Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans (original) (raw)
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).
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).
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).
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 × 106 cells per well in 500 μl culture medium. Cells were stimulated with IL-5 (50 ng/ml), KdO2-Lipid A (100 ng/ml), or TLR2 agonists (a combination 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 KdO2-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 competitors (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/B0). Data are from 1 experiment representative of 3.
Characterization of Rag1–/– recipients adoptively transferred with B-1 cells. (A) Adoptive transfer of B-1 cells into Rag1–/– mice replenishes 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 (IgM+CD43+) were also absent from the spleen (Spleen, left). C57BL/6: Peritoneal macrophages (CD11bhiCD5–) and T cells (CD11b–CD5hi) were intact (PEC, upper middle). B cells could be divided into B-1a (CD19+CD11bintCD5int), B-1b (CD19+CD11bintCD5–), and B-2 cells (CD19+CD11b–CD5–) (PEC, lower middle). In the spleen, B-1 cells were about 2.4% of total splenocytes (Spleen, middle). Rag1–/– + B-1: B-1 cell populations were reconstituted in the peritoneal cavity (PEC, lower right) and spleen (Spleen, right), without B-2 cell or T cell contamination (PEC, right). (B) IgM Abs to oxidation-specific epitopes are present in the plasma of B-1 reconstituted Rag1–/– mice. Plasma collected after 15 weeks from Rag1–/– + B-1 (n = 8) or Rag1–/– + PBS (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/B0). Data are from 1 experiment representative of 3.
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 C57BL/6, Rag1–/– + 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 VH and VL 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 VH rearrangement did not reveal nucleotide variation to germline genes. Sequence analysis of VL rearrangement revealed 1 nucleotide insertion between VL and JL germline gene segments.
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 _Kd_s 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 _Kd_s for MDA-LDL and OxLDL were 9.9 × 10–9 and 1.42 × 10–7 mol/l respectively. These values are similar to the _Kd_s 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 VH and VL rearrangements expressed in one cloned MDA-specific hybridoma (NA-17) displayed complete germline gene usage of the VH rearrangement and only 1 nucleotide insertion (C) at the splice site of the VL and JL 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.
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 Rag1–/– + 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_–/–_Rag1–/– mice (bottom row), but not in PBS-injected Ldlr_–/–_Rag1–/– 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_–/–_Rag1–/– 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/B0).
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).
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).
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).
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 phagocytosed 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 injection into Rag1–/– mice. The phagocytic uptake was significantly different 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 preincubation (RAG) (32% vs. 20% vs. 23%; *P < 0.05, Bonferroni multiple comparison test). Horizontal bars denote means.
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.