in many pathologic conditions [8-121. Infection with Trypanosoma cruzi, an obligate intracellular protozoan parasite, appears to be an excellent model for
the induction of autoimmunity [13, 141. Infection in man causes Chagas disease, which is characterized by chronic autoimmune pathology [13). Experimental infection of the mouse has permitted careful description of the immunopathology [lS, 161. After infection, a T h cell-dependent polyclonal proliferation of both B and T lymphocytes occurs [17, 18, 191. Antibody production is predominantly lgGza and IgGzb [171. Multiple autoantibodies following 7: cruzi infection have been described [20-231. Antitubulin antibodies following 7: cruzi infection of mice have been characterized and may represent an example of deregulation of low-level natural anti-tubulin antibody production [24]. We report here on antibodies from mice chronically infected with T cruzi which bind acidic antigens in a variety of proteins including tubulin.
2 Materials and methods 2.1 Animals Six-week-old female C3H/HeJ mice were purchased from Jackson Laboratory (Bar Harbor, ME). A two-month-old female NZW rabbit was obtained for immunizations.
2.2 Cells [I 106931 *' This study was supported by grants from the National Institute of
Health (HD-2x586 and AI-27803). During part of this study, S.K. was a National Institute of Child Health and Human Developmcnt fellow of the Pediatric Scientist Training Program (HD-22297).
Rat 3T3 cells were maintained in DMEM with 10 % FCS. 7: cruzi CL strain was a recent subclone ("subclone three") [25].Trypomastigotes and amastigotes were obtained from culture supernatants of infected rat 3T3 cells. Epimastigotes were maintained in liver infusion/tryptone medium with 10% FCS.
Correspondence: Stuart Kahn. Department of Pediatrics RD-20, Division of Immunology and Rheumatology, University of Washington, School of Medicine, Seattle, WA. 98195 USA
2.3 Antigens
Abbreviations: CIMS: Chronic infected mouse serum NMS: Normal mouse scrum GST Glutat hione S-transferase Anti1.2N antibodies: Antibodies from normal mouse serum affinitypurified with thc 1.2 peptide Anti-1.2C antibodies: Antibodies from T cvuzi chronic infected mouse serum affinity purified with the 1.2 peptide
Peptides were synthesized on 430A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA). Glutathione S-transferase (GST) and c1.l-GST fusion protein were purified from isopropylthiogalactoside (Sigma Chemical Co., St. Louis, M0)-induced bacterial lysates as described
0 VCH Verlagagesellschaft mbII, D-6940 Weinheim. 1992
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Eur. J. Immunol. 1992. 22: 3051-3056
S . Kahn, M . Kahn and H. Eisen
1261. Tubulin was purified from bovine brain. Briefly, microtubule proteins were purified by cycles of assembly and disassembly 1271. Tubulin was purified from microtubules by phosphocellulose chromatography 1281. Polyglutamate, polyaspartate, and polylysine were purchased from Sigma Chemical Co.
96-well plates, incubated overnight, and blocked with 5 YO nonfat milk in PBS. Then various dilutions of mouse IgM, mouse IgG (Calbiochem, La Jolla, CA) (1000ng/ml to 0.5 ng/ml), and uninfected and chronically infected mouse sera were incubated for 1 h. Bound Ig was detected with goat anti-mouse IgM or IgG coupled to horseradish peroxidase (Zymed).
2.4 Antibodies Eight-week-old female C3H/HeJ mice were infected by intraperitoneal injection of lo5 tissue culture-derived trypomastigotes. Uninfected mice and chronic infected mice were sacrificed at 8 months of age, and sera was obtained. IgM and IgG concentrations of uninfected mice were determined to be 0.547 k 0.053 mg/ml and 2.273 k 0.208 mg/ml, respectively (data not shown). IgM and IgG concentrations of chronic infected mice were determined t o be 1.928 S 0.530 mglml and 11.950 f 0.778 mg/ml, respectively (data not shown). Antibodies were obtained by passing sera over a 1-ml column of peptide Sepharose 4B (Pharmacia Fine Chemicals, Piscataway. NJ). The columns were washed with 20 column volumes of PBS before antibodies were eluted with 3 M NHlSCN in PBS, and dialyzed with PBS. All peptides were coupled directly to CNBr-Sepharose 4B according to the manufacturer’s instructions. Antibodies purified with the 1.2 peptide (Table 1) from normal mouse serum (NMS) are 1.2N antibodies; and those from chronic infected mouse serum (CIMS) are anti-1.2C antibodies. Anti-1.2N antibodies (6 pg) were obtained per ml of uninfected sera. Anti-1.2C antibodies (25 pg) were obtained per ml of CIMS. Anti-cl. 1 antibodies were obtained by immunizing 2-month-old NZW rabbits with 100 pg of affinity-purified c l . 1-GST fusion protein [29]. After five boosts, anti-cl.1 antibodies were affinity purified by sequentially passing the hyperimmunized rabbit sera over a 2-ml column of GSTSepharose 4B column, and then over a cl.1-GST fusion protein column linked to Sepharose 4B, as previously described [29].
2.5 Enzyme-linked immunosorbent assay (ELISA) Antigens (tubulin or c1.l-GST) at a concentration of 5 pg/ml in PBS, were plated onto 96-well plates (Costar, Cambridge, MA) and incubated at room temperature overnight. Plates were washed with PBS containing 0.05 YO Tween (Sigma Chemical Co.),and “blocked” by incubation with 3% BSA in PBS for a minimum of 1 h. Antibodies (50 pl/well) were added and incubated for 1 h at room temperature. If competitions were performed, competitor was added to antibodies at stated concentrations, and preincubated for 30 min, before antibodies and competitor were added to wells. Plates were washed and goat antimouse IgM or IgG coupled to horseradish peroxidase (Zymed, San Francisco, CA) was added and incubated for 1 h. Antibody binding was detected by addition of ophenylenediamine (Zymed), and read on an automated ELISA reader (Bio-Tek Instruments, Burlington, VT). To determine the concentration of TgM and IgG, affinitypurified goat anti-mouse antibodies (IgA, IgG, and IgM; heavy and light chains) (Organon Teknika Corp., West Chester, PA) at a Concentration of 2 pg/ml were plated onto
2.6 Western blots SDS-PAGE was followed by semi-dry transfer of antigens to nitrocellulose membranes (Micron Separations Inc.), “blocked” with 3 % nonfat dry milk: and incubated with antibodies [30]. The blots were developed with alkaline phosphatase-conjugated goat anti-mouse antibodies (Promega, Madison, WI), followed by incubation with bromochloroindolyl phosphatehitro blue tetrazolium
POI. 2.7 Immunostaining
Rat 3T3 cells were grown on slides (Lab-Tek Chamber Slide, Nunc Inc., Naperville, IL). Cells were fixed by incubating in 100 mM MES, pH 6.8, 1mM MgC12, 1mM EGTA (MME) with 4 YO PEG 4000 at 37°C for 5 s, followed by incubation in MME with 4 YOPEG and 10 % Triton at 37°C for 30 s, followed by 0.1 YOglutaraldehyde, 2 % formaldehyde in MME at 37 “C for 15 min, followed by 0.5 Yo NaBHJ in PBS at room temperature for 5 min [31]. Live parasites were adhered to polylysine-coated slides, and then fixed in 4 YOparaformaldehyde, at 4 “C for 15 min [29]. Fixed samples were then preincubated with 3 YOBSA in PBS, incubated with antibodies, and then FITC-labeled goat F(ab‘)z anti-mouse IgG (Tago Inc., Burlingame, CA) ~91.
3 Results Previously, we characterized a family of surface antigens of 7: cruzi (the SASS-1 antigens) using a series of antibodies affinity purified with small peptides from CIMS [29, 321. These antibodies recognized mammalian-stage parasites, and not insect-stage parasites [29]. An example of this binding is shown in Fig. 1A and 1B using antibodies purified with peptide 1.1(Table 1,anti-1.1 antibodies) [29]. Surprisingly, antibodies to an acidic peptide (peptide 1.2, Table 1, anti-1.2C antibodies) derived from a SASS-1 antigen appeared to bind to the flagella of mammalianstage trypomastigotes and insect-stage epimastigotes (Fig. 1C and D). These 1.2C antibodies also appear to recognize the cytoskeleton of the 3T3 cells in which the trypomastigotes were grown (Fig. 1E). This cytoskeletal Table 1. Peptides used for inhibition of tubulin binding
Peptide 1.1
I .2 PTubuli n
Scquencc RETGDGGANGDAVSAYG QFDDDDDGGDDDDEEDSQ EEEEDEGEEAEEE
Eur. J. Inirnunol. 1992. 22; 3051-3056
Autoantibodies to negatively charged epitopes following ‘I: c r u i infection
and flagella binding suggested a cross-reactivity to tubulin, a protein found abundantly in these structurks. In Fig. 2, it can be seen that the 1.2Cantibodies do react with tubulin in both Western blot (Fig. 2A) and ELISA (Fig. 2B). Although tubulin does not contain sequences homologous to the 1.2 peptide, it does contain a run of acidic amino acids at the C terminus [33].The 0-tubulin acidic peptide was synthesized (Table l), and tested as a competitor of anti-1.2C binding to tubulin in an ELlSA (Fig. 3A). The binding of the anti-1.2C antibodies to tubulin was inhibited with the 1.2 peptide, and the (3-tubulin acidic peptide (Fig. 3A). The 1.1 peptide did not inhibit tubulin binding (Fig. 3A). Since the tubulin binding can be inhibited with two small acidic peptides which have different primary sequences
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(1.2, and the C terminal of fi-tubulin, Table 1),we tested whether peptides composed entirely of acidic amino acids could compete in an ELISA. Both polyaspartate and polyglutamate competed the binding of anti-1.2C antibodies to tubulin,whereas polylysine didnot (Fig. 3B). (We do not understand why polylysine augments the antibody binding.) This implies that the anti-1.2C antibodies bind to tubulin through ionic interactions to regions of acidic amino acids. Since ionic interactions were implicated in the recognition of anti-1.2C antibodies to tubulin. we examined the effect of increasing NaCl concentration on this interaction (Fig. 4). The binding was inhibited by 71 % at 300 mM NaCl, and by 85 % at 450 mM NaCl. As a comparison, we examined the effect of salt on a polyclonal affinity-purified
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Figure7 I . Indirect immunofluorescence shows anti-1.2C antibodies react with thc cytoskcleton. Parasites and rat 3T3 cells were stained with 10 pg/ml anti-1.2C or anti-1.1 antibodies and FITCF(ab’)? anti-mouse IgG. (A) 7: cruzi trypomastigotes with anti-1.1 antibodies. (B) 7: c m z i cpimastigotes with anti-1.1 antibodies. (C) 7: c m z i trypomastigotes with anti-1.2C antibodies. (D) 7: cruzi cpimastigotcs with anti-l.2C antibodies. (E) Rat 3T3 cells with anti-1.2C antibodies. (F) Rat 3T3 cells with anti-1.1 antibodies.
Figure 2. Anti-1.2C antibodies bind to tubulin in Western blot and ELISA. (A) Western blot on tubulin (4 &lane) reacted with anti-1.2C antibodies (10 pg/ml). Tubulin was electrophoresed on a 7.5 % SDS-PAGE, transferred to nitrocellulose, reacted with antibodies and bromochloroindolyl phosphate/nitro blue tetrazolium. (B) ELISA detects anti-1.2C and anti-1.2N antibodies binding t o tubulin. Antibodies were affinity purified from normal mouse serum (NMS) and CIMS with the 1.2 peptide (Table I), and their binding t o tubulin detected with peroxidase-conjugated anti-IgM or anti-IgG. Where error bars are not shown, the error was smaller then the symbols.
S. Kahn, M. Kahn and H. Eiscn
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Eur. J. Immunol. 1992. 22: 3051-3056
rabbit antibody generated by a standard immunization protocol (anti-cl. 1 antibodies). The anti-cl.1 antibody was prepared by immunization with C1.1-GST fusion protein. The affinity-purified antibodies were reacted with c l . 1GST in ELISA at various salt concentrations (Fig. 4). The anti-cl. I antibodies were inhibited by only 6 % at 300 mM and 450 mM NaCl; and by < 50 % at 2.0 M NaC1, demonvtrating that the anti-1.2C antibody binding to tubulin was very 5ensitive to salt concentration.These results supported our hypotheqis that the cross-reactive anti-1.2C antibodies were binding to regions of acidic amino acids through ionic interactions.
infected C3H/He mouse sera is shown in Fig. 5. In the serum of uninfected animals, natural anti-tubulin antibodies are detected (Fig. 5 ) . There appears to be a similar amount of IgM antitubulin antibodies in the CIMS. However, in CIMS, there appears to be a large increase of IgG antitubulin antibodies, compared to the NMS anti-tubulin IgG antibodies (Fig. 5 ) .
To determine whether the natural anti-tubulin antibodies also recognized acidic epitopes, we affinity purified antibodies from uninfected mouse serum with the acidic peptide 1.2 (anti-1.2N antibodies). Antibodies were obtained and were found to bind tubulin in ELISA
Previous studies in (CS7BL/6 x BALB/c) F1 mice have demonqtrated the presence of natural anti-tubulin antibodies which appear to expand and mature following 7: cruzi infection 1241. ELISA on uninfected and chronically
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Figure 4. Anti-1.2C binding to tubulin is sensitive to NaC1. Antibody (10 &ml) was incubated in 50 mM phosphatc buffer with increasing concentrations of NaCl as indicated. Binding to tubulin was detected with peroxidase conjugated anti-IgG. The binding to tubulin i s expressed as the percent of maximal binding for each antibody. Mouse antibodies (anti-1.2C) bound maximal at 0 mM NaC1. Rabbit antibodies (anti-cl.1) bound maximal at 150 mM NaCI. (0)equals mouse antibodies; (D) equals rabbit antibodies. Where error bars are not shown, the error is smaller then the symbols.
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Figuw3. Thc binding of anti-1.2C antibodies to tubulin is inhibitcd with peptides rich in acidic amino acids. Anti-1.2C antibody (10 &mi) was incubated with various competitors. Binding to tubulin was detected with pcroxidase-conjugated anti-IgG. Thc binding is expressed as a percent of binding to tubulin in the absence o f competitor.Where error bars are not shown, the error is smallcr then the symbols. (A) Threefold increases in peptide competitor. expressed as mM final concentration of peptide, indicatcd as follows: ( 0 )equals 1.2 peptide; (0)equals b-tubulin pcptidc and (D) equals 1.1 peptide. (B) Threefold increases in peptide competitor. expressed as mglml final concentration of peptide. indicatcd a s follows ( 0 )equals polyglutamate; (0)equals polyaspartate and (D) equals polylysine.
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Figure5. Titration of anti-tubulin IgM and IgG antibodies in NMS, and in ?: cruzi chronic infected mouse sera. NMS and CIMS were diluted with PBS. Binding to tubulin was detected with peroxidase-conjugated anti-IgM or anti-IgG. (0) equals CIMS IgM; (D) equals NMS IgM; (W) equals CIMS IgG and (V)equals NMS 1gG.Where error bars are not shown, the error is smaller then the symbols.
Autoantibodies to negatively charged epitopes following 7: cruzi infection
Eur. J. Imniunol. 1002. 22: 3051-3056 110
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Figure 6. Anti-1.2N antibodies and anti-1.2C antibodies exhibit diffcrent sensitivitiesto pH. Antibody (10 pg/ml) was incubated in 50 mM glycine buffer with the pH adjusted as indicated. Binding to tubulin was detected with pcroxidase-conjugated IgG. Binding to tubulin is cxprcssed as percent of maximal binding. The maximal tubulin binding for this expcrimcnt was with anti-1.2C binding at pH 6.0. (D) equals anti-1.2C antibodies and (0)equals anti-1.2N antibodies.Where error bars are not shown. the error was smaller thcn thc symbols.
(Fig. 2B). There is increased binding of IgG antibodies from 1.2C antibodies from CIMS, as compared to the 1.2N IgC antibodies from NMS (Fig. 2B). These results. and the results of anti-tubulin antibody titers in CIMS and NMS (Fig. S), suggest that during the course of chronic 7: cruzi infection, anti-tubulin IgG antibodies which recognize regions of acidic amino acids are expanded. Both anti-1.2N and anti-1.2C antibodies bound peptides rich in acidic amino acids, suggesting that ionic interactions involving the antigen’s COO- groups of glutamate and aspartate were important. Therefore, we studied the effect of pH on these antibodies’ ability to bind tubulin. This was done by varying the pH of the primary antibody-binding otep during the tubulin ELISA. The affinity-purified antibodies from CIMS exhibited strong tubulin binding (> 80 94 of maximum) at physiological pH (Fig. 6). Maximal binding to tubulin with these antibodies was observed at pH 5 and pH 6, and the binding was greatly inhibited at pI-1 < 5 and > 9 (Fig. 6). I n contrast, the affinity-purified natural tubulin antibodies bound poorly at physiological pH (Fig. 6). Maximal binding was at pH 4 (Fig. 6). These results sugget that the natural tubulin antibodies will bind poorly to acidic antigens encountered when circulating in the mouse. However, in the chronic infected animal, the tubulin antibodies may potentially bind to a wide variety of autoantigens which contain runs of acidic amino acids.
4 Discussion In thi\ report, we describe autoantibodies that bind to ncgatively charged antigens rich in acidic amino acids, including the carboxyl terminal of 0-tubulin. These antibodie5 can he isolated from normal mice and mice chronically infected with 7: cruzi. The antibodies are sensitive to changes in salt concentrations and pH, suggesting that ionic interactions contribute to their antigen binding and poly-
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reactivity. The antibodies (IgG and IgM) from chronically infected animals display increased binding to tubulin (Fig. 2B), and bind it well at physiologic pH (Fig. 6), whereas the antibodies from normal mice bind poorly at physiologic pH (Fig. 6).
A previous report has compared natural antitubulin antibodies and antitubulin antibodies following 7: cruzi infection in mice [24]. Contrary to our experiments, the previous study found no evidence of antibodies that bind to the carboxy terminus of tubulin. Several differences between the studies suggest explanations. First. the earlier study examined the (CS7BL/6 X BALB/c) F1 as opposed to the C3H/He mice used in this study, and the genetic differences may explain these discrepant results. Second, in the present study, the antibodies which bind the carboxy terminus of tubulin were initially affinity purified by a peptide rich in acidic amino acids. Our data suggests that these antibodies are a minor subset of the total anti-tubulin antibodies found in normal and chronically infected animals (Figs. 2B and 5). In the previous study, total anti-tubulin antibodies were examined for the ability to bind to protease-digested fragments of tubulin in Western blot analysis [24]. This assay may not have been sensitive enough to detect a subset of the anti-tubulin antibodies that binds to a carboxyterminal fragment. The anti-1.2N antibodies have different characteristics from the anti-1.2C antibodies. First, the anti-1.2C antibodies (both IgM and IgG) display increased binding to tubulin then the anti-1.2N antibodies (Fig. 2B). Second, the anti1.2C IgG antibody binding to tubulin is increased compared to the anti-1.2N IgC antibody binding (Fig. 2B).Third, the effect of pH on the two different antibody classes is remarkably different (Fig. 6). Together, these results suggest that the interactions between the two antibody subsets and tubulin are different. We speculate that the ability to bind to tubulin is dependent upon interactions between the antibody and peptide acidic amino acids (aspartate pK, of 4.3 and glutamate pK1 of 3.9). The differences in optimal pH for binding anti-1.2N versus anti-1.2C to tubulin suggests that antibody residues such as histidine (pK, of 6.0) may be responsible for binding 1.2N antibodies, while the 1.2C antibodies use residues with higher pK, such as lysine (pK, of 10.5) and arginine (pK1 of 12.5). The mechanisms leading to the generation of the anti-1.2C antibodies are unclear. One poosibility is that the cells secreting the 1.2N antibodies in uninfected mice undergo class switching and/or somatic mutation during the course of 7: cruzc infection leading t o increased production and affinity of 1.2C antibodies. Another possibility is that during the chronic polyclonal proliferation following T cruziinfection, some rare, high-affinity, IgG-producing natural cross-reactive B cell clones are expanded; this may occur without class switching or somatic mutation. Evidence supporting such a possibility exists. First, Abo et al. [34] reported the appearance of “forbidden” T cell clones following antigenic stimulation with E. coli. Second, Shefner et al. [3S]reported the existence of a B cell clone that produces a rare, high-affinity, anti-double-stranded DNA antibody that is encoded by Vn genes containing no somatic mutations. Together these reports and our studies suggest that natural high-affinity anti-self I3 cells exist, and
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S. Kahn, M. Kahn and H. Eiscn
that during infections, difficult to detect anti-self clones can become more prominent. The role of negatively charged antigens in the development of the anti-1.2C antibodies, and in the development of autoantibodies in many human rheumatologic diseases is unclear [3].It is possible that during infection with T cruzi the immune system is chronically exposed to parasite and host intracellular antigens with negatively charged regions such as tubulin, which may lead to an increase in antibodies that bind to negatively charged antigens. Isolation and sequencing of B cell variable regions from chronically infected animals may enable us to define the mechanisms involved in the production of antibodies which recognize charged antigens [3]. We would like to thank R. Scaith and P Andreassen for providing purified tuhulin, C. Wilson, D. Ross, H . Ochs for critical review of the manuscript, and J. Gauthier for helpful discu.wions. Received April 29, 1992; in revised form August 3, 1992.
5 References 1 Peter, J. R . , Use and Interpretation of tests in Clinical Immunology, 8th Edit.. Speciality Laboratories, Inc., Santa Monica, 1YY2, p. 196. 2 Reichlin, M., in Kclley, W. N . , Harris, E. D., Ruddy, S. and Llcdge, C. B. (Eds.), Textbook of Rheumatology, W. B. Saunders Co., Philadelphia 1989, p. 214. 3 Brendel,V, Dohlman, J., Blaisdell, B. E. and Karlin, S., Proc. Natl. Acad. Sci. USA 1991. 88: 1536. 4 Klinman, D. and Steinberg, A. D., J. Exp. Med. 1987. 165: 1755. 5 Shlomchik, M. J., Aucoin, A. H., Pisetsky, D. S. and Weigert, M. G., Proc. Natl. Acad. Sci. USA 1987. 84: 9150. 6 Shlomchik, M., Mascelli, M., Shan, H., Radic, Z . , Pisetsky, D., Marshak-Rothstein. A . and Weigert, M., Exp. Med. 1990.171: 265. 7 van ES, J. H., Meyling, F. H . J. G., van de Akker, W. R. M., Aanstoot, H., Derksen. R. H. W. M. and Logtenberg, T., J. Exp. Med. 1991. 173: 461. 8 Guilbcrt, B., Dighicro, G . and Avrameas, S., J. Immunol. 1982. 128: 2779.
Eur. J. Immunol. 1992. 22: 3051-3056 9 Dighiero, G., Lymberi, P., Mazie, J. C., Rouyre, S., Browne, G. S.,Whalen, R. G. and Avrameas, S., J. Immunol. 1983.131: 3367. 10 Underwood, J. R., Pedersen, J. S., Chalmers, P. J. and Toh, B. H., Clin. Exp. Immunol. 1985. 60: 417. 11 Dighiero. G., Lymberi, P., Holmberg, D., Lundquist, I., Coutinho, A. and Avrameas, S., J. Immunol. 1985. 134: 765. 12 Avrameas, S., Immunol. Today 1991. 12: 154. 13 Petry, K. and Eisen, H., Parisitol. Today 1989. 5: 111. 14 Eisen, H . and Kahn, S., Curr. Opin. Immunol. 1991. 3: 507. 15 Said, G., Joskowicz, M., Barreira, A . A . and Eisen, H., Ann. of Neurol. 1985. 18: 676. 16 Hontebeyrie-Joskowicz, M., Said, G., Milon, G., Marchel, G. and Eisen, H., Eur. J. Immunol. 1987. 17: 1027. 17 D’Imperio Lima, M. R., Joskowicz, M., Coutinho, A., Kipnis, T. and Eisen, H., Eur. J. Immunol. 1985. 15: 201. 18 D’Imperio Lima, M. R., Eisen, H., Minoprio, P., Joskowicz, M. and Coutinho, A., J. Immunol. 1986. 137: 353. 19 Minoprio, P., Eisen, H., Joskowicz, M., Pereira, P. and Coutinho, A , , Immunol. 1987. 139: 545. 20 Wood, J. N., Hudson, L., Jessell, T. M. and Yamamoto, M., Nature 1982. 296: 34. 21 Towbin, H., Rosenfelder, G., Wieslander, J., Avila, J. L., Rojas, M., Szarfman, A., Esser, K., Nowack, H. and Timpl, R., J. Exp. Med. 1987. 166: 419. 22 VanVoorhis,W. C. and Eisen, H., J. Exp. Med. 1989.169: 641. 23 Van Voorhis, W. C., Schlekewy, L. and Trong, H. L., Natl. Acad. Sci. U S A 1991. 88: 5993. 24 Ternynck, T., Bleux, C., Gregoire, J., Avrameas, S. and Kanellopoulos-Langevin, C., .I. Immunol. 1990. 144: 1504. 25 Plata, F., Pons, F. G . andEisen, H., Eur. J. Immunol. 1984.14: 392. 26 Smith, D. B. and Johnson, K. S., Gene 1988. 67: 31. 27 Margolis, R., Rauch, C. T. and Job, D., Proc. Natl. Acad. Sci. USA 1986. 83: 639. 28 Williams, R. C. and Detrich, 111,H. W., Biochemistry 1979.18: 2499. 29 Kahn, S.,VanVoorhis,W. C. and Eisen, H., J. Exp. Med. 1990. 172: 589. 30 Harlow, E. and Lane, D., Antibodies, a laboratory manual, Cold Spring Harbor Laboratory, New York 1988, p. 471. 31 Osborn, M. and Weber, K., Methods Cell Biol. 1982.24: 2598. 32 Kahn, S., Colbert, T. G., Wallace, J. C., Hoagland, N. A. and Eisen, H., Proc. Natl. Acad. Sci. USA 1991. 88: 4481. 33 Cleveland, D. W. and Sullivan, K. F., Annu. Rev. Biochem. 1985. 54: 331. 34 Abo, T., Ohteki, T., Seki, S., Koyamada, N., Yoshikai, Y . , Masnda,T., Rikiishi, H. and Kumagai, K., J. Exp. Med. 1991. 174: 417. 35 Shefner, R., Kleiner, G., Turken, A., Papzian, L. and Diamond, B., J. Exp. Med. 1991. 173: 287.
Stuart KahnO Maria Kahn* and Harvey Eisen" Department of Pediatricso, School of Medicine, University of Washington, Seattle and Division of Basic Sciences", Fred Hutchinson Cancer Research Center, Seattle
Autoantibodies to negatively charged epitopes following 7: cvuzi infection
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Polyreactive autoantibodies to negatively charged epitopes following Trypanosoma cruzi infection* During the course of many human autoimmune diseases, antibodies which recognize negatively charged epitopes on self antigens are detected. Trypanosoma cruzi, an intracellular protozoan parasite capable of infecting a wide variety of vertebrates, is the cause of Chagas disease in humans. Infection with the parasite frequently results in autoimmune and inflammatory pathology.We report here on an affinity-purified population of antibodies that bind to a broad class of antigens that contain runs of acidic amino acids, including tubulin. Although these antibodies can be isolated from both uninfected and 7: cruzi chronically infected C3H/He mice, the antibodies from the normal mice (the natural autoantibodies) bind t o tubulin poorly at physiological pH, whereas the antibodies isolated from the infected animals bind well at physiological pH. We propose that similar processes may occur in humans following other infections accounting for the detection of antibodies to negatively charged epitopes in a variety of autoimmune diseases.
1 Introduction In many autoimmune diseases, antibodies have been described which bind to negatively charged epitopes on common cellular components e.g. anti-phospholipid antibodies [l] and anti-DNA antibodies [2]. In addition, cellular proteins which contain long runs of charged amino acids are the common autoantigens in human rheumatologic diseases 131. The mechanisms leading to the generation of theye and other autoantibodies detected in various disease states remains unclear. A polyclonal proliferation of B lymphocytes has been implicated in the generation of autoantibodies 141. Furthermore, some autoantibodies appear to undergo selection for increased affinity in response to specific self antigens [S-7]. Since low titers of polyreactive autoantibodies (natural autoantibodies) can be detected in normal individuals, it is possible that the deregulation of these natural autoantibody-producing lymphocytes may generate the autoantibodies found in many pathologic conditions [8-121. Infection with Trypanosoma cruzi, an obligate intracellular protozoan parasite, appears to be an excellent model for
the induction of autoimmunity [13, 141. Infection in man causes Chagas disease, which is characterized by chronic autoimmune pathology [13). Experimental infection of the mouse has permitted careful description of the immunopathology [lS, 161. After infection, a T h cell-dependent polyclonal proliferation of both B and T lymphocytes occurs [17, 18, 191. Antibody production is predominantly lgGza and IgGzb [171. Multiple autoantibodies following 7: cruzi infection have been described [20-231. Antitubulin antibodies following 7: cruzi infection of mice have been characterized and may represent an example of deregulation of low-level natural anti-tubulin antibody production [24]. We report here on antibodies from mice chronically infected with T cruzi which bind acidic antigens in a variety of proteins including tubulin.
2 Materials and methods 2.1 Animals Six-week-old female C3H/HeJ mice were purchased from Jackson Laboratory (Bar Harbor, ME). A two-month-old female NZW rabbit was obtained for immunizations.
2.2 Cells [I 106931 *' This study was supported by grants from the National Institute of
Health (HD-2x586 and AI-27803). During part of this study, S.K. was a National Institute of Child Health and Human Developmcnt fellow of the Pediatric Scientist Training Program (HD-22297).
Rat 3T3 cells were maintained in DMEM with 10 % FCS. 7: cruzi CL strain was a recent subclone ("subclone three") [25].Trypomastigotes and amastigotes were obtained from culture supernatants of infected rat 3T3 cells. Epimastigotes were maintained in liver infusion/tryptone medium with 10% FCS.
Correspondence: Stuart Kahn. Department of Pediatrics RD-20, Division of Immunology and Rheumatology, University of Washington, School of Medicine, Seattle, WA. 98195 USA
2.3 Antigens
Abbreviations: CIMS: Chronic infected mouse serum NMS: Normal mouse scrum GST Glutat hione S-transferase Anti1.2N antibodies: Antibodies from normal mouse serum affinitypurified with thc 1.2 peptide Anti-1.2C antibodies: Antibodies from T cvuzi chronic infected mouse serum affinity purified with the 1.2 peptide
Peptides were synthesized on 430A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA). Glutathione S-transferase (GST) and c1.l-GST fusion protein were purified from isopropylthiogalactoside (Sigma Chemical Co., St. Louis, M0)-induced bacterial lysates as described
0 VCH Verlagagesellschaft mbII, D-6940 Weinheim. 1992
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S . Kahn, M . Kahn and H. Eisen
1261. Tubulin was purified from bovine brain. Briefly, microtubule proteins were purified by cycles of assembly and disassembly 1271. Tubulin was purified from microtubules by phosphocellulose chromatography 1281. Polyglutamate, polyaspartate, and polylysine were purchased from Sigma Chemical Co.
96-well plates, incubated overnight, and blocked with 5 YO nonfat milk in PBS. Then various dilutions of mouse IgM, mouse IgG (Calbiochem, La Jolla, CA) (1000ng/ml to 0.5 ng/ml), and uninfected and chronically infected mouse sera were incubated for 1 h. Bound Ig was detected with goat anti-mouse IgM or IgG coupled to horseradish peroxidase (Zymed).
2.4 Antibodies Eight-week-old female C3H/HeJ mice were infected by intraperitoneal injection of lo5 tissue culture-derived trypomastigotes. Uninfected mice and chronic infected mice were sacrificed at 8 months of age, and sera was obtained. IgM and IgG concentrations of uninfected mice were determined to be 0.547 k 0.053 mg/ml and 2.273 k 0.208 mg/ml, respectively (data not shown). IgM and IgG concentrations of chronic infected mice were determined t o be 1.928 S 0.530 mglml and 11.950 f 0.778 mg/ml, respectively (data not shown). Antibodies were obtained by passing sera over a 1-ml column of peptide Sepharose 4B (Pharmacia Fine Chemicals, Piscataway. NJ). The columns were washed with 20 column volumes of PBS before antibodies were eluted with 3 M NHlSCN in PBS, and dialyzed with PBS. All peptides were coupled directly to CNBr-Sepharose 4B according to the manufacturer’s instructions. Antibodies purified with the 1.2 peptide (Table 1) from normal mouse serum (NMS) are 1.2N antibodies; and those from chronic infected mouse serum (CIMS) are anti-1.2C antibodies. Anti-1.2N antibodies (6 pg) were obtained per ml of uninfected sera. Anti-1.2C antibodies (25 pg) were obtained per ml of CIMS. Anti-cl. 1 antibodies were obtained by immunizing 2-month-old NZW rabbits with 100 pg of affinity-purified c l . 1-GST fusion protein [29]. After five boosts, anti-cl.1 antibodies were affinity purified by sequentially passing the hyperimmunized rabbit sera over a 2-ml column of GSTSepharose 4B column, and then over a cl.1-GST fusion protein column linked to Sepharose 4B, as previously described [29].
2.5 Enzyme-linked immunosorbent assay (ELISA) Antigens (tubulin or c1.l-GST) at a concentration of 5 pg/ml in PBS, were plated onto 96-well plates (Costar, Cambridge, MA) and incubated at room temperature overnight. Plates were washed with PBS containing 0.05 YO Tween (Sigma Chemical Co.),and “blocked” by incubation with 3% BSA in PBS for a minimum of 1 h. Antibodies (50 pl/well) were added and incubated for 1 h at room temperature. If competitions were performed, competitor was added to antibodies at stated concentrations, and preincubated for 30 min, before antibodies and competitor were added to wells. Plates were washed and goat antimouse IgM or IgG coupled to horseradish peroxidase (Zymed, San Francisco, CA) was added and incubated for 1 h. Antibody binding was detected by addition of ophenylenediamine (Zymed), and read on an automated ELISA reader (Bio-Tek Instruments, Burlington, VT). To determine the concentration of TgM and IgG, affinitypurified goat anti-mouse antibodies (IgA, IgG, and IgM; heavy and light chains) (Organon Teknika Corp., West Chester, PA) at a Concentration of 2 pg/ml were plated onto
2.6 Western blots SDS-PAGE was followed by semi-dry transfer of antigens to nitrocellulose membranes (Micron Separations Inc.), “blocked” with 3 % nonfat dry milk: and incubated with antibodies [30]. The blots were developed with alkaline phosphatase-conjugated goat anti-mouse antibodies (Promega, Madison, WI), followed by incubation with bromochloroindolyl phosphatehitro blue tetrazolium
POI. 2.7 Immunostaining
Rat 3T3 cells were grown on slides (Lab-Tek Chamber Slide, Nunc Inc., Naperville, IL). Cells were fixed by incubating in 100 mM MES, pH 6.8, 1mM MgC12, 1mM EGTA (MME) with 4 YO PEG 4000 at 37°C for 5 s, followed by incubation in MME with 4 YOPEG and 10 % Triton at 37°C for 30 s, followed by 0.1 YOglutaraldehyde, 2 % formaldehyde in MME at 37 “C for 15 min, followed by 0.5 Yo NaBHJ in PBS at room temperature for 5 min [31]. Live parasites were adhered to polylysine-coated slides, and then fixed in 4 YOparaformaldehyde, at 4 “C for 15 min [29]. Fixed samples were then preincubated with 3 YOBSA in PBS, incubated with antibodies, and then FITC-labeled goat F(ab‘)z anti-mouse IgG (Tago Inc., Burlingame, CA) ~91.
3 Results Previously, we characterized a family of surface antigens of 7: cruzi (the SASS-1 antigens) using a series of antibodies affinity purified with small peptides from CIMS [29, 321. These antibodies recognized mammalian-stage parasites, and not insect-stage parasites [29]. An example of this binding is shown in Fig. 1A and 1B using antibodies purified with peptide 1.1(Table 1,anti-1.1 antibodies) [29]. Surprisingly, antibodies to an acidic peptide (peptide 1.2, Table 1, anti-1.2C antibodies) derived from a SASS-1 antigen appeared to bind to the flagella of mammalianstage trypomastigotes and insect-stage epimastigotes (Fig. 1C and D). These 1.2C antibodies also appear to recognize the cytoskeleton of the 3T3 cells in which the trypomastigotes were grown (Fig. 1E). This cytoskeletal Table 1. Peptides used for inhibition of tubulin binding
Peptide 1.1
I .2 PTubuli n
Scquencc RETGDGGANGDAVSAYG QFDDDDDGGDDDDEEDSQ EEEEDEGEEAEEE
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Autoantibodies to negatively charged epitopes following ‘I: c r u i infection
and flagella binding suggested a cross-reactivity to tubulin, a protein found abundantly in these structurks. In Fig. 2, it can be seen that the 1.2Cantibodies do react with tubulin in both Western blot (Fig. 2A) and ELISA (Fig. 2B). Although tubulin does not contain sequences homologous to the 1.2 peptide, it does contain a run of acidic amino acids at the C terminus [33].The 0-tubulin acidic peptide was synthesized (Table l), and tested as a competitor of anti-1.2C binding to tubulin in an ELlSA (Fig. 3A). The binding of the anti-1.2C antibodies to tubulin was inhibited with the 1.2 peptide, and the (3-tubulin acidic peptide (Fig. 3A). The 1.1 peptide did not inhibit tubulin binding (Fig. 3A). Since the tubulin binding can be inhibited with two small acidic peptides which have different primary sequences
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(1.2, and the C terminal of fi-tubulin, Table 1),we tested whether peptides composed entirely of acidic amino acids could compete in an ELISA. Both polyaspartate and polyglutamate competed the binding of anti-1.2C antibodies to tubulin,whereas polylysine didnot (Fig. 3B). (We do not understand why polylysine augments the antibody binding.) This implies that the anti-1.2C antibodies bind to tubulin through ionic interactions to regions of acidic amino acids. Since ionic interactions were implicated in the recognition of anti-1.2C antibodies to tubulin. we examined the effect of increasing NaCl concentration on this interaction (Fig. 4). The binding was inhibited by 71 % at 300 mM NaCl, and by 85 % at 450 mM NaCl. As a comparison, we examined the effect of salt on a polyclonal affinity-purified
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Figure7 I . Indirect immunofluorescence shows anti-1.2C antibodies react with thc cytoskcleton. Parasites and rat 3T3 cells were stained with 10 pg/ml anti-1.2C or anti-1.1 antibodies and FITCF(ab’)? anti-mouse IgG. (A) 7: cruzi trypomastigotes with anti-1.1 antibodies. (B) 7: c m z i cpimastigotes with anti-1.1 antibodies. (C) 7: c m z i trypomastigotes with anti-1.2C antibodies. (D) 7: cruzi cpimastigotcs with anti-l.2C antibodies. (E) Rat 3T3 cells with anti-1.2C antibodies. (F) Rat 3T3 cells with anti-1.1 antibodies.
Figure 2. Anti-1.2C antibodies bind to tubulin in Western blot and ELISA. (A) Western blot on tubulin (4 &lane) reacted with anti-1.2C antibodies (10 pg/ml). Tubulin was electrophoresed on a 7.5 % SDS-PAGE, transferred to nitrocellulose, reacted with antibodies and bromochloroindolyl phosphate/nitro blue tetrazolium. (B) ELISA detects anti-1.2C and anti-1.2N antibodies binding t o tubulin. Antibodies were affinity purified from normal mouse serum (NMS) and CIMS with the 1.2 peptide (Table I), and their binding t o tubulin detected with peroxidase-conjugated anti-IgM or anti-IgG. Where error bars are not shown, the error was smaller then the symbols.
S. Kahn, M. Kahn and H. Eiscn
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rabbit antibody generated by a standard immunization protocol (anti-cl. 1 antibodies). The anti-cl.1 antibody was prepared by immunization with C1.1-GST fusion protein. The affinity-purified antibodies were reacted with c l . 1GST in ELISA at various salt concentrations (Fig. 4). The anti-cl. I antibodies were inhibited by only 6 % at 300 mM and 450 mM NaCl; and by < 50 % at 2.0 M NaC1, demonvtrating that the anti-1.2C antibody binding to tubulin was very 5ensitive to salt concentration.These results supported our hypotheqis that the cross-reactive anti-1.2C antibodies were binding to regions of acidic amino acids through ionic interactions.
infected C3H/He mouse sera is shown in Fig. 5. In the serum of uninfected animals, natural anti-tubulin antibodies are detected (Fig. 5 ) . There appears to be a similar amount of IgM antitubulin antibodies in the CIMS. However, in CIMS, there appears to be a large increase of IgG antitubulin antibodies, compared to the NMS anti-tubulin IgG antibodies (Fig. 5 ) .
To determine whether the natural anti-tubulin antibodies also recognized acidic epitopes, we affinity purified antibodies from uninfected mouse serum with the acidic peptide 1.2 (anti-1.2N antibodies). Antibodies were obtained and were found to bind tubulin in ELISA
Previous studies in (CS7BL/6 x BALB/c) F1 mice have demonqtrated the presence of natural anti-tubulin antibodies which appear to expand and mature following 7: cruzi infection 1241. ELISA on uninfected and chronically
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Figure 4. Anti-1.2C binding to tubulin is sensitive to NaC1. Antibody (10 &ml) was incubated in 50 mM phosphatc buffer with increasing concentrations of NaCl as indicated. Binding to tubulin was detected with peroxidase conjugated anti-IgG. The binding to tubulin i s expressed as the percent of maximal binding for each antibody. Mouse antibodies (anti-1.2C) bound maximal at 0 mM NaC1. Rabbit antibodies (anti-cl.1) bound maximal at 150 mM NaCI. (0)equals mouse antibodies; (D) equals rabbit antibodies. Where error bars are not shown, the error is smaller then the symbols.
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Figuw3. Thc binding of anti-1.2C antibodies to tubulin is inhibitcd with peptides rich in acidic amino acids. Anti-1.2C antibody (10 &mi) was incubated with various competitors. Binding to tubulin was detected with pcroxidase-conjugated anti-IgG. Thc binding is expressed as a percent of binding to tubulin in the absence o f competitor.Where error bars are not shown, the error is smallcr then the symbols. (A) Threefold increases in peptide competitor. expressed as mM final concentration of peptide, indicatcd as follows: ( 0 )equals 1.2 peptide; (0)equals b-tubulin pcptidc and (D) equals 1.1 peptide. (B) Threefold increases in peptide competitor. expressed as mglml final concentration of peptide. indicatcd a s follows ( 0 )equals polyglutamate; (0)equals polyaspartate and (D) equals polylysine.
0.0 -
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Figure5. Titration of anti-tubulin IgM and IgG antibodies in NMS, and in ?: cruzi chronic infected mouse sera. NMS and CIMS were diluted with PBS. Binding to tubulin was detected with peroxidase-conjugated anti-IgM or anti-IgG. (0) equals CIMS IgM; (D) equals NMS IgM; (W) equals CIMS IgG and (V)equals NMS 1gG.Where error bars are not shown, the error is smaller then the symbols.
Autoantibodies to negatively charged epitopes following 7: cruzi infection
Eur. J. Imniunol. 1002. 22: 3051-3056 110
100
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Figure 6. Anti-1.2N antibodies and anti-1.2C antibodies exhibit diffcrent sensitivitiesto pH. Antibody (10 pg/ml) was incubated in 50 mM glycine buffer with the pH adjusted as indicated. Binding to tubulin was detected with pcroxidase-conjugated IgG. Binding to tubulin is cxprcssed as percent of maximal binding. The maximal tubulin binding for this expcrimcnt was with anti-1.2C binding at pH 6.0. (D) equals anti-1.2C antibodies and (0)equals anti-1.2N antibodies.Where error bars are not shown. the error was smaller thcn thc symbols.
(Fig. 2B). There is increased binding of IgG antibodies from 1.2C antibodies from CIMS, as compared to the 1.2N IgC antibodies from NMS (Fig. 2B). These results. and the results of anti-tubulin antibody titers in CIMS and NMS (Fig. S), suggest that during the course of chronic 7: cruzi infection, anti-tubulin IgG antibodies which recognize regions of acidic amino acids are expanded. Both anti-1.2N and anti-1.2C antibodies bound peptides rich in acidic amino acids, suggesting that ionic interactions involving the antigen’s COO- groups of glutamate and aspartate were important. Therefore, we studied the effect of pH on these antibodies’ ability to bind tubulin. This was done by varying the pH of the primary antibody-binding otep during the tubulin ELISA. The affinity-purified antibodies from CIMS exhibited strong tubulin binding (> 80 94 of maximum) at physiological pH (Fig. 6). Maximal binding to tubulin with these antibodies was observed at pH 5 and pH 6, and the binding was greatly inhibited at pI-1 < 5 and > 9 (Fig. 6). I n contrast, the affinity-purified natural tubulin antibodies bound poorly at physiological pH (Fig. 6). Maximal binding was at pH 4 (Fig. 6). These results sugget that the natural tubulin antibodies will bind poorly to acidic antigens encountered when circulating in the mouse. However, in the chronic infected animal, the tubulin antibodies may potentially bind to a wide variety of autoantigens which contain runs of acidic amino acids.
4 Discussion In thi\ report, we describe autoantibodies that bind to ncgatively charged antigens rich in acidic amino acids, including the carboxyl terminal of 0-tubulin. These antibodie5 can he isolated from normal mice and mice chronically infected with 7: cruzi. The antibodies are sensitive to changes in salt concentrations and pH, suggesting that ionic interactions contribute to their antigen binding and poly-
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reactivity. The antibodies (IgG and IgM) from chronically infected animals display increased binding to tubulin (Fig. 2B), and bind it well at physiologic pH (Fig. 6), whereas the antibodies from normal mice bind poorly at physiologic pH (Fig. 6).
A previous report has compared natural antitubulin antibodies and antitubulin antibodies following 7: cruzi infection in mice [24]. Contrary to our experiments, the previous study found no evidence of antibodies that bind to the carboxy terminus of tubulin. Several differences between the studies suggest explanations. First. the earlier study examined the (CS7BL/6 X BALB/c) F1 as opposed to the C3H/He mice used in this study, and the genetic differences may explain these discrepant results. Second, in the present study, the antibodies which bind the carboxy terminus of tubulin were initially affinity purified by a peptide rich in acidic amino acids. Our data suggests that these antibodies are a minor subset of the total anti-tubulin antibodies found in normal and chronically infected animals (Figs. 2B and 5). In the previous study, total anti-tubulin antibodies were examined for the ability to bind to protease-digested fragments of tubulin in Western blot analysis [24]. This assay may not have been sensitive enough to detect a subset of the anti-tubulin antibodies that binds to a carboxyterminal fragment. The anti-1.2N antibodies have different characteristics from the anti-1.2C antibodies. First, the anti-1.2C antibodies (both IgM and IgG) display increased binding to tubulin then the anti-1.2N antibodies (Fig. 2B). Second, the anti1.2C IgG antibody binding to tubulin is increased compared to the anti-1.2N IgC antibody binding (Fig. 2B).Third, the effect of pH on the two different antibody classes is remarkably different (Fig. 6). Together, these results suggest that the interactions between the two antibody subsets and tubulin are different. We speculate that the ability to bind to tubulin is dependent upon interactions between the antibody and peptide acidic amino acids (aspartate pK, of 4.3 and glutamate pK1 of 3.9). The differences in optimal pH for binding anti-1.2N versus anti-1.2C to tubulin suggests that antibody residues such as histidine (pK, of 6.0) may be responsible for binding 1.2N antibodies, while the 1.2C antibodies use residues with higher pK, such as lysine (pK, of 10.5) and arginine (pK1 of 12.5). The mechanisms leading to the generation of the anti-1.2C antibodies are unclear. One poosibility is that the cells secreting the 1.2N antibodies in uninfected mice undergo class switching and/or somatic mutation during the course of 7: cruzc infection leading t o increased production and affinity of 1.2C antibodies. Another possibility is that during the chronic polyclonal proliferation following T cruziinfection, some rare, high-affinity, IgG-producing natural cross-reactive B cell clones are expanded; this may occur without class switching or somatic mutation. Evidence supporting such a possibility exists. First, Abo et al. [34] reported the appearance of “forbidden” T cell clones following antigenic stimulation with E. coli. Second, Shefner et al. [3S]reported the existence of a B cell clone that produces a rare, high-affinity, anti-double-stranded DNA antibody that is encoded by Vn genes containing no somatic mutations. Together these reports and our studies suggest that natural high-affinity anti-self I3 cells exist, and
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S. Kahn, M. Kahn and H. Eiscn
that during infections, difficult to detect anti-self clones can become more prominent. The role of negatively charged antigens in the development of the anti-1.2C antibodies, and in the development of autoantibodies in many human rheumatologic diseases is unclear [3].It is possible that during infection with T cruzi the immune system is chronically exposed to parasite and host intracellular antigens with negatively charged regions such as tubulin, which may lead to an increase in antibodies that bind to negatively charged antigens. Isolation and sequencing of B cell variable regions from chronically infected animals may enable us to define the mechanisms involved in the production of antibodies which recognize charged antigens [3]. We would like to thank R. Scaith and P Andreassen for providing purified tuhulin, C. Wilson, D. Ross, H . Ochs for critical review of the manuscript, and J. Gauthier for helpful discu.wions. Received April 29, 1992; in revised form August 3, 1992.
5 References 1 Peter, J. R . , Use and Interpretation of tests in Clinical Immunology, 8th Edit.. Speciality Laboratories, Inc., Santa Monica, 1YY2, p. 196. 2 Reichlin, M., in Kclley, W. N . , Harris, E. D., Ruddy, S. and Llcdge, C. B. (Eds.), Textbook of Rheumatology, W. B. Saunders Co., Philadelphia 1989, p. 214. 3 Brendel,V, Dohlman, J., Blaisdell, B. E. and Karlin, S., Proc. Natl. Acad. Sci. USA 1991. 88: 1536. 4 Klinman, D. and Steinberg, A. D., J. Exp. Med. 1987. 165: 1755. 5 Shlomchik, M. J., Aucoin, A. H., Pisetsky, D. S. and Weigert, M. G., Proc. Natl. Acad. Sci. USA 1987. 84: 9150. 6 Shlomchik, M., Mascelli, M., Shan, H., Radic, Z . , Pisetsky, D., Marshak-Rothstein. A . and Weigert, M., Exp. Med. 1990.171: 265. 7 van ES, J. H., Meyling, F. H . J. G., van de Akker, W. R. M., Aanstoot, H., Derksen. R. H. W. M. and Logtenberg, T., J. Exp. Med. 1991. 173: 461. 8 Guilbcrt, B., Dighicro, G . and Avrameas, S., J. Immunol. 1982. 128: 2779.
Eur. J. Immunol. 1992. 22: 3051-3056 9 Dighiero, G., Lymberi, P., Mazie, J. C., Rouyre, S., Browne, G. S.,Whalen, R. G. and Avrameas, S., J. Immunol. 1983.131: 3367. 10 Underwood, J. R., Pedersen, J. S., Chalmers, P. J. and Toh, B. H., Clin. Exp. Immunol. 1985. 60: 417. 11 Dighiero. G., Lymberi, P., Holmberg, D., Lundquist, I., Coutinho, A. and Avrameas, S., J. Immunol. 1985. 134: 765. 12 Avrameas, S., Immunol. Today 1991. 12: 154. 13 Petry, K. and Eisen, H., Parisitol. Today 1989. 5: 111. 14 Eisen, H . and Kahn, S., Curr. Opin. Immunol. 1991. 3: 507. 15 Said, G., Joskowicz, M., Barreira, A . A . and Eisen, H., Ann. of Neurol. 1985. 18: 676. 16 Hontebeyrie-Joskowicz, M., Said, G., Milon, G., Marchel, G. and Eisen, H., Eur. J. Immunol. 1987. 17: 1027. 17 D’Imperio Lima, M. R., Joskowicz, M., Coutinho, A., Kipnis, T. and Eisen, H., Eur. J. Immunol. 1985. 15: 201. 18 D’Imperio Lima, M. R., Eisen, H., Minoprio, P., Joskowicz, M. and Coutinho, A., J. Immunol. 1986. 137: 353. 19 Minoprio, P., Eisen, H., Joskowicz, M., Pereira, P. and Coutinho, A , , Immunol. 1987. 139: 545. 20 Wood, J. N., Hudson, L., Jessell, T. M. and Yamamoto, M., Nature 1982. 296: 34. 21 Towbin, H., Rosenfelder, G., Wieslander, J., Avila, J. L., Rojas, M., Szarfman, A., Esser, K., Nowack, H. and Timpl, R., J. Exp. Med. 1987. 166: 419. 22 VanVoorhis,W. C. and Eisen, H., J. Exp. Med. 1989.169: 641. 23 Van Voorhis, W. C., Schlekewy, L. and Trong, H. L., Natl. Acad. Sci. U S A 1991. 88: 5993. 24 Ternynck, T., Bleux, C., Gregoire, J., Avrameas, S. and Kanellopoulos-Langevin, C., .I. Immunol. 1990. 144: 1504. 25 Plata, F., Pons, F. G . andEisen, H., Eur. J. Immunol. 1984.14: 392. 26 Smith, D. B. and Johnson, K. S., Gene 1988. 67: 31. 27 Margolis, R., Rauch, C. T. and Job, D., Proc. Natl. Acad. Sci. USA 1986. 83: 639. 28 Williams, R. C. and Detrich, 111,H. W., Biochemistry 1979.18: 2499. 29 Kahn, S.,VanVoorhis,W. C. and Eisen, H., J. Exp. Med. 1990. 172: 589. 30 Harlow, E. and Lane, D., Antibodies, a laboratory manual, Cold Spring Harbor Laboratory, New York 1988, p. 471. 31 Osborn, M. and Weber, K., Methods Cell Biol. 1982.24: 2598. 32 Kahn, S., Colbert, T. G., Wallace, J. C., Hoagland, N. A. and Eisen, H., Proc. Natl. Acad. Sci. USA 1991. 88: 4481. 33 Cleveland, D. W. and Sullivan, K. F., Annu. Rev. Biochem. 1985. 54: 331. 34 Abo, T., Ohteki, T., Seki, S., Koyamada, N., Yoshikai, Y . , Masnda,T., Rikiishi, H. and Kumagai, K., J. Exp. Med. 1991. 174: 417. 35 Shefner, R., Kleiner, G., Turken, A., Papzian, L. and Diamond, B., J. Exp. Med. 1991. 173: 287.
