Specificity of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA

Specificity

of Anti-DNA Antibodies Induced Immunization with Bacterial

GARY S. GILKESON, A. JANE PRITCHARD,

in Normal DNA

Mice by

AND DAVID S. PISETSKY

Medical Reseurch Service. Durham VA Medical Cerlter. Dil*ision of Rheumatology und Immunoln~.v. Department of Medicine, Duke Unirjersity Medicul Center. Durham. North Carolinu 27710 To determine the specificity of anti-DNA antibodies induced in normal mice by immunization with bacterial DNA. sera from BALBic mice immunized with single-stranded DNA from Escherichiu coli (EC) were tested for binding to a panel of synthetic DNA and RNA homopolymers as well as duplexes. Results of these studies indicate that sera from EC DNA immunized mice preferentially bind certain DNA and RNA homopolymers as well as DNA duplexes. Furthermore, the specificity of the antibodies from immunized mice resembled those of sera from autoimmune MRL-lpr//pr mice in terms of the synthetic antigens recognized, although some differences were noted in the magnitude of the response to individual duplexes. These results suggest that anti-DNA antibodies induced by bacterial DNA bind to DNA structures dependent on both the base and the sugar phosphate moieties of the nucleic acid antigen and may resemble some anti-DNA antibodies expressed in spontaneous autoimmune disease in these binding properties. ci7 1991 Acadenuc Prcw. Inc.

INTRODUCTION

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE) and markers of diagnostic and prognostic significance (I). These antibodies, moreover, appear to play a direct role in the immunopathologic events of this disease, especially nephritis, since anti-DNA levels frequently parallel measures of clinical activity (2-4). Because of the central role of anti-DNA in the pathogenesis of SLE, the mechanisms underlying this response have been extensively investigated in patients as well as murine models of disease (5). These studies have demonstrated a variety of nonspecific immunoregulatory disturbances of both B and T celIs that could promote polyclonal B cell activation and autoantibody production. The role of these disturbances in contrast to antigenspecific stimulation of anti-DNA antibodies has not yet been elucidated, however. To assess the role of DNA antigen in the induction of autoantibodies, the response of animals to immunization with DNA has been analyzed (6, 7). These studies have demonstrated that, in general, DNA alone is poorly immunogenic. When complexed to a carrier such as methylated bovine serum albumin (mBSA). single-stranded (ss) DNA can induce a limited response. Double-stranded (ds) DNA, even when presented with a carrier in adjuvant, is essentially nonimmunogenic (7). Other DNAs, however, such as chemically modified DNA, glycosylated DNA, and some synthetic polynucleotides, can induce antibodies in normal animals, although these anti-DNAs bind specifically to the immunizing DNA but not mammalian DNA (7, 8). Together, these studies have led to the conclusion 288 0090-1229/91 $1.50 Copyngh! 0 1991 by Academic Pres. Inc. All righta of reproduction m any form reervrd

INDUCED

ANTI-DNA

ANTIBODIES

SPECIFICITY

289

that anti-DNA production in lupus does not result directly from DNA immunization and that this response is induced by some other mechanism, either stimulation by a cross-reactive non-DNA antigen or nonspecific B cell activation (5). Recently, our laboratory has presented evidence that some natural DNAs are, in fact, more immunogenic than previously recognized. We have shown that normal human sera contain high titers of antibodies to ssDNA from bacteria, including Micrococcus lysodeikticus and Staphylococcus epidermidis, and suggested that these antibodies arise during the course of infection (9). We further showed that normal mice immunized with a variety of bacterial DNAs produce antibodies to both ss and ds bacterial DNA forms ( 10, I 1). Some of these induced antibodies also cross-reacted with mammalian ssDNA but not dsDNA (10, 11). These studies have raised the possibility that bacterial DNA can serve as an immunogen in normal as well as aberrant immunity (12). The antigenic specificities of induced anti-DNA antibodies and their relationship to spontaneous autoantibodies have not been analyzed in detail, however. In the present study, we have, therefore, determined the tine specificity of anti-DNA antibodies induced in normal mice by DNA immunization in terms of their binding to synthetic DNA and RNA antigens. The pattern of polynucleotide reactivity has also been compared to the spontaneous anti-DNA response of MRL-lpr/lpr mice, which develop a lupus-like illness ( 13). Our studies indicate that immunization with ssDNA from Escherichia co/i (EC) induces antibodies that bind synthetic ss and ds polynucIeotides in a pattern suggesting recognition of determinants dependent on both the base and sugar phosphate moieties of DNA: some of the induced anti-DNA antibodies resemble spontaneous anti-DNA in their binding properties. MATERIAL

AND METHODS

Mice

Male BALB/c and MRL-lpr/lpr mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were 8 weeks old at the inception of all studies and were housed under standard conditions in the animal facility of the Durham VA Medical Center. Antigens

DNA from EC (purchased from Sigma Chemical Co., St. Louis, MO) was initially dissolved in SCC buffer (0.15 M NaCl, 0.1 M Na citrate) prior to extensive phenol and isoamyl alcohol/chloroform extraction. The DNA was then precipitated with ethanol and redissolved in SSC buffer. ssDNA was obtained by boiling for 10 min followed by rapid immersion in ice. The concentration of DNA was determined by OD260 readings with a LKB uv spectrometer and purity assessed by OD 260/280 ratios. The ssEC DNA preparation had a ratio greater than 1.9. DNA homopolymers used as antigens included poly(dA) (pdA), poly(dT) (pdT), poly(dC) (pdC), poly(dG) (pdG), poly(d1) (pdI), and poly(dU) (pdU). RNA homopoiymers used as antigens were poly(A) (PA), poly(C) (PC), poly(G) (pG), poly(1) (PI), and poly(U) (pU). In the ds polynucleotide assays, the following antigens were used: pdI-pdC, pdIdC-pdIdC, pdG-pdC, pdGme5 dC-pdGmeS dC.

290

<;II.KF.SON.

PRITCHARD.

AND

PlStlSK‘t

pdA-pdT. pdAdT-pdAdT. pdGdC-pdGdC, and pdAdC-pdGdT. All polynucleotides were products of Pharmacia (Piscataway. NJ). Polynucleotides were dihsolved in PBS with concentrations determined by OD260 measurements. The length of all polynucleotides was determined by agarose gel electrophoresis using standards of known length with visualization by ethidium bromide staining. All DNA and RNA homopolymers consisted of strands 20@-500 bases in length. Duplex polynucleotides ranged from 2000 (pdA-pdT) to 5000 (pdIdC-pdIdC) base pairs in length. Lack of ss regions in the ds polynucleotides was confirmed by determining their reactivity with two human sera highly specific for ssDNA. These sera at a I:200 dilution produced OD380 values by ELISA of ~0. I50 for all ds polynucleotides. compared to their reactivity to ssCT DNA. which yielded an OD380 value >1.500. Reactivity of these sera with DNA homopolymers was similar to that of the mouse sera with binding to pdC. pdG, pdi, and pdT, but with poor binding to pdA and pdU.

Fifteen BALB/c mice in two separate immunization groups (5 and 10 animals, respectively) were immunized intraperitoneally with 0.3 ml each of an emulsion containing 50 p,g of ssEC complexed with 75 pg of mBSA (Sigma Chemical Co., St. Louis, MO) in Freund’s complete adjuvant (Difco Co., Detroit, MI). The two groups received antigen complexes prepared separately at different times to ensure reproducibility of the response patterns observed. Control immunization groups received 75 pg of mBSA in Freund’s complete adjuvant. Booster immunizations at weeks 2 and 4 consisted of DNA and mBSA or mBSA alone emulsified in incomplete adjuvant. Mice were bled from the retroorbital sinus prior to immunization and at 2-week intervals until I week after the last immunization. Sera were also obtained from IO unimmunized MRL-lpr/lpr mice at 4-6 months of age. Monoclonal

Antibodies

Monoclonal anti-DNA antibodies were derived from MRL-@r&r mice using the NSI cell line as previously described (14). Positive wells were initially identified by ELJSA using ss calf thymus DNA as antigen. After cloning twice by limiting dilution, lines were expanded and culture fluid obtained as antibody source. Antibodies for these experiments were purified by ammonium sulfate fractionation and affinity chromatography using a Sepharose column of a rabbit anti-mouse IgG or IgM reagent. Subclass assignment was performed by immunodiffusion analysis. Monoclonal antibodies used in this study are denoted IC3 (IgMk), IE8 (IgMk), FlNl (IgGk), and SS12 (IgMk). Assays Anti-DNA antibodies were measured by ELISA as previously described (15). Briefly, 96-well polystyrene microtiter plates (Dynatech, Alexandria, VA) were coated with nucleic acid antigens at 5 I.&ml. ss antigens were incubated for 2 hr at 37°C while ds antigens were coated for 16 hr at 37°C. After incubation, plates were washed with PBS containing 0.05% Tween (PBS-T). Sera or purified mono-

INDUCED

ANTI-DNA

ANTIBODiES

SPECIFICITY

291

clonal antibodies were then added in serial dilutions in PBS-T and incubated for 45 min at room temperature. After washing, peroxidase conjugated goat antimouse IgG (y-chain specific, Sigma) at a l/500 dilution in PBS-T was added, followed by 3,3’,5,5’-tetramethylbenzidine (Organon Teknika, Durham, NC) in 0.1 M citrate with 0.015% H*O,. OD readings at 380 nm were obtained using a Titertek plate reader (Flow Laboratories, McLean, VA). Due to the limited amount of sera available for the duplex assays, variable numbers of sera ranging from 10 to 15 were tested for the different antigens. Inhibition assays were performed by incubating sera (at a dilution yielding an OD380 value of approximately 1 when uninhibited) with varying concentrations of ssEC DNA as well as ss and ds polynucleotides. After 45 min of incubation at room temperature, the sera were added to antigen coated plates and ELISAs performed as described above. Limitations in the amount of sera available prevented complete testing of all possible inhibitor/antigen combinations. Electrophoresis

and Chromatography

The ssEC DNA and mBSA preparations used in these immunizations were analyzed by SDS polyacrylamide gel electrophoresis and Western blotting. Briefly, ssEC DNA (50 ~1 of a 50-pg/ml solution) and mBSA (50 pl of a 12.5~pg/ml solution) were subjected to electrophoresis in 12% polyacrylamide gels in Trisglycine buffer. Gels were stained with Coomassie blue to reveal protein bands. A second gel of DNA or mBSA was electroeluted to nitrocellulose paper for Western blotting. The paper was cut into strips and incubated at a l/200 dilution with sera from BALBlc mice obtained either pre- or postimmunization. Strips were washed and incubated with peroxidase conjugated goat anti-mouse IgG ((H + L)-chain specific, Sigma) followed by HRP substrate (BioRad, Richmond, VA) in methanol. Separate l-ml pools of sera from MRL-lpr/lpr and ssEC DNA immunized BALB/c mice were passed over a 1.75 X 83 cm Sephadex G-200 column, with 3 ml fractions collected with spectrophometric monitoring at 280 nm. Three peaks were observed in both sera pools representing 19S 7S, and albumin fractions. Fractions were assayed for anti-DNA activity as described above. Statistics

All statistical

values were obtained by the Mann-Whitney

U test (two-tailed).

RESULTS

To determine the antigen specificity of antibodies induced by bacterial DNA immunization, set-a from mice immunized with ssEC DNA were tested first on a panel of synthetic DNA homopolymers. As shown in Fig. 1, immune sera bound several of the DNA homopolymers (e.g., pdC, pd1, pdG, and pdT) at levels significantly greater (P < 0.01) than those of control mice immunized with mBSA alone. These sera, however, failed to react appreciably with pdU or pdA. In using synthetic polynucleotides as probes for specificity, it is important to show that various polynucleotides are antigenic and that antibodies of different fine specificity can be distinguished by their binding patterns. To investigate the

392

GILKESON.

PRITCHARD.

,AND

YISE’I‘SK‘r

@A W PdG pdl NT W 1. DNA homopolymer binding of sera from immunized and lupus mice. Binding of sera from ssEC DNA immunized BALBk mice (n = 15, striped bars), MRL-lpr/lpr mice (n = 10, hatched bars), and mBSA immunized animals @I = 5. dotted bars) to DNA homopolymers as tested by ELISA. Values presented are the mean OD380 absorbance c SD of sera at a I : 100 dilution. * denotes values significantly different from control at P < 0.01. FIG.

antigenic properties of the panel of the polynucleotides, the reactivity of four MRL-Zpr/lpr monoclonal antibody antibodies was tested (Table 1). As these data indicate, all of the polynucleotides except poly(dG) were bound well by one or more antibodies and the antibodies differed in their relative binding to the various antigens. These results indicate that the failure of the immune sera to react to pdU or pdA, for example, did not reflect the poor adherence of these polynucleotides to the microtiter plate or lack of antigenically active determinants. Since the molecular weights of all the antigens used in these experiments were also similar (see under Materials and Methods), the contribution of antigen size to antigenicity was minimized.

._ _

TABLE 1 DNA HOMOPOLYMERBINDINGBYMONOCLONAL

~~~ _~ ~

~~

-

-

~-~ -

.

~

ANTI-DNAANTIBODIES

Antibody concentration

w&l) 1c3 IE8 FlNl ss12

50 12 3 50 12 3 SO 12 3 50 12 3

ssEC

pd.4

PdC

PdU

PdT

MI

pdG

1,452 I .097 0.960 1.407 1.208 0.926 >2.2 2.180 0.950 1.850 1.830 1.432

1.716 I .709 1.475 0.097 0.045 0.026

0.193 0.080 0.056 1.153 0.716 0.365
0.256 0.010 10.010 0.901 0.873 0.485 0.808 0.048 0.025 1.961 1.521 1.304

1.417 1.237 0.960 1.409 I.305 1.163 0.418 0.033 0.010 1.736 1.197 0.927

0.202 0.161 0.157 0.278 0.222 0.153
Nore. Monoclonal anti-DNA antibodies were tested by ELISA using the panel of DNA homopolymers. Results are expressed at the OD380 value.

INDUCED

ANTI-DNA

ANTIBODIES

SPECIFICITY

293

Another factor which could influence the interpretation of the binding patterns to DNA antigens is the presence of immune complexes. Such complexes could arise by induction of antibodies to a DNA binding protein in the DNA used for immunization and persistence of soluble complexes in the sera. Depending on the specificity of the DNA binding protein, selective binding to synthetic antigens could be observed. The influence of immune complexes in assays for other nuclear antigens has been described (16, 17). To investigate this possibility in this system, the purity of the EC DNA preparation used for immunization was first evaluated. By Coomassie blue staining, no protein bands were visible when the DNA was analyzed under denaturing conditions by SDS-PAGE (data not shown). Western blotting was therefore used to detect any binding of immune sera to antigenic material in the DNA preparation. As shown in Fig. 2, neither preimmune sera nor hyperimmune sera showed any detectable binding to proteins on blots transferred after electrophoresis of EC DNA. In contrast, the sera bound readily to the mBSA carrier. These results indicate that the DNA preparation was at most minimally contaminated and that antibodies could not be detected to immunogenic or antigenic proteins in the EC DNA preparation. Further evidence that these assays measured IgG anti-DNA was obtained by fractionation of sera by Sephadex G-200 chromatography. When assayed by ELISA, the IgG fraction from both BALB/c immune sera as well as MRL-lpr/lpr sera contained the preponderance of the anti-DNA activity, although some activ-

FIG. 2. Western blot analysis of reactivity of sera from immunized mice. In Panel A, mBSA was subjected to SDS-PAGE before transfer to nitrocellulose. Strips were then incubated with sera at a 1:2BOdilution. Lane 1, serum from a control BALB/c mouse; lanes 2 and 4, sera from BALB/c mice preimmunization; lanes 3 and 5, sera from BALB/c mice postimmunization (Week 7) with ssEC DNA/mBSA complexes in adjuvant. In Panel B, ssEC DNA was similarly electrophoresed and transferred to nitrocellulose. Strips were incubated with the same sera as in panel A.

ity could be demonstrated in the fraction corresponding to IgM (data not shown). If the DNA binding resulted from immune complexes, then the activity would have been expected to occur at a higher molecular weight than that of the IgG fraction. Together, these experiments indicate that binding measured is directly attributable to 1gG anti-DNA antibodies rather than immune complexes with antibodies directed to a bacterial protein. The presence of some immune complexes containing DNA in the sera cannot be excluded, however. All differences in antigen reactivity observed when sera were tested at a I: 100 dilution were confirmed by titration analysis as shown in Fig. 3, which presents representative data obtained using serum obtained from a single ssEC DNA immunized BALB/c mouse. Furthermore. the DNA homopolymer binding of the two separate immunization groups was very similar, suggesting that the binding patterns observed accurately reflected the specificity of antibodies induced to EC DNA and were not the result of a unique set of DNA-mBSA complexes. The reactivity of sera from MRL-lpr/lpr mice was next determined to compare the DNA homopolymer binding of induced and spontaneous anti-DNA antibodies. As shown in Fig. 1. sera from MRL-lpr/lpr mice bound pdC, pdG, pdT, and pdl. but not pdU or pdA. Although some differences among individual mice in the magnitude of the response to different homopolymers were noted, the overall pattern of reactivity of the induced and spontaneous responses was similar. The same comparative assays were next performed using RNA homopolymers as antigens. These data are presented in Fig. 4 and demonstrate that the binding of RNA homopolymers was also selective. Thus, sera from mice immunized with ssEC DNA bound to pA and pl, but not PC. pG. or pU. Similar to sera of immunized mice, MRL-fpr/lpr sera bound only to pA and pl. The antigenicity of the RNA homopolymers was also assessed by binding of the monoclonal antiDNA antibodies (Table 2). Although sera did not react with pU, antibody SS12 bound strongly to this antigen; this result indicates that pU was active in this assay and that the failure of binding by sera reflected the absence of antibodies of certain specificities. SS12 also demonstrated binding to pC and to pG. although at lower levels, while none of the other monoclonals bound these antigens weakly, if at all.

o-

p=Yy l/100

11400

, l/1600

-pdG

@A pdc

-

pdl pdT pdu

.

, 116400

*

FIG. 3. Titration analysis of DNA homopolymer binding. Dilutions of a serum from a ssEC DNA immunized mouse were tested for binding to various DNA homopolymers. Values presented are the OD380 ELISA values.

INDUCED

ANTI-DNA

PA

ANTIBODIES

PC

F

295

SPECIFICITY

PI

dJ

4. RNA homopolymer binding of sera of immunized and lupus mice. Reactivity of sera from ssEC DNA BALB/c immunized (n = 15), MRL-/pr/lpr (n = IO), and mBSA immunized BALB/c mice (n = 5) to RNA homopolymers by ELISA. Data are presented as in Fig. 1. FIG.

Because of evidence that sera from mice immunized with ssEC DNA bind to dsEC DNA (I 11, we tested sera from DNA immunized mice on a panel of synthetic DNA duplexes. As shown in Fig. 5, sera from ssEC DNA immunized mice bound these ds antigens significantly better than control immunized animals. Sera from mBSA immunized animals bound only pdAdC-pdGdT while sera from unimmunized BALB/c mice did not bind any of the ds antigens (data not shown). The reactivity of these sera did not appear to be directed to ss regions in these duplexes, since human sera specific for ssDNA did not show reactivity in these assays. The specificity of these assays for dsDNA determinants was further confirmed using as an antigen pdIdC-pdIdC digested with S, nuclease to remove possible ss regions. This treatment had no effect on the binding of sera from DNA immunized mice (data not shown). Although minor differences in binding to TABLE RNA

HOMOP~LYMER

Antibody concentration ( kg/ml ) lC3 lE8 FIN1

ANTIBODIES

PC

PU

Pl


0.643

0.101

0.215

0.092 0.093

0.027

3


0.010

50 12 3

Note. Monoclonal Table I.

ANTI-DNA

1.599 1.422 1.279 0.065

50 12

anti-DNA

PA

2

OF MONOCLONAL

50 12 3 50 12

3

ss12

BINDING

0.032

0.140

0.350

0.010
0.160

0.046

0.994

0.018 0.010
antibodies were tested by ELISA

0.122 1.350

1.118 1.095 0.073

PG

0.112 0.084 0.020 0.020



0.362

0.042

for RNA homopolymers

0.387

0.071 as in

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p(dldCl’p(dIdC)

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; Ta

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d 6

pidAdCJ’o(dGdTi

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20:

PISE’ISKI

r(dAdTl.p(dAdT)

b(dGdC)‘p(dGdC)

)(dGmdC)*p(dGmdCl

20 .

t

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1

i

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MRL

ctr

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FIG. 5. Duplex binding of sera from immunized and lupus mice. ds polynucleotide binding of individual sera from ssEC DNA BALBic immunized (n = 10-15). MRL-lpr/lpr tn = IO), and mBSA immunized BALBic mice (n = 51 by ELISA. Each point represents the OD380 absorbance of an individual serum at a I:400 dilution with the bar at the mean value 2 SE. Significant differences (P < 0.01) were noted between sera from immunized and MRL-/pr/lpr mice in binding to pdI-pdC, pdGpdC. pdGdC-pdCdG, and pdIdC-pdIdC.

pdGdC-pdGdC, pdI-pdC. and pdAdT-pdAdT were noted between the two immunization groups, none exceeded a two-dilution difference. Sera from MRL-lpr/lpr mice were tested for their reactivity to the ds polynucleotides to compare the binding patterns of the induced and spontaneous antiDNA responses. The MRL-lpritpr sera differed significantly (P < 0.01) from the induced sera in their higher reactivity to pdI-pdC and lower reactivity to pdGdCpdGdC, pdIdC-pdIdC, and pdG-pdC; binding to the other four antigens was similar. Sera of individual MRL-lpr/lpr mice, however, varied in their reactivity with different duplex antigens, and some, like the immunized mice, displayed a higher response to pdGdC-pdGdC and pdIdC-pdIdC than to pdI-pdC. In competitive liquid phase assays, none of the ss or ds polynucleotides tested at 50 kg/ml alone effectively inhibited the binding of sera from either immunized or MRL-lpr/lpr mice to ssEC DNA, whereas ssEC DNA effectively inhibited the

INDUCED

ANTI-DNA

ANTIBODIES

SPECIFICITY

297

binding of immunized and MRL/lpr/lpr sera to all polynucleotides tested (90% inhibition at 50 l&ml). In addition, among the group of polynucleotides analyzed (pd1, p1, pdIdC-pdIdC, pdAdT-pdAdT, pA, pdA), none inhibited binding of either set of sera to another polynucleotide (i.e., pdA did not inhibit binding of sera to pA; data not shown). DISCUSSION

The studies presented herein demonstrate that anti-DNA antibodies induced in normal mice by DNA immunization bind selectively to synthetic DNA and RNA homopolymers and, in addition, react with synthetic DNA duplexes. The reactivity pattern of the immune sera was similar to those of MRL-lpr/fpr mice and suggests that, like spontaneous anti-DNA antibodies, induced anti-DNA antibodies appear to recognize antigenic determinants dependent on both the base and the sugar phosphate moieties rather than a simple backbone structure or charge array. Selective recognition of polynucleotides has also been demonstrated in sera of NZB/NZW mice as well as in patients with SLE (18. 19). This pattern of reactivity is likely to result from a recognition of variations in polynucleotide structure dependent on both the base and the sugar phosphate moieties. These variations could explain, for example, the observed differences in the antibody binding to pdA and pA. These homopolymers differ in the nature of their base stacking and furanose ring puckering and have been shown in previous studies to differ antigenically (20,21). Differences in secondary structure may also account for the enhanced reactivity of certain DNA homopolymers compared to their RNA counterparts. These differences make it unlikely that sera are directed simply to the bases themselves, although there is evidence that some anti-DNA antibodies can recognize nucleotides (22). In addition to binding homopolymers, immune sera bound synthetic duplexes in a pattern suggesting the recognition of unique configurations or conformations that can be displayed by nucleic acids differing in base composition (18, 23-25). The high reactivity of immune sera with duplexes containing dG and dC is noteworthy, suggesting possible immune stimulation by characteristic sequence arrays within the immunizing DNA. It is well documented that bacterial DNAs contain long stretches enriched in dG and dC residues, although shorter segments have also been found in eukaryotic DNA (26). Because of their rarity in mammalian DNA, these sequences could be immunogenic in mice, capable of inducing antibodies cross-reactive with polymers containing these bases. Other sequences in bacterial DNA with unique conformations resembling those of ds polynucleotides would similarly be expected to be immunogenic. The induction of structure-specific antibodies by DNA immunization is substantiated by experiments using ds polynucleotides as immunogens in normal mice (7, 27, 28). In view of the poor immunogenicity of most dsDNA antigens it is of interest that immunization with ssDNA elicited antibodies to dsDNA structures. These antibodies may be stimulated by ds regions within ssDNA used for immunization; preparations of ssDNA have been shown to contain ds regions even after extensive boiling (29). Studies from our laboratory indicate that dsDNA from E. coli is immunogenic, providing plausibility for this explanation (11). Alternatively, ss-

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DNA immunization may induce antibodies that bind both ss and ds antigens (30). Such cross-reactive antibodies are known to be present in the spontaneous antiDNA response (3 I ). Although induced and spontaneous anti-DNA antibodies showed some similarity by direct binding to polynucleotides, we cannot determine the proportion of the total anti-DNA response of either group with these putative common specificities. Inhibition experiments suggest that antibodies binding to individual polynucleotides represent only a component of the total response. It is likely, therefore, that this polynucleotide panel detects only some of the anti-DNA antibodies present in the immune and lupus sera, although their quantitative contribution to the overall anti-DNA response is unknown at this time. In these determinations, we have assumed that the binding to the synthetic polynucleotides represents a direct interaction with anti-DNA antibodies and that the pattern of polynucleotide binding is a reflection of their line specificity. An alternative interpretation of these data is that the binding measured results not from antibodies to DNA but rather from immune complexes containing a DNA binding protein (16, 17). This mechanism would imply that immunization with bacterial DNA induced antibodies to a contaminating DNA binding protein. This protein would have to display the same DNA specificity as either anti-DNA antibodies or similarly generated complexes with a DNA-binding protein from MRL-lpr/lpr sera. Several lines of evidence exclude this possibility. Thus we failed to find a protein contaminant in our DNA by protein or immunoblot analysis. In addition, we showed that the DNA binding activity in the immune BALB/c sera fractionated on Sephadex G-200 chromatography at a position of monomeric IgG. If immune complexes were the source of the anti-DNA activity, fractionation at a higher molecular weight would have been anticipated. Finally, in preliminary experiments, we have generated a panel of monoclonal anti-DNA antibodies from BALB/c mice immunized with ssEC DNA (Gilkeson. preliminary results). The anti-DNA activity of these antibodies is evident despite the lack of exposure to any putative DNA binding protein in the bacterial DNA. Together, these results indicate that the anti-DNA activity measured in immune sera results from induced anti-DNA antibodies whose specificity resembles that of spontaneous anti-DNA antibodies as tested by these assays. Although our findings indicate some similarities in the specificities of induced and spontaneous anti-DNA antibodies, we cannot conclude that they arise from a common mechanism, specifically, that the spontaneous anti-DNA response is stimulated by DNA. It is conceivable that the antibodies detected by the polynucleotide binding result from polyclonal B cell activation in the lupus mice, although they may be specifically induced in the immunized mice. Recent evidence suggests that the anti-DNA response in SLE represents a spectrum of antibodies induced by both polyclonal activation and antigen-specific stimulation (32). While they contain antibodies which bind many DNA and RNA antigens, the sera of DNA immunized mice, nevertheless, lack antibodies directed to mammalian dsDNA (I 1). This specificity is the serological hallmark of SLE and we, as well as others. have been unable to induce its production directly by immunization in mice (6, 7, 1 I), although recent evidence suggests that rabbits immunized with

INDUCED

ANTI-DNA

ANTIBODIES

SPECIFICITY

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