The Binding Mechanisms of Antibodies to DNA from Healthy Subjects and Patients with Systemic Lupus Erythematosus: The Role of Monogamous Bivalency ...
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The Binding Mechanisms of Antibodies to DNA from Healthy
Subjects and Patients with Systemic Lupus Erythematosus:
The Role of Monogamous Bivalency and Fc Dependence
Morgan E. Belina, Diane M. Spencer and David S. Pisetsky
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ImmunoHorizons 2021, 5 (10) 792-801
doi: https://doi.org/10.4049/immunohorizons.2100077
http://www.immunohorizons.org/content/5/10/792
This information is current as of June 30, 2022.
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ISSN 2573-7732.RESEARCH ARTICLE
Clinical and Translational Immunology
The Binding Mechanisms of Antibodies to DNA from Healthy
Subjects and Patients with Systemic Lupus Erythematosus:
The Role of Monogamous Bivalency and Fc Dependence
Morgan E. Belina,* Diane M. Spencer,† and David S. Pisetsky†,‡
*Duke University School of Medicine, Duke University Medical Center, Durham, NC; †Division of Rheumatology and Immunology, Duke University Medical
Downloaded from http://www.immunohorizons.org/ by guest on June 30, 2022
Center, Durham, NC; and ‡Medical Research Service, Durham Veterans Administration Medical Center, Durham, NC
ABSTRACT
Abs to DNA (anti-DNA) are a unique population of Abs that bind structural determinants on the DNA molecule. In systemic lupus
erythematosus (SLE), anti-DNA Abs bind to conserved antigenic determinants, with the phosphodiester backbone being the most
likely. In contrast, otherwise healthy subjects (HS) express anti-DNA that bind selectively to nonconserved sites on certain bacterial
and viral DNA. As shown previously, SLE anti-DNA bind by a mechanism termed Fc-dependent monogamous bivalency. In this
mechanism, both Fab sites interact with determinants on the same extended DNA molecule, reflecting the low affinity of each Fab site;
the requirement for the Fc region suggests some contribution of the C region to increase avidity. In this study, we investigated
whether anti-DNA from HS also bind to bacterial DNA by Fc-dependent monogamous bivalency. For this purpose, we compared the
activity of intact IgG with Fab and F(ab0 )2 fragments prepared from the plasmas of SLE patients and HS using ELISAs with DNA from calf
thymus or Micrococcus luteus. These studies showed that Fab fragments from all plasmas tested, both SLE and HS, failed to bind
significantly to DNA compared with intact IgG. By contrast, some, but not all, F(ab0 )2 preparations from both SLE patients and HS
showed binding to M. luteus DNA; F(ab0 )2 fragments from SLE plasmas, however, did not bind significantly to calf thymus DNA.
Together, these findings suggest that although anti-DNA Abs, whether from SLE or HS, bind by monogamous bivalency, binding to
bacterial DNA does not require the Fc region. ImmunoHorizons, 2021, 5: 792–801.
INTRODUCTION widely shared or conserved antigenic determinants, with the
phosphodiester backbone being the most likely (9, 10).
Abs to DNA (anti-DNA) are a unique population of Abs that The role of anti-DNA in SLE diagnosis and classification
bind structural determinants on the DNA molecule (1, 2). These has suggested that the production of IgG anti-DNA is exclu-
Abs were originally defined in the context of systemic lupus sive to the autoimmune state. However, data assembled over
erythematosus (SLE), a prototypic autoimmune disease charac- the last decades have clearly demonstrated the presence of
terized by the production of autoantibodies to nuclear macro- IgG Abs in the blood of otherwise healthy subjects (HS) to
molecules (36). The serological hallmark of SLE, anti-DNA DNA from certain bacteria and viruses (2, 1114); these IgG
are important biomarkers for disease diagnosis, classification, Abs differ from low-affinity IgM, so-called natural autoanti-
and activity (7). Furthermore, anti-DNA are important media- bodies that can bind DNA (15). Levels of Abs to bacterial
tors of disease manifestations via the formation of immune DNA in the blood of HS can be comparable to those of auto-
complexes (8). The ability of anti-DNA in SLE to bind DNA antibodies to DNA in patients with SLE, indicating a robust
independent of species origin has suggested recognition of immune response (11).
Received for publication August 12, 2021. Accepted for publication August 19, 2021.
Address correspondence and reprint requests to: Dr. David S. Pisetsky, Division of Rheumatology and Immunology, Duke University Medical Center, Durham
Veterans Administration Medical Center, 508 Fulton Street, 151G, Durham, NC 27705. E-mail address: david.pisetsky@duke.edu
D.S.P. is supported by a Veterans Administration Merit Review grant as well as a National Institutes of Health grant (1R01AR073935).
Abbreviations used in this article: anti-DNA, Ab to DNA; cfDNA, cell-free DNA; CT, calf thymus; HS, healthy subject; SLE, systemic lupus erythematosus.
This article is distributed under the terms of the CC BY 4.0 Unported license.
Copyright © 2021 The Authors
792 https://doi.org/10.4049/immunohorizons.2100077
ImmunoHorizons is published by The American Association of Immunologists, Inc.ImmunoHorizons BINDING MECHANISMS OF Abs TO DNA 793
Although the origin of anti-DNA in general has not been SLE plasmas are able to bind M. luteus DNA. These findings
delineated, Abs to foreign DNA likely arise in the context of thus extend the role of monogamous bivalency to HS Abs to
infection or colonization. As now recognized, the microbiome bacterial DNA while indicating some capacity for Fc indepen-
can exert powerful effects on the immune system in both HS dence in the IgG Ab response to bacterial DNA in both HS
and patients with SLE; DNA from biofilms in particular has and patients with SLE.
been shown to be immunogenic (1618). Furthermore, studies
on circulating cell-free DNA (cfDNA) indicate that cfDNA from
MATERIALS AND METHODS
bacteria and fungi, among other species, is present in the blood
of infected individuals (1921). Together, these findings suggest
Source of reagents
that nucleic acids can interact with the immune system at sites
All chemicals were purchased from Sigma-Aldrich (St. Louis,
distinct from any localized sources of infection such as a pneu-
MO), unless otherwise noted. Non-SLE plasmas were obtained
monia or abscess. The production of Abs to foreign DNA may
from Innovative Research (Novi, MI); SLE plasmas were obtained
be one consequence of such interactions.
from Plasma Services Group (Huntingdon Valley, PA). EBV Ag
As our studies have demonstrated, the anti-DNA in HS,
was from Meridian Life Science (Memphis, TN). PBS (Ca21 and
unlike autoantibodies to DNA in SLE, are highly selective in
Mg21 free) and saline sodium citrate solution were purchased
their pattern of Ag interaction and target nonconserved sites
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from Invitrogen, Thermo Fisher Scientific (Waltham, MA).
distinct from the phosphodiester backbone (22). Anti-DNA in
HS also differ from SLE anti-DNA in their pattern of H and L
chain expression (23). In recent studies, we explored the Isolation of IgG
nature of autoantibody-DNA interactions in SLE, delineating Purification of IgG from plasmas as well as a pooled, technical
the role of monogamous bivalency in anti-DNA binding (24). grade human IgG preparation was accomplished using 0.2 ml
In monogamous bivalency, both Fab regions of an IgG mole- NAb Protein A/G Spin Columns (Thermo Fisher Scientific/
cule must contact sites along the same extended polynucleo- Pierce Biotechnology, Rockford, IL) following the suppliers
tide chain for stable interaction; the binding of each Fab alone protocol. Binding buffer used was 0.01 M sodium phosphate
is too weak to allow univalent binding (2527). This mode of and 0.15 M sodium chloride (pH 7.2); elution buffer used was
binding requires a piece of DNA with a minimum size of 0.1 M glycine (pH 2.5). Eluted fractions of IgG were neutralized
3540 bp, which is sufficiently long to span the 140-Å dis- by addition of 1/10 volume of neutralization buffer (1 M Tris
tance between the two Fab sites on an intact IgG molecule HCl [pH 8.9]). Concentrations of IgG were determined from
(26, 28). measurements of A280 with a Thermo Scientific NanoDrop
As expected, we showed that Fab fragments of IgG purified 1000 spectrophotometer, read in undiluted form; blanking solu-
from the blood of patients with SLE failed to bind DNA in an tion was chosen to be as similar as possible to the solvent of
ELISA using calf thymus (CT) DNA as the source of mamma- IgG samples. IgG preparations were stored at 4 C until use.
lian dsDNA (24). Our work produced an additional, surprising
result: we found that F(ab0 )2 fragments of SLE IgG failed to Generation of Fab fragments
bind DNA, even though such fragments should be capable of a Monovalent Fab fragments were prepared from purified IgG by
monogamous bivalent interaction. Previous studies have dem- papain digestion using the Pierce Fab Micro Preparation Kit
onstrated the importance of the Fc region to the binding of (Thermo Fisher Scientific/Pierce Biotechnology) according to
anti-DNA and other Ags, although such studies involved molec- the suppliers protocol as follows. IgG was diluted to 2 mg/ml
ular constructs with the same variable regions joined to differ- and buffer exchanged to digestion buffer (supplied proprietary
ent IgG H chains (2931). In our studies, by contrast, we used buffer with cysteine added to 20 mM) with desalting columns.
F(ab0 )2 fragments lacking an Fc region. To describe the lack of IgG was then incubated with immobilized papain at 37 C for
both Fab fragment and F(ab0 )2 fragment reactivity to native 5 h with end-over-end rotation. The digest was removed from
DNA in SLE, we termed this pattern of anti-DNA binding as the immobilized papain by centrifugation; Fab fragments were
Fc-dependent monogamous bivalency (24). isolated using a protein A spin column. Fc fragments and undi-
In this study, we further investigated the binding of HS gested IgG were eluted from the protein A spin column with
anti-DNA to bacterial DNA. Specifically, we produced Fab and elution buffer (supplied proprietary buffer [pH 2.8], containing
F(ab0 )2 fragments of IgG from the plasmas of HS to test primary amine) and neutralized by addition of 1/10 volume of
whether HS anti-DNA display monogamous bivalency and, if neutralization buffer (1 M Tris HCl [pH 8.9]). To assess the
so, whether such bivalency is Fc dependent. In addition, we completeness of the digestion, Fab fragments, and undigested
compared the binding activities of HS anti-DNA and SLE IgG and Fc fragments were evaluated via nonreducing SDS-
anti-DNA with DNA from Micrococcus luteus, previously PAGE using NuPAGE 412% Bis Tris gels (Invitrogen, Thermo
known as M. lysodeikticus. In the results presented in this Fisher Scientific). Samples were mixed with LDS sample buffer
study, we show that Fab fragments of HS IgG, like those from (Invitrogen, Thermo Fisher Scientific) and heated for 10 min at
SLE IgG, fail to bind to M. luteus DNA. In contrast, we dem- 70 C before loading. Fab fragments were quantitated and
onstrate that F(ab0 )2 fragments from some, but not all, HS and stored using the methods outlined above.
https://doi.org/10.4049/immunohorizons.2100077794 BINDING MECHANISMS OF Abs TO DNA ImmunoHorizons
Generation of F(ab0 )2 fragments for 1 h except when otherwise noted. Secondary Ab used was
F(ab0 )2 fragments were prepared from purified IgG by pepsin anti-human IgG (Fab specific)peroxidase diluted 1/300 in 50
digestion using the Pierce F(ab0 )2 Micro Preparation Kit mM Tris (pH 7.4) containing 0.1% BSA and 0.05% Tween 20
(Thermo Fisher Scientific/Pierce Biotechnology) according to (ELISA Dilution Buffer).
the suppliers protocol as follows. IgG was diluted to 2 mg/ml Wells of plates were coated with 5 mg/ml M. luteus or CT
and buffer exchanged to digestion buffer (20 mM sodium ace- DNA diluted in 1 SSC buffer, 1 mg/ml tetanus toxoid diluted
tate [pH 4.4] and 0.05% sodium azide) with desalting columns. in ELISA coating buffer (0.1 M sodium phosphate buffer [pH
IgG was then incubated with immobilized pepsin at 37 C for 9]), or 6.6 mg/ml EBV Ag diluted in 1 PBS and incubated over-
2 h with end-over-end rotation. The digest was removed from night at 4 C. Plates were then washed and blocked with 200
the immobilized pepsin by centrifugation; F(ab0 )2 fragments ml/well PBS containing 2% BSA and 0.05% Tween 20 (Block
were isolated using a protein A spin column. Fc fragments and Buffer II) for 2 h. Plates were washed, and wells were incu-
undigested IgG were eluted from the protein A spin column bated with serial 2-fold dilutions of IgG, Fab, or F(ab0 )2 frag-
with elution buffer (supplied proprietary buffer [pH 2.8], con- ments in ELISA Dilution Buffer. Plates were then washed, and
taining primary amine) and neutralized by addition of 1/10 vol- wells were incubated with secondary Ab. Finally, plates were
ume of neutralization buffer (1 M Tris HCl [pH 8.9]). To assess washed and incubated with HRP substrate (0.015% 30 30 ,5,50 -tet-
digestion completion, fractions containing pepsin digest, F(ab0 )2
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ramethylbenzidine dihydrochloride, 0.01% H2O2 in 0.1 M cit-
fragments, and undigested IgG and Fc fragments were evalu- rate buffer [pH 4]) for 30 min. Color development was halted
ated via nonreducing SDS-PAGE. Samples were mixed with with addition of 2 M H2SO4, and the absorbance of wells at
LDS sample buffer (Invitrogen, Thermo Fisher Scientific) and 450 nm was measured with a UVmax spectrophotometer
heated for 10 min at 70 C before loading. F(ab0 )2 fragments (Molecular Devices, San Jose, CA).
were quantitated and stored using the methods outlined above.
RESULTS
Calculation of concentrations of IgG, Fab, and F(ab0 )2 for
use in ELISAs The binding of IgG, Fab, and F(ab0 )2 fragments from HS
As the purpose of these experiments was to assess the relative Previous studies have indicated that SLE anti-DNA bind to DNA
binding activity of intact IgG, Fab fragments, and F(ab0 )2 frag- by a mechanism termed monogamous bivalency, in which both
ments, the initial concentration of each reagent was calculated Fab sites must contact antigenic determinants on the same poly-
to provide equivalent numbers of Ag-binding sites. The molecu- nucleotide chain (24, 26). In other studies, we showed that
lar mass of an intact IgG molecule was assumed to be 160 kDa F(ab0 )2 fragments of SLE IgG, like Fab fragments, failed to bind
and the molecular mass of one Fab fragment to be 50 kDa. The mammalian DNA even though these fragments should be capa-
weight of a preparation of Fab fragments containing the same ble of monogamous bivalency. We designated this mode of bind-
number of Ag-binding sites as an intact IgG molecule (2) was ing as Fc-dependent monogamous bivalency (24). We thus
calculated as two Fab fragments 50 kDa, hence 100 kDa. wished to determine whether HS anti-DNA also bind by this
Therefore, for example, the binding activity of 20 mg/ml intact mechanism in their interaction with bacterial DNA.
IgG was compared with the binding activity of 12.5 mg/ml Fab In these experiments, we determined the relative binding
fragment preparation (calculated as 20 mg/ml 100 kDa/160 activities of IgG, Fab, and F(ab0 )2 preparations isolated from
kDa) in the experiments of this study. the plasmas of otherwise healthy individuals (HS); we also
Each F(ab0 )2 fragment theoretically contains the same num- tested a pooled IgG preparation that was commercially pur-
ber of Ag-binding sites as an intact IgG molecule. Preparations of chased. Figs. 1 and 2 present these results, with Fig. 1 showing
F(ab0 )2 contained not only F(ab0 )2 fragments but also Fc frag- two HS samples selected to show representative data of IgG
ments remaining after pepsin digestion. Assuming that the con- and fragment binding to M. luteus DNA.
centration of Fc fragments remaining in F(ab0 )2 preparations was As these data indicate, the Fab fragments from all HS sam-
not negligible, the weight of a preparation of F(ab0 )2 fragments ples failed to bind to M. luteus DNA compared with intact IgG.
could be concluded to have an identical number of Ag-binding These findings indicate that HS anti-DNA, like SLE anti-DNA,
sites as the same weight of intact IgG (24). Thus, for example, bind by a monogamous bivalent mechanism. Thus, even though
the binding activity of 20 mg/ml intact IgG was compared with the antigenic determinants recognized by HS and SLE DNA
the binding activity of 20 mg/ml F(ab0 )2 fragment preparation. are different, the Fab fragments of both types of anti-DNA lack
sufficient affinity for monovalent binding.
ELISAs We next tested the activity of the F(ab0 )2 fragments. As
ELISAs were conducted as previously described (24). Briefly, these data indicate, F(ab0 )2 fragments from two out of five HS
high-binding, flat-bottom, 96-well microtiter plates (Immulon plasmas as well as from the pooled IgG showed similar anti-
2HB; Thermo Fisher Scientific) were used. All washes were DNA binding activity compared with that of intact IgG (Figs. 1,
performed three times with 1 PBS. All reagent volumes were 2). Thus, in the Ab response of the individuals tested, the Fc
100 ml/well and all incubations at room temperature (2123 C) region is not essential.
https://doi.org/10.4049/immunohorizons.2100077ImmunoHorizons BINDING MECHANISMS OF Abs TO DNA 795
A B
2.5 H14 IgG 2.5 H19 IgG
H14 Fab H19 Fab
2.0 H14 F(ab')2 2.0 H19 F(ab')2
OD 450 nm
1.5 1.5
1.0 1.0
0.5 0.5
0 0
1 2 3 4 5 6 1 2 3 4 5 6
Antibody Dilution
0
FIGURE 1. Binding of representative HS IgG, Fab, and F(ab )2 fragment preparations to M. luteus DNA.
The binding of IgG (circle), Fab (triangle), and F(ab0 )2 (square) fragments from two representative HS plasmas (H14 and H19) to M. luteus DNA was
Downloaded from http://www.immunohorizons.org/ by guest on June 30, 2022
examined by ELISA. Each point shown is the average OD450 of two wells, with error bars indicating SD. Serial 2-fold dilutions of IgG and fragments
were tested, with initial concentrations as follows. For plasma H14 (A): IgG and F(ab0 )2, 20 mg/ml; Fab, 12.5 mg/ml. For plasma H19 (B): IgG and
F(ab0 )2, 80 mg/ml; Fab, 50 mg/ml.
The interpretation of these experiments depends on the M. luteus DNA and whether the binding of these Abs requires
activity of the fragments. To determine whether the Fab and the Fc region.
F(ab0 )2 fragments of HS IgG retained their Ag-binding activity, As shown in Figs. 3 and 4, we found that Fab fragments
we tested the relative binding activity of IgG, Fab, and F(ab0 )2 from SLE plasmas had negligible binding activity to M. luteus
preparations to EBV Ag and tetanus toxoid. Fab and F(ab0 )2 DNA similar to that observed with HS fragments. By contrast,
fragments prepared from HS plasmas and from the pooled IgG F(ab0 )2 fragments from two out of five SLE plasmas showed
demonstrated levels of binding activity to EBV Ag and tetanus significant binding activity to M. luteus DNA, albeit lower than
toxoid like those of intact IgG (Fig. 2). that of intact IgG. Together, these findings indicate that, whe-
reas the autoantibody response to DNA of SLE IgG requires
Binding of IgG, Fab, and F(ab0 )2 fragments from SLE the presence of the Fc region, the binding to bacterial DNA
patients does not have this requirement whether in HS or SLE patients.
Because our previous study examined SLE anti-DNA binding to
mammalian DNA (i.e., CT DNA) (24), we decided to expand
DISCUSSION
our investigation of anti-DNA in SLE. Specifically, we wanted
to determine the binding activity of both Fab and F(ab0 )2 frag-
The results of these experiments, to our knowledge, provide
ments of SLE IgG with nonconserved determinants on bacterial
new insight into the mechanisms of anti-DNA binding in nor-
DNA. Figs. 3 and 4 present these results.
mal as well as aberrant immunity. As shown in previous stud-
As has been previously shown, Fab and F(ab0 )2 fragments
ies, autoantibodies to DNA in patients with SLE interact with
from all SLE plasmas demonstrated negligible binding activity
to CT DNA compared with intact IgG (24). Also consistent DNA by a binding mode termed monogamous bivalency. In this
with earlier work, Fab and F(ab0 )2 fragments prepared from mode, both Fab sites contact antigenic determinants on the
SLE sera displayed binding activity to both EBV and tetanus same extended polynucleotide chain (24, 26). The necessity for
toxoid. These results confirm and extend our previous findings bivalency reflects the low affinity of each Fab site for DNA.
on the role of Fc-dependent monogamous bivalency, with all Whereas prior work established monogamous bivalent binding
SLE plasmas tested thus far displaying this binding mechanism. of autoantibodies to DNA in SLE, this study demonstrates that
Having confirmed our previous results on autoantibodies to Abs to bacterial DNA in otherwise healthy individuals also use
DNA, we then investigated the binding of fragments from SLE this mechanism.
plasmas to M. luteus DNA. Prior studies have indicated that The necessity for monogamous bivalency in HS anti-DNA is
blood from some patients with SLE contains at least two popu- perhaps surprising because these Abs arise in otherwise healthy
lations of anti-DNA (32). Whereas one population (i.e., autoan- individuals, likely in response to foreign DNA from bacterial
tibodies) binds to conserved sites on mammalian and bacterial infection or colonization. HS anti-DNA bind to nonconserved
DNA, another population binds specifically to bacterial DNA; sites on foreign DNA and might be expected to have high affin-
specific binding to bacterial DNA suggests retention of the ordi- ity comparable with that of Abs to foreign proteins. Neverthe-
nary mode of binding to nonconserved DNA sites. We there- less, as our experiments demonstrated, Fab fragments of HS
fore wanted to test whether SLE plasma contains Abs to IgG failed to bind to M. luteus DNA under the assay conditions
https://doi.org/10.4049/immunohorizons.2100077796 BINDING MECHANISMS OF Abs TO DNA ImmunoHorizons
MC DNA EBV Antigen Tetanus Toxoid
2.0
H13
1.0
0
2.0
H14
1.0
0
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2.0
H16 ND
1.0
OD 450 nm
0
2.0
H19
1.0
0
2.0
H20 ND
1.0
0
2.0
Pool
1.0
0
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Antibody Dilution
FIGURE 2. Binding of HS IgG, Fab, and F(ab0 )2 fragment preparations to M. luteus DNA and controls.
The binding of IgG (circle), Fab (triangle), and F(ab0 )2 (square) fragments from five HS plasmas (H13, H14, H16, H19, and H20) and preisolated,
pooled IgG (Pool) to M. luteus DNA, EBV Ag, and tetanus toxoid was examined by ELISA. ND, data not determined for a particular control. Each
point shown is the average OD450 of two wells, with error bars indicating SD. Serial 2-fold dilutions of IgG and fragments were tested, with initial
concentrations as follows. For plasmas H13, H14, H16, H20, and Pool: IgG and F(ab0 )2, 20 mg/ml; Fab, 12.5 mg/ml. For plasma H19: IgG and F(ab0 )2,
80 mg/ml; Fab, 50 mg/ml.
https://doi.org/10.4049/immunohorizons.2100077ImmunoHorizons BINDING MECHANISMS OF Abs TO DNA 797
A B
2.5 #32 IgG 2.5 #35 IgG
#32 Fab #35 Fab
2.0 #32 F(ab')2 2.0 #35 F(ab')2
1.5 1.5
1.0 1.0
0.5 0.5
0 0
1 2 3 4 5 6 1 2 3 4 5 6
OD 450 nm
C D
2.5 2.5
#32 IgG #35 IgG
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#32 Fab #35 Fab
2.0 #32 F(ab')2 2.0 #35 F(ab')2
1.5 1.5
1.0 1.0
0.5 0.5
0 0
1 2 3 4 5 6 1 2 3 4 5 6
Antibody Dilution
FIGURE 3. Binding of representative SLE IgG, Fab, and F(ab0 )2 fragment preparations to M. luteus and CT DNA.
The binding of IgG (circle), Fab (triangle), and F(ab0 )2 (square) fragments from two representative SLE plasmas (32 and 35) to M. luteus DNA (A and
B) and to CT DNA (C and D) was examined by ELISA. Each point is the average OD450 of two wells, with error bars indicating SD. Serial 2-fold dilu-
tions of IgG and fragments were tested, with initial concentrations as follows. For plasma 32 (A and C): IgG and F(ab0 )2, 100 mg/ml, Fab, 62.5 mg/ml.
For plasma 35 (B and D): IgG and F(ab0 )2, 80 mg/ml, Fab, 50 mg/ml.
used. This finding suggests that there are limitations in the gen- In our previous work, we showed that SLE autoantibodies to
eration of high-affinity anti-DNA, whether in HS in their DNA display a unique type of bivalent interaction that requires
response to foreign DNA or in patients with SLE in their the presence of the Fc region (24). Even though the F(ab0 )2 frag-
response to self-DNA. ments of IgG anti-DNA are capable of bivalent binding, in our
The lack of Abs capable of monovalent interaction suggests prior studies, the fragments failed to bind to CT DNA. Control
restrictions in Ag selection whether in normal or aberrant studies showed that the F(ab0 )2 fragments, as well as Fab frag-
immunity. Perhaps the preimmune repertoire lacks precursors ments, were functional because they bound both tetanus toxoid
that can be mutated to form a high-affinity binding site capable and an EBV Ag preparation. We thus termed this observed bind-
of a monovalent interaction. An alternative explanation for the ing mechanism Fc-dependent monogamous bivalency. Our prior
pattern of binding observed may relate to the properties of study involved samples from five patients. We have now
DNA as an immunogen, with susceptibility to DNase digestion observed similar results in samples from another five patients
limiting the amount of DNA that can contact surface receptors with SLE to bring the total number of samples to 10, strongly
on B cells to drive an Ag-specific response (33, 34). Based on suggesting that Fc-dependent monogamous bivalency is the
studies of cfDNA, circulating DNA from bacterial sources has a usual binding mode for autoantibodies to DNA.
short half-life in blood as well as a low molecular mass (35, 36). The impact of Fc on Ag binding has been previously charac-
In this regard, the pathways of B cell activation may affect the terized in studies on the binding of murine monoclonal Abs to
avidity of anti-DNA induced. Of note, studies in murine infec- fungal capsular polysaccharides, bacterial polysaccharides, and
tion and lupus models have shown that pathogenic autoanti- DNA (2931, 39, 40). Rather than using fragments produced by
bodies may develop by T celldependent germinal center enzyme digestion, these studies characterized the binding activity
reactions as well as extrafollicular reactions (37, 38). of molecular constructs in which the same variable regions were
https://doi.org/10.4049/immunohorizons.2100077798 BINDING MECHANISMS OF Abs TO DNA ImmunoHorizons
MC DNA CT DNA EBV Antigen Tetanus Toxoid
2.0
#31
1.0
0
2.0
#32
1.0
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0
2.0
OD 450 nm
#35
1.0
0
2.0
#36
1.0
0
2.0
#37
1.0
0
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Antibody Dilution
FIGURE 4. Binding of SLE IgG, Fab, and F(ab0 )2 fragment preparations to M. luteus DNA, CT DNA, and controls.
The binding of IgG (circle), Fab (triangle), and F(ab0 )2 (square) preparations from five SLE plasmas (31, 32, and 35–37) to M. luteus DNA, CT DNA,
EBV Ag, and tetanus toxoid was examined by ELISA. Each point is the average OD450 of two wells, with error bars indicating SD. Serial 2-fold dilu-
tions of IgG and fragments were tested, with initial concentrations as follows. For plasmas 31 and 36: IgG and F(ab0 )2, 120 mg/ml; Fab, 75 mg/ml. For
plasma 32: IgG and F(ab0 )2, 100 mg/ml; Fab, 62.5 mg/ml. For plasmas 35 and 37: IgG and F(ab0 )2, 80 mg/ml; Fab, 50 mg/ml.
joined with H chains of different isotypes. Importantly, these in solution have indicated that sequence changes to the Fc region
constructs varied in their strength and specificity of Ag binding, influence the structures of AbAg complexes (41, 42).
pointing to some type of structural connection or communication In addition to induced allosteric changes in Ab structure, the
from the Fc region to the variable regions. For anti-DNA Abs, H Fc region may also influence Ab binding through other means,
chain isotype also influenced cross-reactivity with laminin and such as FcFc interactions. Greenspan and colleagues (43) sho-
collagen IV as well as pathogenicity (41). Furthermore, com- wed that murine Abs to streptococcal polysaccharides can bind
puter-generated models and spectroscopy of AbAg complexes cooperatively, with the expression of IgG3 in particular
https://doi.org/10.4049/immunohorizons.2100077ImmunoHorizons BINDING MECHANISMS OF Abs TO DNA 799
enhancing binding avidity. Crystal structures of IgG3 in solution These motifs can serve as adjuvants (50), and in vitro studies
indicate that the single disulfide bond in the hinge region of have demonstrated that CpG oligonucleotides can promote
IgG3 confers greater flexibility and allows for increased interac- native B cell progression to Ab-secreting plasma cells without
tion with other IgG molecules compared with other isotypes requiring BCR cross-linking (51). The presence of this adjuvant
(44). The Fc dependence of anti-DNA in SLE suggests that auto- activity may enable the generation of higher affinity IgG Abs
antibodies to DNA have low binding avidity without FcFc inter- than would occur with mammalian DNA even in the setting of
actions, with cooperativity needed to increase binding activity. A autoimmunity. Because of their increased affinity, Abs to for-
direct interaction of Fc with DNA is also possible, creating an eign DNA, although binding by monogamous bivalent interac-
additional binding site. In preliminary experiments (data not tions, may nevertheless not require the presence of the Fc
shown), however, we did not observe binding of Fc fragments to region for stable interaction.
M. luteus DNA under the conditions of the ELISA. Together, our findings suggest that anti-DNA Abs bind by
We have thus far studied only a limited number of both HS monogamous bivalency to both foreign and self-DNA; the
and SLE preparations because of availability of samples in ade- response to foreign DNA in both SLE patients and HS, how-
quate amounts to prepare fragments, which is labor intensive ever, does not invariably require the Fc region. This study also
and requires sufficient titers of anti-DNA. Although these stud- provides further evidence that patients with SLE can produce
ies can be considered as pilot experiments, we nevertheless
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different types of anti-DNA: variably Fc-dependent Abs to for-
showed that F(ab0 )2 fragments from the blood of both groups eign DNA like those found in otherwise healthy individuals and
can bind M. luteus DNA. F(ab0 )2 fragments prepared from the consistently Fc-dependent Abs to self-DNA. Future studies will
pooled IgG, which we presume came from many individuals, define further the binding properties of these two types of anti-
also demonstrated significant binding activity to M. luteus DNA and delineate in greater detail how the presence of the Fc
DNA. In this property, the SLE samples resembled those from region influences anti-DNA binding.
HS, suggesting that the binding to nonconserved sites on for-
eign DNA differs from that of SLE anti-DNA, for which a fully
DISCLOSURES
intact IgG structure is required for binding to self-DNA.
In these experiments, we have used an ELISA, which is a
The authors have no financial conflicts of interest.
solid phase assay that potentially could affect the antigenicity
of DNA. In previous experiments, we compared the antigenic-
ity of DNA either coated directly to plates with that of biotiny- REFERENCES
lated DNA bound to the solid phase via streptavidin; binding of
biotinylated DNA to streptavidin assures that the DNA is solu- 1. Stollar, B. D. 1989. Immunochemistry of DNA. Int. Rev. Immunol. 5:
ble and freely mobile in the fluid phase (24). In those experi- 122.
2. Pisetsky, D. S. 2016. Anti-DNA antibodiesquintessential bio-
ments, we did not observe differences with respect to the markers of SLE. Nat. Rev. Rheumatol. 12: 102110.
activity of the Fab and F(ab0 )2 fragments. Both were inactive in 3. Tan, E. M., P. H. Schur, R. I. Carr, and H. G. Kunkel. 1966. Deoxybo-
the conventional ELISA as well as the sandwich ELISA with nucleic acid (DNA) and antibodies to DNA in the serum of patients
streptavidin. We have hypothesized that, with high molecular with systemic lupus erythematosus. J. Clin. Invest. 45: 17321740.
mass DNA as we used in the current experiments, much of the 4. Tsokos, G. C., M. S. Lo, P. Costa Reis, and K. E. Sullivan. 2016. New
insights into the immunopathogenesis of systemic lupus erythema-
DNA is effectively in the fluid phase and capable of any rear-
tosus. Nat. Rev. Rheumatol. 12: 716730.
rangements or topologic changes needed for antigenicity. In 5. Wang, X., and Y. Xia. 2019. Anti-double stranded DNA antibodies:
contrast, with low molecular mass DNA, DNA is likely more origin, pathogenicity, and targeted therapies. Front. Immunol. 10:
adherent to the solid phase, and interaction with Ab can be 1667.
affected (45). 6. Pisetsky, D. S., and P. E. Lipsky. 2020. New insights into the role of
antinuclear antibodies in systemic lupus erythematosus. Nat. Rev.
As demonstrated previously by adsorption experiments,
Rheumatol. 16: 565579.
anti-DNA in patients with SLE are mixtures of autoantibodies 7. Mummert, E., M. J. Fritzler, C. Sj€ owall, C. Bentow, and M. Mahler.
to conserved sites (i.e., DNA backbone) on all DNA as well as 2018. The clinical utility of anti-double-stranded DNA antibodies
Abs to nonconserved sites on foreign DNA (32). In the current and the challenges of their determination. J. Immunol. Methods.
study, the binding of F(ab0 )2 fragments to M. luteus, but not CT, 459: 1119.
8. Rekvig, O. P., and J. Van der Vlag. 2014. The pathogenesis and diag-
DNA provides further evidence for the presence of Abs to for-
nosis of systemic lupus erythematosus: still not resolved. Semin.
eign DNA in the blood of SLE patients. Immunopathol. 36: 301311.
The differences in the role of Fc dependence on the interac- 9. Emlen, W., D. S. Pisetsky, and R. P. Taylor. 1986. Antibodies to
tion of anti-DNA with self-DNA and foreign DNA have implica- DNA. A perspective. Arthritis Rheum. 29: 14171426.
tions for the mechanisms of DNA Ag selection. An important 10. Rekvig, O. P. 2015. The anti-DNA antibody: origin and impact, dog-
mas and controversies. Nat. Rev. Rheumatol. 11: 530540.
distinction between mammalian and bacterial DNA relates to
11. Karounos, D. G., J. P. Grudier, and D. S. Pisetsky. 1988. Spontaneous
their immunological properties. Whereas mammalian DNA is expression of antibodies to DNA of various species origin in sera of
immunologically inactive if not inhibitory (1), bacterial DNA is normal subjects and patients with systemic lupus erythematosus. J.
immunologically active by virtue of its CpG motifs (4649). Immunol. 140: 451455.
https://doi.org/10.4049/immunohorizons.2100077800 BINDING MECHANISMS OF Abs TO DNA ImmunoHorizons
12. Fredriksen, K., T. Traavik, and O. P. Rekvig. 1990. Anti-DNA anti- 29. Torres, M., N. Fernandez-Fuentes, A. Fiser, and A. Casadevall. 2007.
bodies induced by BK virus inoculations. Demonstration of the spe- The immunoglobulin heavy chain constant region affects kinetic
cificities for eukaryotic dsDNA and synthetic polynucleotides. and thermodynamic parameters of antibody variable region interac-
Scand. J. Immunol. 32: 197203. tions with antigen. J. Biol. Chem. 282: 1391713927.
13. Robertson, C. R., and D. S. Pisetsky. 1992. Specificity analysis of anti- 30. Xia, Y., A. Janda, E. Eryilmaz, A. Casadevall, and C. Putterman.
bodies to single-stranded micrococcal DNA in the sera of normal 2013. The constant region affects antigen binding of antibodies to
human subjects and patients with systemic lupus erythematosus. DNA by altering secondary structure. Mol. Immunol. 56: 2837.
Clin. Exp. Rheumatol. 10: 589594. 31. Janda, A., A. Bowen, N. S. Greenspan, and A. Casadevall. 2016. Ig
14. Fredriksen, K., A. Skogsholm, T. Flaegstad, T. Traavik, and O. P. Rekvig. constant region effects on variable region structure and function.
1993. Antibodies to dsDNA are produced during primary BK virus infec- Front. Microbiol. 7: 22.
tion in man, indicating that anti-dsDNA antibodies may be related to 32. Hamilton, K. J., G. Schett, C. F. Reich III, J. S. Smolen, and D. S.
virus replication in vivo. Scand. J. Immunol. 38: 401406. Pisetsky. 2006. The binding of sera of patients with SLE to bacterial
15. Hahn, B. H. 1998. Antibodies to DNA. N. Engl. J. Med. 338: and mammalian DNA. Clin. Immunol. 118: 209218.
13591368. 33. Suurmond, J., J. Calise, S. Malkiel, and B. Diamond. 2016. DNA-
16. Azzouz, D., A. Omarbekova, A. Heguy, D. Schwudke, N. Gisch, B. H. reactive B cells in lupus. Curr. Opin. Immunol. 43: 17.
Rovin, R. Caricchio, J. P. Buyon, A. V. Alekseyenko, and G. J. Silver- 34. Soni, C., and B. Reizis. 2019. Self-DNA at the epicenter of SLE: immu-
man. 2019. Lupus nephritis is linked to disease-activity associated nogenic forms, regulation, and effects. Front. Immunol. 10: 1601.
expansions and immunity to a gut commensal. Ann. Rheum. Dis. 78: 35. Burnham, P., M. S. Kim, S. Agbor-Enoh, H. Luikart, H. A. Valantine,
Downloaded from http://www.immunohorizons.org/ by guest on June 30, 2022
947956. K. K. Khush, and I. De Vlaminck. 2016. Single-stranded DNA library
17. Qiu, C. C., R. Caricchio, and S. Gallucci. 2019. Triggers of autoimmu- preparation uncovers the origin and diversity of ultrashort cell-free
nity: the role of bacterial infections in the extracellular exposure of DNA in plasma. Sci. Rep. 6: 27859.
lupus nuclear autoantigens. Front. Immunol. 10: 2608. 36. Han, D., R. Li, J. Shi, P. Tan, R. Zhang, and J. Li. 2020. Liquid biopsy
18. Pachucki, R. J., C. Corradetti, L. Kohler, J. Ghadiali, P. M. Gallo, L. for infectious diseases: a focus on microbial cell-free DNA sequenc-
Nicastro, S. A. Tursi, S. Gallucci, Ç. T€ukel, and R. Caricchio. 2020. ing. Theranostics. 10: 55015513.
37. Wei, C., J. Anolik, A. Cappione, B. Zheng, A. Pugh-Bernard, J.
Persistent bacteriuria and antibodies recognizing curli/eDNA com-
Brooks, E. H. Lee, E. C. Milner, and I. Sanz. 2007. A new population
plexes from Escherichia coli are linked to flares in systemic lupus
of cells lacking expression of CD27 represents a notable component
erythematosus. Arthritis Rheumatol. 72: 18721881.
of the B cell memory compartment in systemic lupus erythematosus.
19. Hong, D. K., T. A. Blauwkamp, M. Kertesz, S. Bercovici, C. Truong,
J. Immunol. 178: 66246633.
and N. Banaei. 2018. Liquid biopsy for infectious diseases: sequenc-
38. Jenks, S. A., K. S. Cashman, M. C. Woodruff, F. E. Lee, and I. Sanz.
ing of cell-free plasma to detect pathogen DNA in patients with
2019. Extrafollicular responses in humans and SLE. Immunol. Rev.
invasive fungal disease. Diagn. Microbiol. Infect. Dis. 92: 210213.
288: 136148.
20. Guimaraes, A. O., J. Gutierrez, S. A. Maskarinec, Y. Cao, K. Hong, F.
39. Cooper, L. J., A. R. Shikhman, D. D. Glass, D. Kangisser, M. W. Cun-
Ruffin, M. Carrasco-Triguero, M. C. Peck, V. G. Fowler, A. Baruch,
ningham, and N. S. Greenspan. 1993. Role of heavy chain constant
and C. M. Rosenberger. 2019. Prognostic power of pathogen cell-
domains in antibody-antigen interaction. Apparent specificity differ-
free DNA in Staphylococcus aureus bacteremia. Open Forum Infect.
ences among streptococcal IgG antibodies expressing identical vari-
Dis. 6: ofz126.
able domains. J. Immunol. 150: 22312242.
21. Hogan, C. A., S. Yang, O. B. Garner, D. A. Green, C. A. Gomez, J.
40. McLean, G. R., M. Torres, N. Elguezabal, A. Nakouzi, and A. Casa-
Dien Bard, B. A. Pinsky, and N. Banaei. 2021. Clinical impact of devall. 2002. Isotype can affect the fine specificity of an antibody
metagenomic next-generation sequencing of plasma cell-free DNA for a polysaccharide antigen. J. Immunol. 169: 13791386.
for the diagnosis of infectious diseases: a multicenter retrospective 41. Xia, Y., R. D. Pawar, A. S. Nakouzi, L. Herlitz, A. Broder, K. Liu, B.
cohort study. Clin. Infect. Dis. 72: 239245. Goilav, M. Fan, L. Wang, Q. Z. Li, et al. 2012. The constant region
22. Pisetsky, D. S. 1997. Specificity and immunochemical properties of contributes to the antigenic specificity and renal pathogenicity of
antibodies to bacterial DNA. Methods. 11: 5561. murine anti-DNA antibodies. J. Autoimmun. 39: 398411.
23. Robertson, C. R., G. S. Gilkeson, M. M. Ward, and D. S. Pisetsky. 42. Janda, A., and A. Casadevall. 2010. Circular dichroism reveals evi-
1992. Patterns of heavy and light chain utilization in the antibody dence of coupling between immunoglobulin constant and variable
response to single-stranded bacterial DNA in normal human sub- region secondary structure. Mol. Immunol. 47: 14211425.
jects and patients with systemic lupus erythematosus. Clin. Immu- 43. Greenspan, N. S., D. A. Dacek, and L. J. Cooper. 1989. Cooperative
nol. Immunopathol. 62: 2532. binding of two antibodies to independent antigens by an Fc-depen-
24. Stearns, N. A., and D. S. Pisetsky. 2016. The role of monogamous dent mechanism. FASEB J. 3: 22032207.
bivalency and Fc interactions in the binding of anti-DNA antibodies 44. Eryilmaz, E., A. Janda, J. Kim, R. J. Cordero, D. Cowburn, and A.
to DNA antigen. Clin. Immunol. 166167: 3847. Casadevall. 2013. Global structures of IgG isotypes expressing iden-
25. Hornick, C. L., and F. Karuch. 1972. Antibody affinity. 3. The role of tical variable regions. Mol. Immunol. 56: 588598.
multivalance. Immunochemistry. 9: 325340. 45. Pisetsky, D. S., and C. F. Reich. 1994. The influence of DNA size on
26. Papalian, M., E. Lafer, R. Wong, and B. D. Stollar. 1980. Reaction of the binding of anti-DNA antibodies in the solid and fluid phase.
systemic lupus erythematosus antinative DNA antibodies with Clin. Immunol. Immunopathol. 72: 350356.
native DNA fragments from 20 to 1,200 base pairs. J. Clin. Invest. 46. Yamamoto, S., T. Yamamoto, T. Kataoka, E. Kuramoto, O. Yano, and
65: 469477. T. Tokunaga. 1992. Unique palindromic sequences in synthetic oli-
27. Emlen, W., R. Ansari, and G. Burdick. 1984. DNA-anti-DNA immune gonucleotides are required to induce IFN [correction of INF] and
complexes. Antibody protection of a discrete DNA fragment from augment IFN-mediated [correction of INF] natural killer activity. J.
DNase digestion in vitro. J. Clin. Invest. 74: 185190. Immunol. 148: 40724076.
28. Pisetsky, D. S., and T. C. Gonzalez. 1999. The influence of DNA size 47. Klinman, D. M., A. K. Yi, S. L. Beaucage, J. Conover, and A. M.
on the binding of antibodies to DNA in the sera of normal human Krieg. 1996. CpG motifs present in bacteria DNA rapidly induce
subjects and patients with systemic lupus erythematosus (SLE). lymphocytes to secrete interleukin 6, interleukin 12, and interferon
Clin. Exp. Immunol. 116: 354359. gamma. Proc. Natl. Acad. Sci. USA. 93: 28792883.
https://doi.org/10.4049/immunohorizons.2100077ImmunoHorizons BINDING MECHANISMS OF Abs TO DNA 801
48. Sato, Y., M. Roman, H. Tighe, D. Lee, M. Corr, M. D. 50. Pisetsky, D. S., K. S. Wenk, and C. F. Reich III. 2001. The role of cpg
Nguyen, G. J. Silverman, M. Lotz, D. A. Carson, and E. Raz. sequences in the induction of anti-DNA antibodies. Clin. Immunol.
1996. Immunostimulatory DNA sequences necessary for 100: 157163.
effective intradermal gene immunization. Science. 273: 51. Huggins, J., T. Pellegrin, R. E. Felgar, C. Wei, M. Brown, B. Zheng,
352354. E. C. Milner, S. H. Bernstein, I. Sanz, and M. S. Zand. 2007. CpG
49. Krieg, A. M. 2002. CpG motifs in bacterial DNA and their immune DNA activation and plasma-cell differentiation of CD27- naive
effects. Annu. Rev. Immunol. 20: 709760. human B cells. Blood. 109: 16111619.
Downloaded from http://www.immunohorizons.org/ by guest on June 30, 2022
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