Oxidation-specific epitopes are dominant targets of innate natural antibodies in 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 previously observed (8), prominent IgM titers to oxidation-specific epitopes, 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 consistently 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 pathogens. Moreover, we found that oxidation-specific IgM levels were also present in T cell receptor–deficient (Tcra^–/–) 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-specific 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-acetaldehyde adduct) is a specific and prominent chemical moiety generated 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-hydroxynonenal–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 bacteria (“conventionalized mice”) for only 2 weeks led to increases in many — but not all — of the oxidation-specific IgM levels (Figure 1D), strongly suggesting molecular mimicry between many endogenous oxidation-specific epitopes and gut bacterial epitopes. While IgM responses to PC and 4-HNE, as well as to α1,3-dextran, 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 background (Figure 1D). Note again that oxidation-specific IgM Abs constitute a major fraction of total IgM Abs, which were not different 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 preparation 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-1a and 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 (KdO₂-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 Myd88^–/– mice, we observed that the responses to both TLR4 and TLR2 agonists were in large part MyD88 dependent (data not shown). Interestingly, the basal secretion of IgM to OxLDL and MDA-LDL was strikingly more prominent than that of IgM against α1,3-dextran (Figure 2A), and in response to stimulation with the TLR agonists, the oxidation-specific IgM increased to a greater extent than did the total IgM, or the IgM to α1,3-dextran, for example (Supplemental Figure 2). Basal titers to PC-BSA were relatively low and increased only in response to IL-5. PC is an epitope for some OxLDL-specific IgM, but not all.

By analogy to the robust anti-MDA responses in vivo, MDA-specific 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 K_ds for MDA-LDL and OxLDL were 1.46 × 10^–7 mol/l and 4.03 × 10^–9 mol/l, 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 Rag1^–/– recipient mice, which lack functional B and T cells. This led to the selective reconstitution of only B-1 cells in the peritoneum of Rag1^–/– recipients (Figure 3A, top row), and both B-1a (CD5⁺CD19⁺CD11b⁺) and B-1b cells (CD5^–CD19⁺CD11b⁺) were detected (Figure 3A, middle row), whereas conventional B-2 cells and T cells were typically not found in recipient mice. The average percentages of B-1a and B-1b cells among total cells analyzed in the peritoneum of Rag1^–/– B-1 recipients were 11.0% ± 2.6% and 8.5% ± 1.0% respectively (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 Rag1^–/– 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 2.7% (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 Rag1^–/– B-1 recipients (n = 9) and C57BL/6 mice (n = 7), respectively.

The B-1 cell–reconstituted Rag1^–/– mice developed readily detectable plasma titers of IgM by the tenth week after transfer (Figure 3B), while Rag1^–/– 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 Figure 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 predominantly secreted by B-1 cells (24), we found that adoptive B-1 cell transfer gave rise to E06/T15-idiotypic IgM in Rag1^–/– mice as well (Figure 3B, bottom middle panel).

Whereas the extent of plasma IgM measured at 10 weeks was generally lower in B-1 cell–reconstituted Rag1^–/– 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 contamination 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 Rag1^–/– plasma for oxidation-specific epitopes as described above. The apparent K_ds for MDA-LDL and OxLDL were 9.9 × 10^–9 and 1.42 × 10^–7 mol/l respectively. These values are similar to the K_ds of 6.85 × 10^–8 mol/l determined for the MDA-LDL–specific natural mAb NA-17, cloned from the spleen of a B-1 cell reconstituted Rag1^–/– mice as described below. Only very low titers of IgG against oxidation-specific epitopes (or any other antigen) 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 Rag1^–/– 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 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 Rag1^–/– 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), demonstrating that a large percentage of all splenic ISCs have specificity 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 Rag1^–/– 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_H and V_L rearrangements expressed in one cloned MDA-specific hybridoma (NA-17) displayed complete germline gene usage of the V_H rearrangement and only 1 nucleotide insertion (C) at the splice site of the V_L and J_L 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 B-1 cell–reconstituted Rag1^–/– mice recognized surface epitopes on apoptotic cells, as demonstrated by immunocytochemistry. Similarly, the MDA-LDL–specific NAb NA-17 strongly stained apoptotic cells. Neither plasma from PBS-injected Rag1^–/– mice nor a keyhole limpet hemocyanin–specific (KLH-specific) control IgM bound apoptotic cells.

We also tested IgM binding to apoptotic cells by flow cytometry in relation to measures of cellular apoptosis (Figure 5A). Plasma IgM from reconstituted Rag1^–/– 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 Ldlr^–/–Rag^–/– mice that had been fed a high-cholesterol diet for 10 weeks to induce atherosclerotic lesions, which accumulate apoptotic cells and OxLDL (28, 29). The mice were maintained on the atherogenic diet for an additional 6 weeks and then sacrificed, and atherosclerotic lesions were assessed for the deposition of endogenous Abs (Figure 5B). As expected, neither IgM nor IgG Abs were present in lesions of Ldlr^–/–Rag1^–/– mice injected 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 atherosclerotic lesions of Ldlr^–/–Rag^–/– mice (Figure 5C), whereas a control natural IgM did not. We also found that NA-17 could substantially 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 epitopes.

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. Importantly, umbilical cord plasma contained prominent IgM titers against MDA-LDL and OxLDL, but not native LDL or KLH (Figure 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 placenta. As the total IgM titers were found to be generally higher in maternal samples, we calculated the ratios of oxidation-specific 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 adaptive 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 binding 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 oxidation-specific epitopes on apoptotic cells (Figure 5A). When not promptly cleared, apoptotic cells are immunogenic and proinflammatory (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 apoptotic cell uptake by macrophages in vivo in Rag1^–/– 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 uptake of apoptotic thymocytes was significantly enhanced in B-1 cell–reconstituted Rag1^–/– mice compared with PBS controls (33% vs. 27%, P < 0.05; Figure 7A).

To directly test the impact of an oxidation-specific mAb, we preincubated the apoptotic cells with NA-17 or with a control IgM (specific for KLH) before injection into Rag1^–/– mice. The percentage of macrophages that had taken up apoptotic thymocytes was similar in Rag1^–/– mice receiving apoptotic thymocytes preincubated with control IgM or untreated apoptotic thymocytes (20% vs. 23%, P = NS; Figure 7B). In contrast, the phagocytic uptake increased to 32% when the apoptotic cells were preincubated with NA-17 (P < 0.05), demonstrating that binding of NA-17 facilitated apoptotic cell uptake by macrophages in vivo.

Animals.

C57BL/6 and Rag1^–/– mice (The Jackson Laboratory) and Ldlr^–/–Rag1^–/– mice (a gift from Godfrey Getz and Katherine Reardon, University 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 receptor α^–/– (Tcra^–/–) 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. Conventionally 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 Committee 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 fluorescently 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^–, CD19⁺, and CD23^– population.

B-1 cell cultures.

Purified B-1 cells were seeded at 1 × 10⁶ cells per well in 24-well flat-bottom plates in culture medium (RPMI 1640 medium containing 10% heat-inactivated FCS, 10 mM HEPES buffer, 2mM l-glutamine, 0.05 mM 2-mercaptoethanol, 50 μg/ml gentamicin) in the presence and absence of the specific TLR4 agonist KdO₂-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% CO₂ 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 × 10⁶ cells in 200 μl were injected into the peritoneal cavity of 6- to 15-week-old Rag1^–/– mice. Three to 4 donor mice were used for each recipient. Control Rag1^–/– mice received 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 resuspended in staining buffer. After blocking with a specific anti–Fcγ receptor mAb (2.4G2) for 15 minutes at 4°C, 10⁶ cells were stained with fluorescently 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, followed by extensive washing. Cell populations were analyzed on a BD FACS­Calibur or FACScan instrument. More than 0.5 × 10⁵ 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 supernatant, 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 measure 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 Biosciences — 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-conjugated 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 following antigens were prepared as described previously (29): copper sulfate–oxidized 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 previously (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 supernatants 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 limiting 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 concentration 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–secreting splenocytes were quantified by ELISpot assay as described previously (14). Splenocytes were suspended in culture medium at 2 × 10⁵ and 1 × 10⁵ cells/100 μl and cultured in triplicate in washed and blocked 96-well Multi­Screen-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% CO₂, 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 incubated 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-conjugated goat anti-human IgM (Jackson ImmunoResearch Laboratories Inc.). Apoptosis of Jurkat cells was induced with UV irradiation at 20 mJ/cm², followed by further incubation of the cells in medium for 14–16 hours before use. For immunofluorescence microscopy studies, cells were incubated with 2 μg/ml Hoechst dye (Sigma-Aldrich) for 10 minutes, fixed with 2% paraformaldehyde, and spun down on glass slides using a cytospin (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 Ldlr^–/–Rag1^–/– 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^–/– mice reconstituted with B-1 cells were used to prepare B-1–derived hybridomas 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_H and V_L was accomplished as previously described (12).

Human samples.

Blood samples from healthy pregnant women and their newborn infants delivering at Magee-Womens Hospital (Pittsburgh, Pennsylvania, 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. Maternal samples were collected in EDTA before delivery (mean, 10.5 hours) and cord venous samples by sterile aspiration of the umbilical vein after delivery of the infant. Samples were processed and aliquoted within 3 hours of collection and stored at –70°C until use. Ten uncomplicated nulliparous 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 clearance 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% thioglycollate to induce sterile peritonitis 15–19 weeks after B-1 cell transfer. Four days later, the mice were injected i.p. with 20 × 10⁶ fluorescently labeled 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 supernatant 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^–/– mice.

Macrophage binding assay.

Binding of biotinylated MDA-LDL to J774 macrophages plated in microtiter wells was assessed by a chemiluminescent 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 analyzed with InStat 3 for Macintosh (GraphPad Software). A P value less than 0.05 was considered significant.