Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans
Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of oxidized lipoproteins and apoptotic cells. Adaptive immune responses to various oxidation-specific epitopes play an important role in atherogenesis. However, accumulating evidence suggests that these epitopes are also recognized by innate receptors, such as scavenger receptors on macrophages, and plasma proteins, such as C-reactive protein (CRP). Here, we provide multiple lines of evidence that oxidation-specific epitopes constitute a dominant, previously unrecognized target of natural Abs (NAbs) in both mice and humans. Using reconstituted mice expressing solely IgM NAbs, we have shown that approximately 30% of all NAbs bound to model oxidation-specific epitopes, as well as to atherosclerotic lesions and apoptotic cells. Because oxidative processes are ubiquitous, we hypothesized that these epitopes exert selective pressure to expand NAbs, which in turn play an important role in mediating homeostatic functions consequent to inflammation and cell death, as demonstrated by their ability to facilitate apoptotic cell clearance. These findings provide novel insights into the functions of NAbs in mediating host homeostasis and into their roles in health and diseases, such as chronic inflammatory diseases and atherosclerosis.
The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009 1335
Oxidation-specific epitopes are dominant
targets of innate natural antibodies
in mice and humans
Lotte F. Hansen,
Peter X. Shaw,
Yury I. Miller,
Joseph L. Witztum,
and Christoph J. Binder
Department of Medicine and
Department of Reproductive Medicine, UCSD, La Jolla, California, USA.
Department of Periodontology,
Pusan National University, Pusan, Republic of Korea.
Center for Molecular Medicine (CeMM) of the Austrian Academy of Sciences and
Department of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria.
Sahlgrenska Centre for Cardiovascular and Metabolic Research, Wallenberg Laboratory, University of Göteborg, Göteborg, Sweden.
Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of oxidized lipoproteins
and apoptotic cells. Adaptive immune responses to various oxidation-specific epitopes play an important role
in atherogenesis. However, accumulating evidence suggests that these epitopes are also recognized by innate
receptors, such as scavenger receptors on macrophages, and plasma proteins, such as C-reactive protein (CRP).
Here, we provide multiple lines of evidence that oxidation-specific epitopes constitute a dominant, previously
unrecognized target of natural Abs (NAbs) in both mice and humans. Using reconstituted mice expressing
solely IgM NAbs, we have shown that approximately 30% of all NAbs bound to model oxidation-specific epi-
topes, as well as to atherosclerotic lesions and apoptotic cells. Because oxidative processes are ubiquitous, we
hypothesized that these epitopes exert selective pressure to expand NAbs, which in turn play an important
role in mediating homeostatic functions consequent to inflammation and cell death, as demonstrated by their
ability to facilitate apoptotic cell clearance. These findings provide novel insights into the functions of NAbs in
mediating host homeostasis and into their roles in health and diseases, such as chronic inflammatory diseases
Although hypercholesterolemia is necessary for the initiation
and progression of atherosclerosis, there is now abundant evi
dence that immune mechanisms are also central to all phases of
lesion development (1–3). We and others have documented that,
among several suggested immunogens present in the atheroscle
rotic plaque, oxidation-specific epitopes, as occur in oxidized LDL
(OxLDL), are immunodominant. In turn, these lead to profound
immune responses, including autoantibody generation, that mod
ulate lesion formation (4).
Many of these responses are adaptive in nature, responding to
the myriad of new moieties generated in response to the complex
neoepitopes formed when lipid peroxidation occurs. Surprisingly,
innate immune recognition of these oxidation-specific epitopes
is also prominent, and various macrophage scavenger receptors
bind to epitopes of OxLDL (5–7). In addition, we previously found
that innate natural Abs (NAbs) bind to oxidized phospholipids
(OxPLs) of OxLDL. For example, cholesterol-fed apoE-deficient
mice have very high IgM titers to OxLDL, which enabled clon
ing of IgM-secreting hybridomas from the spleens of these mice
with specificity for OxLDL (8). A large number of these bound
to both the lipid and apoB moieties of OxLDL, and specifically
to the phosphocholine (PC) headgroup of OxPL, such as 1-pal
present as either a lipid or as an adduct bound to protein via the
ε-amino group of lysine. They did not bind to the PC of native
phospholipids (9). Importantly, these antibodies, as represented
by the prototypic antibody E06, inhibited the uptake of OxLDL by
macrophage scavenger receptors CD36 and SR-BI (5, 6, 10), as did
POVPC linked to BSA or a peptide. This demonstrates that the PC
moiety of OxPL is a ligand for macrophage scavenger receptors,
which are innate, cellular pattern recognition receptors (PRRs).
Because all of these cloned autoantibodies were IgM Abs, which
are thought in large part to represent NAbs in uninfected mice (11),
we sequenced the complementarity-determining regions (CDRs)
determining their antigen-binding sites, which revealed them all to
be genetically identical to a well-characterized B-1 cell clone, T15,
described more than 30 years ago (12). T15 NAbs bind to PC cova
lently linked to the cell wall polysaccharide (C-PS) of pathogens
and provide optimal protection to mice from lethal infection with
Streptococcus pneumoniae (13). Furthermore, immunization of cho-
mice with heat-killed S. pneumoniae led to a nearly
exclusive expansion of E06/T15 NAbs and atheroprotection (14).
Because NAbs are postulated to be conserved by natural selec
tion, it was not apparent what the selecting agent might be, as
Conflict of interest: Joseph L. Witztum is named as inventor in patents and patent
applications from the UCSD for the potential commercial use of antibodies to oxi-
Nonstandard abbreviations used: AP, alkaline phosphatase; CuOx-LDL, copper
sulfate–oxidized LDL; ELISpot, enzyme-linked immunospot; FACS, fluorescence-acti
vated cell sorting; 4-HNE-LDL, 4-hydroxynonenal–modified LDL; 4-HNE-MSA,
4-hydroxynonenal–modified mouse serum albumin; ISC, IgM-secreting cell;
MAA-BSA, malondialdehyde-acetaldehyde–modified BSA; MAA-MSA,
malondialdehyde-acetaldehyde–modified mouse serum albumin; MDA-LDL,
malondialdehyde-modified LDL; NAb, natural Ab; OxLDL, oxidized LDL; OxPL,
oxidized phospholipid; PAMP, pathogen-associated molecular pattern; PC, phospho
choline; PC-BSA, PC-conjugated BSA; PC-KLH, PC-conjugated keyhole limpet hemo-
cyanin; PEC, peritoneal exudate cell; PRR, pattern recognition receptor; SPF, specific
Citation for this article:
J. Clin. Invest. 119:1335–1349 (2009). doi:10.1172/JCI36800.
1336 The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009
oxidation of LDL and atherosclerosis per se should not exert any
positive selective pressure. We postulated that apoptotic cells,
similar to OxLDL, would also display oxidation-specific epitopes
on their surface, as cells undergoing programmed cell death are
known to undergo enhanced oxidative processes (15, 16) and if
not promptly cleared are likely to be proinflammatory (17, 18).
Indeed, using mass spectroscopy, we demonstrated that apoptotic
cells contained an enhanced content of OxPL in their membranes
and that E06 bound prominently to their cell surface, consistent
with this hypothesis (18, 19). We also demonstrated that C-reactive
protein (CRP), an innate acute-phase protein, recognized the same
PC moiety on OxLDL and apoptotic cells (20). These data strongly
suggest that the PC moiety of OxPL, apoptotic cells, and the cell
wall of bacteria constitute a pathogen-associated molecular pat
tern (PAMP) recognized by multiple arcs of innate immunity and
that each could exert positive selective pressure.
A variety of such oxidation-specific epitopes, besides PC of OxPL,
are likely to occur in abundance not only on apoptotic cells, but
on shed microparticles, and in general on membranes and even
bacteria during inflammatory responses. We postulated that they
might constitute a previously unrecognized but important class
of PAMPs and in turn would be a major target of innate NAbs. In
IgM Abs to oxidation-specific antigens are present in germ-free and conventional mice. (A) Conventional and SPF C57BL/6 mice have similar
IgM titers to oxidation-specific antigens. Plasma from 11-week-old female conventionally raised (n = 4) and SPF (n = 4) C57BL/6 mice were
tested by ELISA. Values are mean and SEM. (B) MDA-LDL–specific ISCs are dominant in the spleens of conventionally raised C57BL/6 mice.
Splenocytes from conventionally raised 12-week-old female C57BL/6 mice (n = 4) were tested by ELISpot assay for frequencies of ISCs as
described in Methods. Values represent the number of ISCs to indicated antigen as a percentage of total ISCs (mean and SD). Data are from 1
experiment representative of 3. **P < 0.01 compared with all other antigens (1-way ANOVA with Tukey-Kramer multiple comparison test). (C)
Binding curves of plasma IgM from germ-free Swiss-Webster mice to indicated antigens. Plasma samples were from 14- to 16-week old female
and male mice (n = 9). Values are mean and SEM. (D) Titers of IgM Abs to oxidation-specific epitopes are present in conventional and germ-free
Swiss Webster mice. Serum from 14- to 16-week-old female and male conventionally raised (n = 7), conventionalized (germ-free colonized with
bacterial flora) (n = 11), and germ-free (n = 9) mice were diluted 1:400 and tested for binding to the indicated antigens. Values are mean and
SEM. *P < 0.05, **P < 0.01, ***P < 0.002 compared with α1,3-dextran (1-way ANOVA with Tukey-Kramer multiple comparison test).
The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009 1337
this article, we provide multiple lines of evidence suggesting that
oxidation-specific epitopes are a dominant target of innate NAbs
in both mice and humans.
IgM Abs against oxidation-specific epitopes are present in normal and germ-
free mice. To characterize the murine humoral IgM responses to
defined oxidation-specific epitopes, we assessed specific IgM titers
in plasma of naive, nonatherosclerotic C57BL/6 mice. As previ
ously observed (8), prominent IgM titers to oxidation-specific epi
topes, such as OxLDL (>1:1,350) and malondialdehyde-modified
LDL (MDA-LDL) (>>1:1,350), and to 4-hydroxynonenal–modified
mouse serum albumin (4-HNE-MSA) and PC-conjugated BSA
(PC-BSA; 1:1,350), can be detected even in normal, conventionally
housed mice, whereas IgM titers to “native LDL” are minimal or
undetectable (Figure 1A) (see comment on apparent binding to
native LDL below under the subhead
IgM binding to native LDL).
Among these, the IgM responses to MDA modifications were con
sistently found to be the most robust, and the titers were many fold
higher than the titer (1:1,350) to the prototypic B-1 cell antigen
α1,3-dextran. In addition, when comparing the plasma IgM titers
to those in age-matched mice bred under specific pathogen–free
(SPF) conditions, a similar response pattern was observed (Figure
1A), suggesting that the basal titers of these IgM Abs are largely
independent of noncommensal exposure to microbial patho
gens. Moreover, we found that oxidation-specific IgM levels were
also present in T cell receptor–deficient (
) mice — although
slightly lower — indicating that in large part these responses do not
require T cells (see Supplemental Figure 1; supplemental material
available online with this article; doi:10.1172/JCI36800DS1).
In mice, IgM Abs are in large part derived from Ab-secreting cells
in the spleen (21). Using enzyme-linked immunospot (ELISpot)
analysis, we tested the frequencies of IgM-secreting cells (ISCs)
against candidate oxidation-specific epitopes in the spleens of
conventionally housed C57BL/6 mice. ISCs with specificity for
oxidation-specific epitopes were equally prominent in the spleen,
as was observed for the IgM in plasma, with up to 15% of all ISCs
having specificity for MDA-LDL (Figure 1B).
Our data suggest that IgM titers to an array of oxidation-spe
cific epitopes may in fact represent IgM generated even in the
absence of response-eliciting antigen exposure. In confirmation
of this, we demonstrated robust IgM titers to oxidation-specific
epitopes in the serum of “germ-free mice,” which are completely
free of gut bacteria (Figure 1C). In particular, IgM titers to MDA-
LDL (>1:1,600) and MAA-BSA (>1:1,600) were the most prominent
among all IgM titers measured: MAA (malondialdehyde-acetalde
hyde adduct) is a specific and prominent chemical moiety gener
ated from 2 MDA and 1 acetaldehyde molecules reacting with the
ε-amino group of lysine to form an adduct; in this case forming
adducts with BSA. Titers to OxLDL (>1:1,250) and 4-hydroxynon
enal–modified LDL (4-HNE-LDL; 1:800) were also much higher
than those to
α1,3-dextran (1:400), while titers to PC-BSA, which
shares molecular identity to the PC of OxPL (as found in OxLDL),
were approximately 1:400.
Furthermore, reconstitution of germ-free mice with gut bacte
ria (“conventionalized mice”) for only 2 weeks led to increases in
In vitro stimulation of B-1 cells induces increased natural
IgM Ab titers to oxidation-specific antigens. (A) Purified
B-1 cells were cultured in 24-well plates in triplicate at
a cell density of 1 × 10
cells per well in 500 μl culture
medium. Cells were stimulated with IL-5 (50 ng/ml),
-Lipid A (100 ng/ml), or TLR2 agonists (a combi-
nation of Pam3CSK4 [300 ng/ml] and FSL-1 [1 μg/ml])
and incubated at 37°C for 7 days. Control B-1 cells were
cultured in medium alone. Cell culture supernatants were
harvested after 7 days and IgM Ab titers analyzed by
ELISA at 1:45 dilution. Results were normalized to cell
number recovered after 7 days. Values are mean and
SEM. Data are from 1 experiment representative of 3.
*P < 0.05, **P < 0.01, ***P < 0.002 compared with α1,3-
dextran (repeated-measures ANOVA with Tukey-Kramer
multiple comparison test). (B) Natural IgM Abs produced
in vitro show specificity to MDA-LDL and CuOx-LDL. For
competition immunoassay, supernatants from purified
B-1 cell cultures stimulated with KdO
-Lipid A (100 ng/
ml) or IL-5 (50 ng/ml) were diluted to 1:20 and incubated
in the presence of the indicated concentrations of com-
petitors (Competitor conc.) overnight. After incubation,
IgM binding to MDA-LDL and CuOx-LDL was tested by
ELISA. Data are the mean of triplicate determinations,
expressed as ratio of IgM binding to MDA-LDL or CuOx-
LDL in the presence or absence of competitor (B/B
Data are from 1 experiment representative of 3.
1338 The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009
The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009 1339
many — but not all — of the oxidation-specific IgM levels (Figure
1D), strongly suggesting molecular mimicry between many endog
enous oxidation-specific epitopes and gut bacterial epitopes.
While IgM responses to PC and 4-HNE, as well as to
tran, were increased (more than 2-fold) in these mice, the MDA-
specific responses were found to be similar to those in germ-free
mice. Moreover, similar specific IgM responses were measured in
age-matched conventionally raised mice of the same genetic back
ground (Figure 1D). Note again that oxidation-specific IgM Abs
constitute a major fraction of total IgM Abs, which were not differ
ent among wild-type, germ-free, or colonized mice (Figure 1D).
IgM binding to native LDL. In these studies, we tested IgM binding
to antigens coated on microtiter wells using standard solid-phase
ELISA techniques (22). In some experiments, we saw low to modest
levels of IgM binding to native LDL, which appeared to vary with
different LDL preparations. However, in all cases, the binding to
plated native LDL could not be competed by the same prepara
tion of native LDL in solution (data not shown), suggesting that
the LDL became modified in some way during the plating process.
It should be emphasized that in competition immunoassays, we
never observed native LDL competing for binding to any of the
modified LDL preparations (e.g., as shown in Figure 2B, Figure
3A, and Figure 4A).
B-1 cells secrete IgM NAbs against oxidation-specific epitopes in vitro. To
directly demonstrate that oxidation-specific IgM Abs are derived
from innate B-1 cells, we isolated B-1 cells (both CD5
B-1b) from naive mice by fluorescence-activated cell sorting
(FACS), stimulated them in vitro with various stimuli, and tested
the culture supernatants for specific IgM Abs. IL-5, a TLR4 ligand
-Lipid A), as well as a combination of TLR2 ligands (FSL-1
and Pam3CSK4) all induced B-1 cells to secrete IgM against MDA-
LDL, OxLDL (Figure 2A), and 4-HNE-LDL (data not shown); but
also against the prototypic B-1 cell antigen
α1,3-dextran (Figure 2A).
Utilizing B-1 cells from
mice, we observed that the respons-
es to both TLR4 and TLR2 agonists were in large part MyD88 depen
dent (data not shown). Interestingly, the basal secretion of IgM to
OxLDL and MDA-LDL was strikingly more prominent than that of
α1,3-dextran (Figure 2A), and in response to stimula-
tion with the TLR agonists, the oxidation-specific IgM increased to
a greater extent than did the total IgM, or the IgM to
for example (Supplemental Figure 2). Basal titers to PC-BSA were
relatively low and increased only in response to IL-5. PC is an epit
ope for some OxLDL-specific IgM, but not all.
By analogy to the robust anti-MDA responses in vivo, MDA-spe
cific IgM Abs were the dominant set of IgM Abs secreted by B-1
cells in vitro. Although we tested only a narrowly selected set of
antigens, the anti-MDA Abs constituted up to 30% of total IgM
secreted. These Abs were highly specific for MDA modifications, as
only MDA-LDL, but neither OxLDL nor native LDL, competed for
the binding (Figure 2B), while IgM Abs bound to plated OxLDL
were competed by both OxLDL and MDA-LDL. We also calculated
the binding avidities of the IgM in the supernatants for MDA-LDL
and OxLDL using the Klotz method (23).The calculated
MDA-LDL and OxLDL were 1.46
mol/l and 4.03 × 10
respectively, similar to values we previously determined for IgM in
plasma of mice immunized against these epitopes (18).
B-1 cells secrete IgM NAbs against oxidation-specific epitopes in vivo. To
test whether innate B-1 cells can also secrete oxidation-specific
IgM in vivo, peritoneal B-1 cells from naive C57BL/6 mice were
adoptively transferred into the peritoneum of
mice, which lack functional B and T cells. This led to the selective
reconstitution of only B-1 cells in the peritoneum of
ients (Figure 3A, top row), and both B-1a (CD5
and B-1b cells (CD5
) were detected (Figure 3A,
middle row), whereas conventional B-2 cells and T cells were typi
cally not found in recipient mice. The average percentages of B-1a
and B-1b cells among total cells analyzed in the peritoneum of
B-1 recipients were 11.0% ± 2.6% and 8.5% ± 1.0% respec-
n = 12), compared with 18.2% ± 3.2% and 8.3% ± 2.3% (n = 4)
in wild-type C57BL/6 mice.
The adoptive transfer also reconstituted the B-1 cell population
in the spleens of
B-1 recipients. B-1 cells (IgM- and CD43-
positive; Figure 3A, bottom row) constituted an average of about
1% of total splenocytes analyzed (data ranged from 0.4% to 1.4%;
n = 9), while in wild-type C57BL/6 mice, B-1 cells averaged about
n = 3). Moreover, this reconstitution was also demonstrated
by the number of splenic ISCs as measured by ELISpot: 60 ± 15
ISCs vs. 152 ± 21 ISCs per 200,000 splenocytes in
n = 9) and C57BL/6 mice (n = 7), respectively.
The B-1 cell–reconstituted
mice developed readily detect-
able plasma titers of IgM by the tenth week after transfer (Figure
mice that received only PBS did not have any
plasma IgM. In a series of 8 similar transfer experiments, the extent
of reconstitution, as indicated by the levels of plasma IgM, varied
and in part appeared to be related positively to the number of B-1
cells transferred and the time after transfer studied. Remarkably, in
all of the transfers, the recipient mice exhibited robust IgM titers
against oxidation-specific epitopes (Figure 3B), but not native LDL
(data not shown), and the prevalence of oxidation-specific IgM
appeared similar to that observed in naive wild-type mice. In Fig
ure 3B, we show Ab binding dilution curves to oxidation-specific
epitopes and other antigens for a typical transfer of approximately
99% pure B-1 cells. The titers ranged from 1:800 to 1:6,400 for
oxidation-specific epitopes versus 1:400 for
α1,3-dextran and PC-
BSA. Moreover, consistent with the fact that T15-idiotypic Abs are
Characterization of Rag1
recipients adoptively transferred with B-1
cells. (A) Adoptive transfer of B-1 cells into Rag1
B-1 cell population. Rag1
mice were injected with PBS (Rag1
PBS) or with B-1 cells (Rag1
+ B-1). Rag1
+ PBS: Lymphocyte
populations were absent in the peritoneal cavity (PEC, left). B-1 cells
) were also absent from the spleen (Spleen, left). C57BL/6:
Peritoneal macrophages (CD11b
) and T cells (CD11b
were intact (PEC, upper middle). B cells could be divided into B-1a
), B-1b (CD19
), and B-2 cells
) (PEC, lower middle). In the spleen, B-1 cells
were about 2.4% of total splenocytes (Spleen, middle). Rag1
B-1 cell populations were reconstituted in the peritoneal cavity (PEC,
lower right) and spleen (Spleen, right), without B-2 cell or T cell con-
tamination (PEC, right). (B) IgM Abs to oxidation-specific epitopes are
present in the plasma of B-1 reconstituted Rag1
mice. Plasma col-
lected after 15 weeks from Rag1
+ B-1 (n = 8) or Rag1
(n = 6) and age-matched C57BL/6 mice (n = 7) were tested. Data
shown are from 1 transfer experiment representative of 6. Values
are mean and SEM. Numbers in the upper-right corner represent the
IgM titer to each antigen. (C) Natural IgM Abs produced in vivo show
specificity to MDA-LDL and CuOx-LDL. Data are the mean of triplicate
determinations, expressed as the ratio of IgM binding to MDA-LDL or
CuOx-LDL in the presence or absence of competitor (B/B
). Data are
from 1 experiment representative of 3.
1340 The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009
predominantly secreted by B-1 cells (24), we found that adoptive
B-1 cell transfer gave rise to E06/T15-idiotypic IgM in
as well (Figure 3B, bottom middle panel).
Whereas the extent of plasma IgM measured at 10 weeks was gen
erally lower in B-1 cell–reconstituted
than in wild-type mice
in all the experiments noted above, in which greater than 99% pure
B-1 cells were transferred, in one experiment, in which a small con
tamination of T cells inadvertently occurred (estimated to be <3% of
all peritoneal cells at sacrifice), the IgM levels actually equaled those
of wild-type C57BL/6 mice (Supplemental Figure 3). Presumably,
cotransferred T cells promoted IgM secretion in the recipient mice,
possibly in part by secretion of cytokines such as IL-5, which, as we
have previously shown, augments secretion of OxLDL-specific IgM
by B-1 cells in a non-cognate manner (Figure 2A and ref. 25).
Again, oxidation-specific IgM Abs were a major fraction of the
total IgM. Competition immunoassays with pooled plasma of
recipient mice demonstrated high specificity of the MDA-specific
IgM Abs, as neither OxLDL nor native LDL competed for binding to
MDA-LDL (Figure 3C). We also calculated binding avidities for the
IgM in the B-1 cell–reconstituted
plasma for oxidation-spe-
cific epitopes as described above. The apparent
s for MDA-LDL
and OxLDL were 9.9
and 1.42 × 10
mol/l respectively. These
values are similar to the
s of 6.85 × 10
mol/l determined for the
MDA-LDL–specific natural mAb NA-17, cloned from the spleen of a
B-1 cell reconstituted
mice as described below. Only very low
titers of IgG against oxidation-specific epitopes (or any other anti
gen) were found in the plasma of recipient mice, and these Abs were
predominantly of the IgG3 isotype, which are known to be secreted
by B-1 cells in a T cell–independent fashion (26) (data not shown).
Oxidation-specific epitopes are dominant targets of natural IgM Abs. To
directly address the extent to which oxidation-specific IgM Abs
contribute to the total IgM NAb pool, we performed absorption
studies using pooled plasma from
mice reconstituted with
B-1 cells. In these experiments, specific IgM Abs were absorbed
from plasma with selected oxidation-specific model antigens and
the amount of remaining IgM measured. Strikingly, MDA-LDL as
well as OxLDL (containing a variety of oxidation-specific epitopes)
absorbed out approximately 10% of all IgM, while native LDL did
Oxidation-specific epitopes are dominant targets of NAbs. (A) Preabsorption of plasma from Rag1
+ B-1 mice with oxidation-specific antigens
shows that oxidation-specific epitopes (OxEpitopes) are dominant targets for NAbs. Plasmas from Rag1
+ B-1 mice were preincubated in the
absence or presence of the indicated antigens (250 μg/ml total antigen) overnight and antigen-immune complexes pelleted by centrifugation.
Total IgM levels were then tested by ELISA. *P < 0.05, **P < 0.01, ***P < 0.002 compared with native LDL (ANOVA with Tukey-Kramer multiple
comparisons test). Data are means (and SEM) from 5 separate experiments, each using 3–7 plasma samples obtained from 5 different transfer
experiments, with each sample assayed in triplicate. (B) ELISpot assay of frequencies of MDA-LDL–specific ISCs in the spleens of wild-type
+ B-1, and Rag1
+ PBS mice. Results are from individual mice, and data are from 3 separate B-1 cell transfer experiments.
Horizontal bar represents the mean for the group.
P < 0.002 compared with Rag1
+ PBS (unpaired t test). (C) B-1 cell–derived natural mAb
NA-17. DNA sequences of VDJ splice sites of the V
rearrangements expressed in NA-17 B-1 cell hybridoma and their relationship to the
most homologous germline V, D, J gene segments. Sequence analysis of NA-17 V
rearrangement did not reveal nucleotide variation to germline
genes. Sequence analysis of V
rearrangement revealed 1 nucleotide insertion between V
germline gene segments.
The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009 1341
Natural IgM Abs recognize oxidation-specific epitopes present on apoptotic cells and atherosclerotic lesions (A) Natural IgM Abs bind to apoptotic
thymocytes but not normal thymocytes. Apoptotic thymocytes from C57BL/6 mice were incubated with plasma from Rag1
+ B-1 or Rag1
PBS mice at 1:10 dilution, NA-17 at 2.5 μg/ml, or control IgM at 5 μg/ml. Top row: Deconvolution microscopy shows that NAbs in Rag1
+ B-1 as
well as NA-17 bound to apoptotic thymocytes. Bottom row: None of the IgM bound to normal thymocytes (quadrant 1 [Q1). NAbs in plasma from
+ B-1 bound to both early (Q2) and late apoptotic thymocytes (Q3), while NA-17 bound prominently to late apoptotic cells (Q3). Scale
bar: 5 μm. 2°Ab, secondary Ab; Anti-ms-IgM-FITC, FITC-labeled anti-mouse IgM. (B) Natural IgM Abs are present in atherosclerotic lesions.
Endogenous IgM Abs were detected in aortic sections from cholesterol-fed B-1 cell–reconstituted Ldlr
mice (bottom row), but not in
mice (top row). Sections were also stained with MDA2 (5 μg/ml) for the presence of MDA epitopes. Red indicates
positive staining. Original magnification, ×160. (C) NA-17 recognizes oxidation-specific epitopes present in atherosclerotic lesions. Sections
of the brachiocephalic artery from cholesterol-fed Ldlr
mice were stained with NA-17 (0.85 μg/ml) or a control natural IgM Ab, EN-2
(1.6 μg/ml). Original magnification, ×200. (D) NA-17 inhibits MDA-LDL binding to macrophages. Increasing concentrations of NA-17 or control
IgM were added with a fixed amount of biotinylated MDA-LDL (Bt-MDA-LDL; 2 μg/ml) to macrophages. Data are the average of 2 experiments,
expressed as the ratio of biotinylated MDA-LDL binding to macrophages in the presence or absence of IgM (B/B
1342 The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009
not (Figure 4A). Furthermore, the MDA-specific MAA epitope,
conjugated to mouse serum albumin (MAA-MSA), was capable of
absorbing up to 25% of all IgM. Moreover, a combination of all
model antigens removed 35% of the NAbs in these plasmas (Figure
4A). Thus, a surprisingly large percentage of B-1 cell–derived NAbs
are directed against various oxidation-specific epitopes.
The prominent representation of oxidation-specific IgM in
plasma was also reflected by the frequency of MDA-specific ISCs
in the spleens of reconstituted
mice. As expected, no ISCs
were detected in the spleens of mice injected with PBS (Figure 4B).
In contrast, in B-1 cell–reconstituted mice, approximately 12% of
all ISCs were found to have specificity for MDA-LDL (Figure 4B).
Interestingly, the frequency of MDA-LDL–specific ISCs in spleens
of wild-type mice was found to be similarly high (Figure 4B), dem
onstrating that a large percentage of all splenic ISCs have specific
ity for MDA modifications, and a majority of these ISCs are likely
derived from B-1 cells.
We further corroborated this notion by the characterization
of mAbs derived from hybridomas prepared from the spleens
of B-1 cell–reconstituted
mice. From 2 separate fusions,
we observed that 20%–30% of all IgM-secreting hybridomas had
reactivity for MDA-LDL (data not shown). For example, the DNA
sequence of VDJ splice sites of the V
expressed in one cloned MDA-specific hybridoma (NA-17) dis
played complete germline gene usage of the V
and only 1 nucleotide insertion (C) at the splice site of the V
germline gene segments (Figure 4C; the complete sequence is
presented in Supplemental Figure 4).
B-1 cell–derived natural IgM Abs recognize oxidation-specific epitopes on
apoptotic cells and in atherosclerotic lesions. Oxidation-specific epitopes
are ubiquitously present in inflammatory settings and are present
on apoptotic cells (19, 27). As shown in Figure 5A, plasma IgM from
mice recognized surface epitopes on
apoptotic cells, as demonstrated by immunocytochemistry. Simi
larly, the MDA-LDL–specific NAb NA-17 strongly stained apop
totic cells. Neither plasma from PBS-injected
mice nor a
keyhole limpet hemocyanin–specific (KLH-specific) control IgM
bound apoptotic cells.
Human umbilical cord blood contains natural IgM Abs against oxidation-specific epitopes. (A) Left: Plasma titers of IgM in maternal and umbilical
cord plasma to native LDL, KLH, and oxidation-specific antigens measured by ELISA. Right: Data are plotted as ratio of antigen-specific IgM to
total IgM. ***P < 0.002 compared with maternal blood (Wilcoxon matched-pairs test and paired t test). Data shown are from 10 paired maternal-
infant samples, and each sample was assayed in triplicate. Values are mean and SEM. (B) Umbilical cord IgM binds to apoptotic cells in part via
binding to MDA. Apoptotic Jurkat cells, induced by UV exposure, were incubated with representative umbilical cord plasma (1:50 dilution) in the
absence and presence of MDA-LDL and native LDL (1 mg/ml). Abs bound were detected by FITC-conjugated anti-human IgM. Umbilical cord
IgM binding to apoptotic Jurkat cells (median fluorescence intensity [MFI], 1,917) was inhibited 45% by MDA-LDL (MFI, 1,047) while minimally
affected by native LDL (MFI, 1,514).
The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009 1343
We also tested IgM binding to apoptotic cells by flow cytometry
in relation to measures of cellular apoptosis (Figure 5A). Plasma
IgM from reconstituted
mice bound to apoptotic cells with
early (quadrant 2 [Q2]) and late (Q3) stages of apoptosis, but not
viable cells (Q1). Monoclonal NA-17 stained almost the entire
population of late apoptotic cells. To further test whether these
IgM NAbs recognize their cognate epitopes in vivo, we transferred
B-1 cells into
mice that had been fed a high-cholester-
ol diet for 10 weeks to induce atherosclerotic lesions, which accu
mulate apoptotic cells and OxLDL (28, 29). The mice were main
tained on the atherogenic diet for an additional 6 weeks and then
sacrificed, and atherosclerotic lesions were assessed for the depo
sition of endogenous Abs (Figure 5B). As expected, neither IgM
nor IgG Abs were present in lesions of
with PBS alone (Figure 5B, top row). In contrast, IgM accumulated
in lesions of B-1 cell–reconstituted mice, at sites likely reflecting
the edges of lesions at the time of engraftment of the transferred
B-1 cells (Figure 5B, bottom row). In part, these IgM Abs were
binding to endogenous MDA epitopes in the lesions, which were
found in comparable (but not identical) sites, as demonstrated
by immunostaining of adjacent sections using MDA2, an MDA-
specific murine monoclonal IgG we previously cloned (30) (Figure
5B). Presumably MDA epitopes bound by endogenous IgM would
also not be available to bind MDA2. Moreover, consistent with our
earlier observation that only low IgG titers are secreted by B-1 cells,
no IgG Abs were found in lesions of these mice.
Monoclonal NA-17 also recognized MDA epitopes in atheroscle
rotic lesions of
mice (Figure 5C), whereas a control
natural IgM did not. We also found that NA-17 could substan
tially inhibit the binding of MDA-LDL to J774 macrophages in a
dose-dependent fashion, whereas a KLH-specific IgM showed only
nonspecific inhibition (Figure 5D).
Natural IgM Abs in human cord blood recognize oxidation-specific epi-
topes. We previously showed that IgG and IgM titers to oxidation-
specific epitopes are present in nearly all adult human plasmas
tested (4). However, we wanted to know whether the human IgM
NAb repertoire displays binding to oxidation-specific epitopes
similar to those observed in mice. IgM Abs found in umbilical
cord blood are exclusively from the infant and are considered to
represent the human equivalent of naive NAbs (31). Therefore,
we characterized the binding properties of IgM in umbilical cord
blood and in the respective maternal plasma samples. Impor
tantly, umbilical cord plasma contained prominent IgM titers
against MDA-LDL and OxLDL, but not native LDL or KLH (Fig
ure 6A). In contrast, maternal plasma also contained IgM against
KLH, presumably due to exogenous antigen exposure (Figure 6A).
Unlike IgM titers, IgG titers were found to be similar in maternal
and umbilical cord plasma (data not shown), consistent with the
ability of maternal IgG to be actively transferred across the pla
centa. As the total IgM titers were found to be generally higher
in maternal samples, we calculated the ratios of oxidation-spe
cific IgM to total IgM in individual samples. This revealed that
MDA-LDL– and OxLDL-specific IgM — but not KLH-specific IgM
— are relatively enriched in umbilical cord plasmas compared with
maternal plasmas (Figure 6A), which typically also contain adap
tive IgM Abs (e.g., against KLH).
We further showed that human umbilical cord IgM also bound
to apoptotic cells (Figure 6B). This binding was at least in part
mediated through the recognition of MDA epitopes, as, in the
example shown, MDA-LDL competed for up to 45% of the bind
ing of the umbilical cord IgM to apoptotic cells. Thus, oxidation-
specific epitopes also appear to be a dominant target for natural
IgM Abs in humans.
NAbs facilitate apoptotic cell uptake by macrophages in vivo. Natural
IgM Abs and the MDA-LDL–specific NAb NA-17 recognize oxi
dation-specific epitopes on apoptotic cells (Figure 5A). When not
promptly cleared, apoptotic cells are immunogenic and proinflam
matory (17, 18). To test the hypothesis that these NAbs maintain
homeostasis against oxidatively modified structures, we examined
their ability to mediate enhanced clearance of apoptotic cells in
vivo. Using a previously described model (32), we compared apop
totic cell uptake by macrophages in vivo in
mice 10 weeks
after reconstitution with B-1 cells or with PBS. Mice were injected
i.p. with fluorescently labeled apoptotic thymocytes 4 days after
induction of sterile peritonitis with thioglycollate. Macrophage
Natural IgM Abs against oxidation-specific epitopes facilitate apoptotic
cell uptake by macrophages in vivo. (A) Percentage of macrophages
that contained fluorescently labeled apoptotic thymocytes following i.p.
injection. In RAG + PBS mice, about 27% of macrophages phago-
cytosed apoptotic cells. The percentage was significantly increased,
to 33%, in RAG + B-1 mice (*P < 0.05, unpaired t test). Horizontal
bars denote means. (B) Apoptotic thymocytes were preincubated with
NA-17 or a control IgM that does not bind apoptotic cells before injec-
tion into Rag1
mice. The phagocytic uptake was significantly differ-
ent between the 3 groups (P = 0.01, 1-way ANOVA). The percentage
of macrophages taking up apoptotic cells was significantly increased
when the apoptotic cells were preincubated with NA-17 (RAG + NA-17),
compared with control IgM (RAG + control IgM) or without preincu-
bation (RAG) (32% vs. 20% vs. 23%; *P < 0.05, Bonferroni multiple
comparison test). Horizontal bars denote means.
1344 The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009
uptake of apoptotic thymocytes was significantly enhanced in B-1
mice compared with PBS controls (33%
P < 0.05; Figure 7A).
To directly test the impact of an oxidation-specific mAb, we pre
incubated the apoptotic cells with NA-17 or with a control IgM
(specific for KLH) before injection into
mice. The percent-
age of macrophages that had taken up apoptotic thymocytes was
mice receiving apoptotic thymocytes preincu-
bated with control IgM or untreated apoptotic thymocytes (20%
P = NS; Figure 7B). In contrast, the phagocytic uptake
increased to 32% when the apoptotic cells were preincubated with
P < 0.05), demonstrating that binding of NA-17 facilitated
apoptotic cell uptake by macrophages in vivo.
Innate immune responses provide a vital and nonredundant role
in the initial defense against invading pathogens and in maintain
ing homeostasis against a variety of self-antigens. The mediators
of innate immunity are germline encoded, preformed, and utilize a
limited set of PRRs to recognize a set of common PAMPs. It is now
widely recognized that one class of PRRs, the so-called scavenger
receptors, such as SRA-1 and -2, CD36, SR-B1, LOX-1, PSOX, and
others, all recognize various oxidation-specific epitopes present on
OxLDL and apoptotic cells (7). Indeed, recent evidence suggests
that members of the innate TLR family also recognize such epit
opes as well (33, 34). The soluble innate protein CRP also binds to
the PC of OxPL present on OxLDL and apoptotic cells, as well as
S. pneumoniae (20). Thus, oxidation-specific epitopes — as a class
— constitute an important PAMP recognized by innate immunity,
consistent with the oxygen-centric nature of life itself.
NAbs are part of the humoral arc of innate immunity. They are
the product of natural selection, exhibiting a genetically deter
mined and stable repertoire that is largely independent of exter
nal antigenic stimuli (35–37). NAbs have been defined in various
ways, for example, as the IgM present in normal plasma (38), but
more stringently, as germline-encoded Abs that arise without any
exogenous immune exposure or bacterial colonization of the gut
(39). They are produced at tightly regulated and stable levels in
healthy individuals and are found in all vertebrate species (11, 40).
In normal, uninfected mice, most plasma IgM Abs are NAbs that
are derived from innate B-1 cells, which differ from conventional
B-2 cells in surface phenotype; anatomic location; restricted use
genes that are minimally edited and reflective of germline
usage; and, importantly, their capacity for self renewal (reviewed
in refs. 11, 41, 42). Despite this, relatively little is known about
B-1 cell ontogeny, development, and, surprisingly, function.
Only recently has evidence been presented to support a lineage
independent from that of B-2 cells (43–46). Although the mecha
nisms leading to selection and expansion of B-1 cells are unclear,
it is now appreciated that antigen selection during the fetal and
neonatal period leads to
positive selection (11, 47, 48). Since this
appears to occur equally well in mice raised in germ-free environ
ments (35, 49, 50), this selection is of necessity made by endog
enous “self-antigens.” This is in contrast to developing, self-reac
tive B-2 cells, in which such antigen encounter early in life leads
to negative selection via apoptosis (a process known as clonal
deletion) or anergy. Thus, the repertoire of B-1 cells is selected to
bind to evolutionarily important epitopes. Furthermore, because
many, if not most, NAbs have dual specificities (11), typically with
molecular epitopes on pathogens, an encounter later in life with
such external pathogens could further serve to expand a given
B-1 cell clone (12, 51). The example given in the Introduction that
E06/T15, which binds OxPL of OxLDL and apoptotic cells, also
binds to the PCs present on the cell wall of many pathogens nicely
illustrates this paradigm. We report now that as a class, oxidation-
specific neoepitopes constitute a disproportionately large portion
of such self-antigens.
In this article, we provide multiple lines of evidence suggesting
that oxidation-specific epitopes are a dominant target of innate
NAbs in both mice and humans. First, consistent with the view that
the IgM pool of uninfected mice comprises NAbs (11), we show that
SPF-maintained mice as well as germ-free mice maintained under
strict gnotobiotic conditions contained prominent IgM titers to
oxidation-specific epitopes compared with titers against recognized
TI-2 antigens, such as
α1,3-dextran. Consistent with this, there was
a high frequency of ISCs to oxidation-specific epitopes, chiefly
MDA, in spleens of wild-type mice. Of interest, the introduction of
normal gut flora into the germ-free mice (conventionalized mice
in Figure 1D) increased the titers to OxLDL, PC-BSA, 4-HNE-LDL,
α1,3-dextran. After birth, B-1 cells are typically thought to
have minimal response to exogenous antigenic stimuli, but some,
such as E06/T15 (which binds to the PC of OxPL in OxLDL and
PC-BSA), are known to be responsive (11, 14).
Second, B-1 cells in culture, purified from naive uninfected mice,
gave rise to an array of oxidation-specific IgM Abs, which were
much more prevalent than IgM to the classic antigen
tran (42). Indeed, such IgM Abs were already detectable in super
natants of unstimulated B-1 cells in culture, and the production
of these IgM Abs in response to stimulation, especially that of
TLRs, increased to a greater extent than that of total IgM or IgM
α1,3-dextran. Moreover, by analogy to the robust anti-MDA
responses in vivo, MDA-specific IgM Abs were the most dominant
set of IgM Abs secreted in vitro, constituting up to 30% of total
IgM. A similar specificity was seen for the IgM found in plasma of
mice following adoptive transfer of B-1 cells. Absorption
studies demonstrated that 20%–35% of these IgM Abs had specific
ity for combinations of oxidation-specific epitopes, predominantly
MDA-related epitopes. These data were further corroborated by
ELISpot analysis of splenocytes of
B-1 recipients, which
demonstrates that ISCs against MDA-LDL accounted for 10%–12%
of all ISCs in the spleens of the reconstituted mice, similar to the
frequency found in spleens of wild-type mice. In addition, study
of hybridomas generated from the spleens of the B-1 cell–recon
stituted mice indicated that 20%–30% of all IgM-secreting clones
bound to oxidation-specific epitopes, mostly MDA. Cloning and
sequencing of a number of these confirmed their germline origin,
as exemplified by the MDA-specific B-1 clone NA-17.
Third, these NAbs bound prominently to biologically relevant
oxidation-specific epitopes. B-1 cell–derived IgM in the plasma of
the reconstituted mice bound prominently to epitopes on apoptotic
cells and to atherosclerotic lesions and colocalized with oxidation-
specific epitopes in vivo in atherosclerotic lesions. Furthermore,
monoclonal NA-17 bound prominently to apoptotic cells and to
atherosclerotic lesions. These data strongly support the hypothesis
that oxidation-specific epitopes are a dominant target of NAbs in
mice, which in turn bind to biologically relevant self-antigens.
Fourth, IgM Abs in umbilical cord blood are solely of fetal origin
and represent such naive NAbs in humans (31). The presence of an
enriched titer of oxidation-specific IgM Abs in human umbilical
cord blood, which bind to apoptotic cells in part via MDA-LDL
The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009 1345
epitopes, strongly suggests that oxidation-specific epitopes are an
important target of NAbs in humans as well. Interestingly, Merbl
et al., using a sensitive antigen-array screening technique, recently
tested the specificities of autoreactive NAbs in human cord blood
(31) and reported that the antigen to which such NAbs most
commonly bound was native LDL. However, as noted in Results,
though we often find low levels of binding to plated LDL, this can
not be competed by the same LDL in solution, suggesting that the
LDL undergoes modification during the plating procedure (either
structural, e.g., via oxidation, or even conformational). Indeed, the
LDL used in the studies of Merbl et al. was bought from a com
mercial source, and we speculate that it may have been oxidized at
the time of use. Thus, their data may be consistent with our obser
vations that oxidation-specific epitopes are a dominant target of
NAbs found in humans.
The apparent high prevalence of oxidation-specific epitopes as
targets of NAbs in mice and humans likely reflects the ubiquitous
presence of these epitopes consequent to oxidative events. Cells
undergoing apoptosis and the microparticles shed from them are
rich in oxidation-specific epitopes (15, 18, 52). Because this process
is universal, we speculate that they could be fundamental select
ing antigens. Further, inflammatory events are associated with
enhanced oxidative stress, and our different oxidation-specific
Abs have been used to demonstrate the presence of these epitopes
not only in atherosclerotic tissue, but in a variety of inflammatory
settings, including renal, liver, and pulmonary disease (53–55),
rheumatoid arthritis (our unpublished observation), CNS lesions
found in multiple sclerosis (56), and Alzheimer disease (57), all of
which would serve to provide stimulation later in life for particu
lar B-1 cell clones. Finally, many if not most NAbs also react with
microbes that contain the same or similar antigenic self-determi
nants, leading to cross-reactivity between self-determinants and
microbial antigens. The classic natural IgM E06/T15, for example,
binds PC of OxPL as well as PC as part of the capsular polysaccha
ride of pneumococci and many other microbes. Thus, postnatal
stimulation of particular B-1 cell clones likely occurs upon expo
sure to molecular equivalents on pathogens.
Although the selection and expansion of B-1 cell clones clearly
occurs in both fetal and postnatal life, the mechanisms leading
to the activation of B-1 cells, conversion to plasma cells, and gen
eration of NAbs are poorly understood. The mode of presenta
tion of antigen that results in productive B-1 cell expansion is
generally considered to be independent of cognate T cell help.
Although some TI-2 multivalent antigens may lead to direct
crosslinking of B cell receptor on B-1 cells and IgM production,
in general most B-1 cell antigens do not (58). However, B-1 cells
are known to be responsive to several non-cognate stimuli that
also stimulate B-2 cells, including LPS and T cell cytokines such
as IL-5. In our studies, we also found that NAbs specific for oxi
dation-specific epitopes are partially dependent on T cell help.
First, we demonstrated in culture that IL-5 stimulated B-1 cells
to secrete IgM to oxidation-specific epitopes. Second, we found
that anti-OxLDL IgM titers were lower in age-matched
T cell–deficient mice compared with wild-type mice (Supple
mental Figure 1) and that the B-1 cell transfer experiments with
contaminating T cells resulted in more robust IgM titers in the
recipients, including titers to OxLDL. Likely, the in vivo data can
be explained in part by the secretion of IL-5, which we have pre
viously shown to provide non-cognate help for the production
of E06/T15 IgM in vivo in atherosclerotic mice (25). However,
both T cell–deficient mice and
recipients of pure B-1 cells
exhibited IgM Abs against oxidation-specific epitopes, indicating
that T cell cytokines were not obligatory.
We also demonstrated that TLR4 and TLR2 agonists were par
ticularly effective in stimulating the generation of IgM by B-1 cells
in culture, an effect that was in large part MyD88 dependent. A
similar observation was reported by Genestier et al. while this arti
cle was in preparation (59). Remarkably, however, our data indi
cate that TLR activation seems to have preferentially expanded
the number of B-1 clones secreting IgM to oxidation-specific epi
topes as compared with those secreting total IgM or IgM to
dextran (Figure 2A). This would imply that B-1 clones secreting
such IgM are more responsive to activation by TLRs, suggesting
evolutionary pressure linking generation of NAbs to oxidation-
specific epitopes and activation of innate PRRs. This is intriguing,
as we and others have provided data suggesting that aside from
products of exogenous pathogens, there are endogenous antigens
capable of stimulating such TLRs, including the oxidized moi
eties of OxLDL (60, 61). Obviously much study will be needed to
explore such speculations.
NAbs have been said to be polyreactive and to bind to many auto
antigens nonspecifically. However, we suggest that these observa
tions may be confounded by the fact that a given NAb against a
specific oxidation-specific epitope may bind to the same structural
modification on many different proteins or even lipids. For exam
ple, E06 binds to the PCs moiety on the surface of bacteria, as well
as to the PC headgroup of OxPL, which is found in OxLDL in both
the lipid phase and covalently bound to apoB. E06 binds to OxPL
covalently bound to apo(a) as well as OxPL in the lipid phase of
Lp(a) (62). It also binds to the OxPLs present on the cell surface
of apoptotic cells (19) and to the OxPLs generated in the lungs
of mice and humans infected with viruses (55). Thus, it binds not
only to atherosclerotic lesions, but to a large number of tissues in
which inflammation exists, presumably to the PC epitope gener
ated as result of lipid peroxidation and/or apoptosis. If one did not
know the true identity of the epitope of the NAb E06, one would
say this Ab was “polyreactive.” In this article, we identify an even
more prominent set of natural IgM directed against MDA epitopes
(which are in fact a complex set of related epitopes) that are gener
ated as a consequence of lipid peroxidation. The highly reactive
MDA may similarly modify a wide range of substrates, including
proteins, lipids, and even DNA (63), generating MDA neoepitopes
in a variety of pathophysiological events, such as ischemia and
reperfusion, diabetes, and atherosclerosis, as well as inflammatory
events in the brain (64). Indeed, we speculate that many of the so-
called self-antigens that have been reported to be targets of NAbs
may well be modified self-antigens.
Functionally, NAbs play an important role in providing the
first line of defense against viral and bacterial pathogens (65–67).
NAbs also have important activities in homeostasis (68), includ
ing immunoregulatory functions, tumor surveillance, and rec
ognition and removal of senescent cells, cell debris, and other
self-antigens, which if persisting would be proinflammatory and
immunogenic. Indeed, mice that cannot secrete IgM Abs and
thus lack natural IgM have been found to more readily develop
autoimmune disease when crossed on a susceptible background
(69). Here we demonstrate that both murine natural IgM as well
as human umbilical cord IgM bind apoptotic cells, but not nor
mal cells, in part through binding to oxidation-specific epitopes
on their cell surface. IgM Abs in general have been shown to be
1346 The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009
required in complement-mediated clearance of apoptotic cells
(70). In the present work we explicitly demonstrate that pure IgM
NAbs facilitate macrophage uptake of apoptotic cells in vivo and,
specifically, that the MDA-specific NAb NA-17 similarly promotes
clearance. Previously, the OxPL-specific NAb E06/T15 was also
shown to facilitate efficient complement-mediated phagocytosis
of apoptotic cells in vivo (71). These data demonstrate an impor
tant role for oxidation-specific natural IgM in mediating clearance
of apoptotic cells. There are likely many other roles as well. Oxi
dized lipids have increasingly been shown to be proinflammatory.
Apoptotic cells and their apoptotic blebs can activate endothelial
cells, and E06 can block these effects (19, 52). Recently it was
shown that multiple lung pathogens, such as chemical agents,
H5N1 avian flu, or SARS, which are associated with high lethality
due to acute respiratory distress syndrome, induce robust OxPL
formation in the lung. In turn, the OxPLs induce lung injury and
cytokine production by lung macrophages, and this latter effect
was ameliorated by E06/T15 in vitro (55). As another example,
we have previously shown that enhancing the titer of E06/T15
is atheroprotective (14), in part through inhibiting the uptake
of OxLDL by macrophages. We now show that NA-17 also has
the ability to inhibit binding of MDA-LDL in a similar manner. A
great deal of work will be needed to assess the overall functional
role of such oxidation-specific NAbs in atherosclerosis in particu
lar and in homeostasis in general.
In conclusion, oxidation-specific epitopes constitute a previ
ously unrecognized but important target of NAbs, and of innate
immunity in general. NAbs may be beneficial not only for defense
against pathogens but also in identifying altered-self produced as a
result of oxidative stress under inflammatory events. Understand
ing their role in health and disease may lead to novel diagnostic
and possibly therapeutic approaches to deal with consequences of
oxidative stress such as atherogenesis.
Animals. C57BL/6 and Rag1
mice (The Jackson Laboratory) and Ldlr
mice (a gift from Godfrey Getz and Katherine Reardon, Univer-
sity of Chicago, Chicago, Illinois, USA), all on C57BL/6 background, were
bred and maintained in our colony under SPF conditions unless otherwise
noted. Plasma was obtained from 6-week-old female C57BL/6 T cell recep-
) mice (courtesy of Stephen Hedrick, UCSD).
Fourteen- to 16-week-old germ-free Swiss Webster mice were maintained
at the Göteborg University vivarium under strict gnotobiotic conditions.
Sterility was monitored by stool culturing and PCR for bacterial DNA. Con-
ventionally raised mice were transferred to gnotobiotic isolators at weaning
and fed the same autoclaved chow diet. To obtain conventionalized mice,
13- to 14-week-old germ-free mice were colonized with cecal content from
12-week-old conventionally raised Swiss Webster donors for 14 days.
All experimental protocols were approved by the Animal Subjects Com-
mittee at UCSD; care and use of the germ-free mice was approved by the
Göteborg University Animal Studies Committee.
B-1 cell isolation. Peritoneal exudate cells (PECs) from 6 -to 15-week-old
naive C57BL/6 mice were harvested by peritoneal lavage using ice-cold
PBS supplemented with 1% heat-inactivated FCS (Invitrogen). PECs were
incubated with an anti–Fcγ receptor mAb (clone 2.4G2; BD Biosciences
— Pharmingen) for 15 minutes at 4°C before being stained with fluo-
rescently labeled mAbs to block nonspecific binding. PECs were stained
with R-PE–labeled anti-CD19 (1D3), FITC-labeled anti-CD23 (B3B4),
and in some experiments PE-Cy5–labeled anti-CD3 (clone 145-2C11) (all
from BD Biosciences — Pharmingen). B-1 cells were sorted to greater than
99% purity using a FACSVantage SE cell sorter (BD) as the CD3
B-1 cell cultures. Purified B-1 cells were seeded at 1 × 10
cells per well in
24-well flat-bottom plates in culture medium (RPMI 1640 medium con-
taining 10% heat-inactivated FCS, 10 mM HEPES buffer, 2mM l-gluta-
mine, 0.05 mM 2-mercaptoethanol, 50 μg/ml gentamicin) in the presence
and absence of the specific TLR4 agonist KdO
-Lipid A (100 ng/ml; Avanti
Polar Lipids), a combination of the TLR2 agonists Pam3CSK4 (300 ng/ml)
and FSL-1 (1 μg/ml) (Invivogen), or vehicle control in triplicate in a final
volume of 500 μl. Cells were incubated at 37°C/5% CO
for up to 7 days.
Adoptive transfer of B-1 cells. B-1 cells were isolated from the peritoneum of
6- to 15-week-old female donor C57BL/6 mice as described above. Purified
B-1 cells were resuspended in PBS, and 0.5 or 1 × 10
cells in 200 μl were
injected into the peritoneal cavity of 6- to 15-week-old Rag1
to 4 donor mice were used for each recipient. Control Rag1
an equal volume of PBS. Blood was collected via the retro-orbital plexus
from recipient mice before and 4 and 10 weeks after transfer.
Flow cytometry. At the time of sacrifice, PECs and splenocytes were resus-
pended in staining buffer. After blocking with a specific anti–Fcγ receptor
mAb (2.4G2) for 15 minutes at 4°C, 10
cells were stained with fluores-
cently labeled mAbs specific for various surface markers (FITC-labeled
anti–CD11b/Mac-1 [M1/70], PE-labeled anti-CD5, PerCP-Cy5.5–labeled
anti-CD19 [1D3]; PE-labeled anti–mouse CD43 [S7], and APC- or FITC-
labeled anti-mouse IgM [II/41]; all from BD Biosciences — Pharmingen) in
100-μl volumes of staining buffer for 30 minutes at 4°C in darkness, fol-
lowed by extensive washing. Cell populations were analyzed on a BD FACS-
Calibur or FACScan instrument. More than 0.5 × 10
cells were analyzed
per sample, with dead cells excluded by forward and side scatter. Surface
marker analysis was performed using FlowJo software (Tree Star Inc.).
Measurement of Ab titers. Specific Ab titers to given antigens in plasma or
cell culture supernatants were determined by chemiluminescent ELISA as
previously described (14, 23). Ab dilution curves of Ab binding to plated
antigens were determined by serial dilutions of plasma or culture superna-
tant, and a titer was defined as the highest dilution that yielded binding
that was 2-fold greater than the background level. Purified rat anti-mouse
IgM (II/41; BD Biosciences — Pharmingen) was used as capture Ab to mea-
sure total IgM levels. AP-labeled goat anti-mouse IgM (μ chain specific) and
anti-mouse IgG (γ chain specific) (Sigma-Aldrich) were used as detection
Abs, as well as biotinylated rat anti-mouse IgM (R6-60.2; BD Biosciences
— Pharmingen). To detect other Ig isotypes, rat anti-mouse IgG1 (A85-3),
IgG2a/c (R11-89), IgG2b (R9-91), IgG3 (R2-38), and IgA (C10-3) were used
as capture Abs; biotin-conjugated rat anti-mouse IgG1 (A85-1), IgG2a/c
(R19-15), IgG2b (R12-3), IgG3 (R40-82), and IgA (C10-1) (all from BD Bio-
sciences — Pharmingen) were used as secondary Abs. To detect the levels of
E06, a T15-specific anti-idiotype Ab (AB1-2) (72) was used as capture Ab,
followed by incubation with AP-labeled goat anti-mouse IgM. Biotin-con-
jugated Abs were then detected with AP-conjugated neutravidin (Pierce,
Thermo Scientific). Mouse anti-human IgG (G18-145) and IgM (G20-127;
BD Biosciences — Pharmingen) were used as capture Abs to measure total
IgM and IgG levels in humans. AP-labeled goat anti-human IgG and IgM
(A3187 and A3437; Sigma-Aldrich) were used as detection Abs. The follow-
ing antigens were prepared as described previously (29): copper sulfate–oxi-
dized LDL (CuOx-LDL), 4-HNE-LDL, MDA-LDL prepared from human
LDL, and 4-HNE-MSA prepared from MSA. MAA-BSA was prepared as
described previously (73). α1,3-dextran was a gift from John F. Kearney
(University of Alabama at Birmingham, Birmingham, Alabama, USA). PC-
BSA and PC-KLH were from Biosearch Technologies Inc., and KLH was
from Pierce Biotechnology.
Immunocompetition assays. The specificity of IgM Abs binding to MDA-
LDL was determined by competition immunoassays as described previ-
The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009 1347
ously (14, 23). Plasma from B-1 cell–recipient Rag1
mice was pooled and
diluted to 1:100 for MDA-LDL and 1:200 for CuOx-LDL, or B-1 cell super-
natants were incubated overnight at 4°C in the presence and absence of
increasing concentrations of competitors. Samples were then centrifuged
at 15,800 g for 45 minutes at 4°C and supernatants analyzed for binding
to the respective antigen by chemiluminescent ELISA.
To determine the percentage of total IgM in plasma binding to specific
antigens, plasma samples were diluted in 1% BSA-TBS to yield a limit-
ing dilution for each antigen, as determined in preliminary experiments.
Diluted plasma samples were incubated overnight at 4°C in the absence
or presence of individual or combinations of antigens at a final concen-
tration of 250 μg/ml. Thereafter, samples were centrifuged at 15,800 g
for 45 minutes at 4°C to pellet immune complexes and supernatants
analyzed for total IgM content by chemiluminescent ELISA, using a
monoclonal rat anti-mouse IgM to capture and a polyclonal alkaline
phosphatase–labeled (AP-labeled) goat anti-mouse IgM (μ chain specific)
for detection. Data were calculated as the amount of IgM remaining
in the supernatant after absorption, expressed as a percentage of total
IgM. In some experiments, we calculated the apparent Ab avidity of IgM
binding to a given antigen by analysis of the competition assays using
the Klotz method (23).
ELISpot assay. The frequencies of total and antigen-specific IgM–secret-
ing splenocytes were quantified by ELISpot assay as described previously
(14). Splenocytes were suspended in culture medium at 2 × 10
and 1 × 10
cells/100 μl and cultured in triplicate in washed and blocked 96-well Multi-
Screen-HA sterile nitrocellulose plates (Millipore) that had been coated
overnight with 4 μg/ml of rat anti-mouse IgM (II/41), MDA-LDL, OxLDL,
4-HNE-LDL, or α1,3-dextran. After 22 hours incubation at 37°C/5% CO
cells were removed by washing, and ISCs were detected using biotinylated rat
anti-mouse IgM, followed by HRP-streptavidin (Zymed Laboratories Inc.,
Invitrogen). Plates were developed using a tetramethylbenzidine membrane
substrate system (KPL), and spots were quantified using an automated
ImmunoSpot Image Analyzer (Cellular Technology Ltd.).
Apoptotic cells and immunofluorescence microscopy. Thymocytes harvested
from C57BL/6 mice were cultured in cell culture medium and induced
to undergo apoptosis by 10 ng/ml PMA (Sigma-Aldrich) for 16 hours as
described previously (18). Plasma, NA-17, or control IgM (C48-6, anti-
KLH; BD Biosciences — Pharmingen) diluted in 1% BSA-PBS was incu-
bated with apoptotic thymocytes for 30 minutes at 4°C. Cells were washed
and incubated with FITC-labeled rat anti-mouse IgM (II/41) in 1% BSA-
PBS for 30 minutes and then washed again. For FACS analysis, cells were
incubated with annexin V and 7-AAD (BD Biosciences — Pharmingen)
for 15 minutes and immediately analyzed by BD FACSCanto. Umbilical
plasma IgM binding to apoptotic Jurkat cells was detected by FITC-conju-
gated goat anti-human IgM (Jackson ImmunoResearch Laboratories Inc.).
Apoptosis of Jurkat cells was induced with UV irradiation at 20 mJ/cm
followed by further incubation of the cells in medium for 14–16 hours
before use. For immunofluorescence microscopy studies, cells were incu-
bated with 2 μg/ml Hoechst dye (Sigma-Aldrich) for 10 minutes, fixed
with 2% paraformaldehyde, and spun down on glass slides using a cyto-
spin (StatSpin). Images were captured by deconvolution microscopy using
a DeltaVision deconvolution microscopic system operated by softWoRx
software (Applied Precision) as described previously (18).
Immunocytochemistry. Frozen-embedded sections (embedded in Tissue-
Tek OCT compound [Sakura Finetek]) of aortic origin from cholesterol-fed
mice were fixed with methanol, blocked with 2% goat serum,
and stained with 5 μg/ml MDA2, 1.6 μg/ml EN2, or 0.8 μg/ml NA-17
(29), followed by a biotinylated goat anti-mouse IgG or anti-mouse IgM
(Jackson ImmunoResearch Laboratories Inc.) to detect endogenous NAbs
in the lesions. A Vectastain ABC-AP kit and a Vector Red AP chromogenic
substrate (Vector Laboratories) were used to visualize the Ab staining.
Slides were counterstained with Weigert’s iron hematoxylin (Richard-Allan
Scientific, Thermo Scientific). Immunostaining of adjacent sections in the
absence of primary Ab was used as a negative control.
Cloning and genetic analysis of hybridoma NA-17. Spleens of Rag1
reconstituted with B-1 cells were used to prepare B-1–derived hybrid-
omas using techniques established in our laboratory (29). Productive
hybridomas, by default, should only secrete NAbs. In brief, splenocytes
from 4 Rag1
B-1 recipients were fused with the P3 × 63Ag8.653.1
myeloma cell line. Primary screening of supernatants was performed
after 10 days of growth for the ability to secrete IgM and subsequently
for IgM binding to MDA-LDL by chemiluminescent ELISA. Selected
hybridomas were then cloned by limiting dilution (8). Here we report
on clone NA-17, which was confirmed by DNA sequencing to be a NAb.
Cloning and sequence analysis of IgM NA-17 hybridoma V
accomplished as previously described (12).
Human samples. Blood samples from healthy pregnant women and their
newborn infants delivering at Magee-Womens Hospital (Pittsburgh, Penn-
sylvania, USA) were collected as part of a prospective study of preeclampsia,
approved by the Institutional Review Board of the University of Pittsburgh,
for which written informed consent was received from the mothers. Mater-
nal samples were collected in EDTA before delivery (mean, 10.5 hours) and
cord venous samples by sterile aspiration of the umbilical vein after deliv-
ery of the infant. Samples were processed and aliquoted within 3 hours
of collection and stored at –70°C until use. Ten uncomplicated nullipa-
rous maternal-infant pairs were selected for this preliminary analysis. The
mothers (50% of mixed European descent and 50% African American) were
23.4 ± 5.4 years of age, 39.9 ± 1.2 weeks gestational age (values are mean
and SD). There were 6 male and 4 female infants.
In vivo uptake of apoptotic cells by peritoneal macrophages. The in vivo clear-
ance of apoptotic cells was assessed using a modification of the method
described by Taylor et al. (32). Rag1
mice, adoptively transferred with
B-1 cells or with PBS, were injected i.p. with 1 ml of sterile 3% thioglycol-
late to induce sterile peritonitis 15–19 weeks after B-1 cell transfer. Four
days later, the mice were injected i.p. with 20 × 10
apoptotic thymocytes (using 5-CMFDA; Molecular Probes, Invitrogen)
in 200 μl PBS. The mice were sacrificed 40 minutes after injection, and
peritoneal cells were recovered by lavage with 10 ml of ice-cold PBS with
1% heat-inactivated FBS/10 mM EDTA. Macrophages were labeled with
PE-conjugated F4/80 (BM8; eBioscience) and macrophage-specific uptake
of apoptotic cells analyzed by FACS. Phagocytosis was expressed as the
percentage of macrophages ingesting apoptotic cells. To test the ability
of NA-17 to mediate enhanced uptake, fluorescently labeled apoptotic
thymocytes were preincubated with cultured NA-17 hybridoma superna-
tant or anti-KLH IgM (C48-6; BD Biosciences — Pharmingen) at 5 μg/ml
for 1 hour, washed, and then injected into 8- to 10-week-old Rag1
Macrophage binding assay. Binding of biotinylated MDA-LDL to J774
macrophages plated in microtiter wells was assessed by a chemilumines-
cent binding assay as described previously (60). Biotinylated MDA-LDL
(2 μg/ml) was incubated in the absence or presence of NA-17 or anti-KLH
IgM (C48-6; BD Biosciences — Pharmingen) at different concentrations
overnight. Samples were then centrifuged at 15,800 g for 45 minutes at
4°C. The harvested supernatants were then added to macrophages and the
binding of biotinylated MDA-LDL determined by ELISA.
Statistics. Statistical tests used to analyze for significance are described in
the figure legends. The tests used were: 1-way or repeated measures ANOVA
with Tukey-Kramer multiple comparison tests; Wilcoxon matched-pairs
test; and paired t test, using 2-tailed levels of significance. Results were ana-
lyzed with InStat 3 for Macintosh (GraphPad Software). A P value less than
0.05 was considered significant.
1348 The Journal of Clinical Investigation http://www.jci.org Volume 119 Number 5 May 2009
This work was supported by American Heart Association (AHA)
Scientist Development Awards 0530159N (to Y.I. Miller),
0630228N (to K. Hartvigsen), and 0430127N (to P.X. Shaw) and
AHA Postdoctoral Award 0625133Y (to L.F. Hansen); NIH grants
HL086559 (to J.L. Witztum), P50 HL056989 (to J.L. Witztum),
and P01 HL088093 (to J.L. Witztum, Y.I. Miller, and C.J. Binder);
the Austrian Academy of Sciences (to C.J. Binder); the Fondation
Leducq (to J.L. Witztum and C.J. Binder); and grants from the
Swedish Heart and Lung Foundation, the Swedish Society of Med
icine, and the Gothenburg Medical Society (to L. Fogelstrand).
Received for publication July 16, 2008, and accepted in revised
form February 25, 2009.
Address correspondence to: Joseph L. Witztum, University of Cali
fornia, San Diego, Basic Science Building, Room 1080, 9500 Gil
man Drive, La Jolla, California 92093, USA. Phone: (858) 534-4347;
Fax: (858) 534-2005; E-mail: email@example.com. Or to: Christoph
J. Binder, CeMM and Department of Medical and Chemical Labo
ratory Diagnostics, Medical University of Vienna, Währinger Gür
tel 18-20, A-1090 Vienna, Austria. Phone: 43-1-40400-6441; Fax:
43-1-40400-2097; E-mail: firstname.lastname@example.org.
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