Secreted IgM is predominantly found as pentameric molecules, but IgM can also be secreted as hexamers by B cell lines. Murine hexamers activate the complement cascade more efficiently than pentamers, but the physiologic significance of hexameric IgM remains unknown. Here, we report that IgM hexamers and pentamers are cleared from the circulation with similar kinetics, suggesting that the predominance of pentameric IgM in vivo reflects the regulation of polymer assembly and secretion in responding B cells. Normal IgM-secreting B cells, particularly those from the peritoneal cavity, are capable of secreting abundant hexameric IgM in vitro. The disparity between the ability of B cells to secrete IgM hexamers in vitro and the paucity of this polymer in vivo suggest that IgM hexamers might be deleterious. In support of this, we demonstrate that the autoantibodies from a number of patients with cold agglutinin (CA) disease include both IgM hexamers and pentamers. The CA IgM hexamers lyse human erythrocytes in the presence of human complement more efficiently than CA IgM pentamers, suggesting a potential role for hexameric IgM in the pathogenesis of this autoimmune syndrome.

Immunoglobulin M is the first Ab produced in primary immune responses and the predominant isotype secreted in T cell-independent immune responses. Like all Abs, IgM consists of heavy (μ) and light chains that are assembled into H2L2 structures, but secretory IgM is further assembled into higher order polymers (reviewed in Refs. 1 and 2). The predominant form of IgM present in serum is pentameric in structure (i.e., (μ2L2)5) (3, 4, 5, 6, 7, 8). However, a number of IgM-secreting cell lines can also secrete hexameric molecules, which contain an additional μ2L2 monomeric subunit ((μ2L2)6) (9, 10, 11, 12, 13, 14). These hexamers do not contain J chain (10, 11, 12, 14), a polypeptide frequently associated with pentameric IgM (15, 16).

Hexameric IgM is 15- to 20-fold more efficient than pentameric IgM in activating complement (10, 11). Despite this impressive activity, the physiologic relevance of IgM hexamers is unknown. While it is clear that hexamers can be secreted by B cell lines, it is not known whether hexameric IgM is produced at significant levels by normal B cells, nor is it known whether this polymer is stable in vivo. There is some evidence, however, that IgM hexamers can exist in vivo (6, 8), raising the possibility that hexamers could play some role in immune responses.

We report here that although hexameric IgM is not abundant in vivo, normal B cells are capable of producing significant amounts of IgM hexamers in vitro. We also demonstrate that IgM pentamers and hexamers exhibit very similar half-lives in vivo, suggesting that the predominance of circulating IgM pentamers in normal animals reflects the relative levels of assembly of the two types of polymers by IgM-secreting cells. However, hexameric IgM can be found at significant levels in the serum IgM autoantibodies of a number of patients with an IgM-mediated autoimmune condition, cold agglutinin (CA)7 disease. Importantly, the hexameric CA IgM exhibits an enhanced ability to activate the complement cascade compared with its pentameric counterparts, as measured by the ability to mediate complement-dependent hemolysis of human erythrocytes in the presence of human complement. These data provide evidence that human IgM hexamers are biologically potent, raising the possibility that they can be a deleterious component of the autoantibodies found in patients with IgM-mediated autoimmune diseases.

All cells were cultured in DMEM with 10% FBS, supplemented as previously described (17). CH12 is an inducible B cell lymphoma passaged in vivo as previously described (17). CH12 cells were stimulated with LPS (Escherichia coli 055:B5; Difco, Detroit, MI) at 50 μg/ml for 48 h to induce high rate Ab secretion (18). Peritoneal cells (19) and spleen cells (20) were prepared from C57BL/6 or BALB/c mice (The Jackson Laboratory, Bar Harbor, ME). Cells (1 × 106 cells/ml) were cultured alone or in the presence of LPS (10 μg/ml).

CH12 cells were stimulated with LPS to induce high rate secretion of pentameric and hexameric IgM (13). Stimulated CH12 cells were then metabolically labeled with [35S]methionine and [35S]cysteine (see below), and the radiolabeled secreted IgM was isolated from the culture supernatant using a goat anti-mouse IgM affinity column (Sigma, St. Louis, MO) as previously described (11). Approximately 10 to 20 μg of 35S-labeled IgM (∼5 × 106 total cpm) in a volume of 100 μl was introduced into each mouse by i.p. injection. Control experiments demonstrated that similar results (i.e., similar clearance times) were obtained when IgM was injected i.v. In preliminary experiments we monitored the half-life of injected IgM in both B6 and B10.A mice and found it to be 24 and 30 h, respectively. Subsequent experiments used only B10.A mice. At the time of injection and at intervals thereafter, blood samples were obtained, serum was separated, and aliquots from each bleed were analyzed by scintillation spectroscopy. The half-life of total CH12 IgM was determined by plotting the counts per minute/volume vs time postinjection. To distinguish between the half-life of pentameric and hexameric IgM, an equal volume of each serum sample was analyzed by nonreducing, nondenaturing (native) 4% PAGE (see below). After autoradiography, the slices of the dried gel corresponding to the respective IgM pentamer and hexamer bands were excised from the gel and quantitated directly for radioactivity, and the counts per minute/polymer type was then plotted vs time postinjection. The background for 35S in these experiments was 34 ± 4 cpm.

The monoclonal IgM (μ, κ) autoantibodies of seven patients with chronic CA syndrome were analyzed. CA syndrome is characterized by the monoclonal outgrowth of IgM-producing cells. All IgM Abs had anti-I specificity, as they agglutinated adult red cells to a greater degree than neonatal cells (21). The monoclonality of the CA IgM was confirmed by demonstration of a single light chain on Ouchterlony analysis and of restricted heavy chain mobility on gel electrophoresis (21). Blood from these patients was collected into prewarmed syringes and allowed to clot at 37°C. Serum was separated and incubated at 56°C for 30 min to inactivate complement. The IgM anti-I Ab was purified by three sequential cycles of adsorption and elution from washed normal adult O+ red cells (22). The supernatant fluid obtained from the final elution was removed and chromatographed on Sephadex G-200. The fractions demonstrating the strongest agglutination but without hemoglobin were pooled, concentrated, and stored at −90°C.

The IgM concentration of supernatant samples was determined by quantitative ELISA as previously described (11). For CA samples, purified rabbit anti-human μ-chain Ab (Sigma) was adsorbed to the solid phase, and the bound human IgM was detected using a peroxidase-conjugated goat anti-human μ-chain specific Ab (Sigma). Purified human IgM (Sigma) was used as a standard.

Cell lysates were prepared in 1.0% Nonidet P-40 exactly as previously described (23). Nonreducing 4% PAGE under nondenaturing (native) conditions (11), electrophoresis of IgM under nonreducing and denaturing conditions by agarose/SDS-PAGE (23), and standard reducing 10.5% SDS-PAGE using prestained molecular size markers (13) have been described. Denaturing agarose/SDS-PAGE gels separate IgM based on size, and we have not found any evidence that V region structure or charge has any influence on the migration of IgM polymers in these gels. For example, we have fractionated IgM Abs with V regions that vary in length (by 10 amino acids) and charge (calculated pI values ranging from 4.6–9.6) (24) and have found no differences in their migration on these agarose/SDS-PAGE gels (14). In contrast, nondenaturing PAGE gels separate based on charge. In the latter gels, different polymeric forms of IgM can only be distinguished in monoclonal IgM (11) (our unpublished observations).

Gels were soaked in buffer containing 0.5% 2-ME to facilitate transfer and were prepared for Western blotting or autoradiography as previously described (13, 23). Western blotting for IgM μ heavy chains was performed using an 125I-labeled goat anti-mouse μ heavy chain Ab (Sigma) or rabbit anti-human μ (Sigma). J chain was detected using either a rabbit anti-mouse J chain Ab (25) that also cross-reacts with human J chain (our unpublished observation) or a rabbit anti-human J chain Ab (Biogenex Laboratories, San Ramon, CA) followed by an 125I-labeled goat anti-rabbit IgG Ab for detection (13).

In some experiments, films were quantitated by densitometry (Molecular Dynamics, Sunnyvale, CA). Boxes of identical sizes were used, and backgrounds were determined from blank portions of the gels. In the experiment described in Figure 1, portions of gels corresponding to bands of interest were quantitated directly by excising them from the dried gel, solubilizing them in Aquasol-2 (DuPont, Wilmington, DE), and analyzing them by scintillation spectroscopy.

FIGURE 1.

IgM pentamers and hexamers exhibit similar kinetics of clearance in vivo. 35S-labeled secreted IgM, containing a mixture of pentamers and hexamers, was obtained from the supernatants of metabolically labeled LPS-stimulated CH12 cells. IgM was affinity purified and then injected into mice. Blood samples were collected from the tail vein at the various times indicated postinjection. Serum was separated from the whole blood and analyzed for total content of radiolabeled IgM by scintillation spectroscopy. Equal aliquots of serum were subjected to nonreducing native PAGE to separate the monoclonal CH12 radiolabeled IgM pentamers and hexamers. After autoradiography, the portions of the gel corresponding to pentamers and hexamers, respectively, were excised, solubilized, and quantitated for radioactivity by scintillation spectroscopy. A plot of the counts per minute of the various samples vs time yielded the in vivo half-lives as shown.

FIGURE 1.

IgM pentamers and hexamers exhibit similar kinetics of clearance in vivo. 35S-labeled secreted IgM, containing a mixture of pentamers and hexamers, was obtained from the supernatants of metabolically labeled LPS-stimulated CH12 cells. IgM was affinity purified and then injected into mice. Blood samples were collected from the tail vein at the various times indicated postinjection. Serum was separated from the whole blood and analyzed for total content of radiolabeled IgM by scintillation spectroscopy. Equal aliquots of serum were subjected to nonreducing native PAGE to separate the monoclonal CH12 radiolabeled IgM pentamers and hexamers. After autoradiography, the portions of the gel corresponding to pentamers and hexamers, respectively, were excised, solubilized, and quantitated for radioactivity by scintillation spectroscopy. A plot of the counts per minute of the various samples vs time yielded the in vivo half-lives as shown.

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Purified CA-IgM samples were fractionated on an 11-ml step gradient (5–20% sucrose) as previously described (23). Before pouring gradients, gradient tubes were precoated overnight with PBS containing 10 mg/ml BSA. Gradients were centrifuged at 285,000 × g for 6.75 h at 20°C using an SW41Ti rotor in a Beckman L8-80 M ultracentrifuge. Following centrifugation, gradients were manually tapped from the bottom, and fractions of approximately 300 μl were collected. Aliquots from individual fractions were screened for IgM by quantitative ELISA as described below.

Blood from a single patient with paroxysmal nocturnal hemoglobinuria (PNH) or from rabbits was used as a source of erythrocytes for hemolytic assays. The PNH erythrocytes consisted of 98% PNH type III cells, as determined by the complement lysis sensitivity test (26). Rabbit erythrocytes, which also express the I Ag (27), were obtained from Colorado Serum (Denver, CO).

Frozen guinea pig serum (Hazelton Research Products, Denver, PA) and human serum from healthy donors were used as sources of complement. Serum was absorbed three times on ice with a 1/10th volume of washed rabbit or human PNH cells and was stored at −90°C until use.

Hemolytic assays measuring the lysis of rabbit or human erythrocytes were conducted in a biphasic reaction as previously described (28). Briefly, in the first phase, a standard suspension of red cells (1%), CA IgM, and human or guinea pig serum were incubated together for 30 min at 4°C. In the second stage, the reaction mixture was slowly warmed over a 10-min period and then incubated for 1 h at 37°C. At the end of the incubation, ice-cold PBS was added, and the red cells were removed by centrifugation. The OD of the supernatant fluid at 405 nm was determined spectrophotometrically, and the percentage of cells lysed was determined using appropriate controls (11). Hemolytic assays were conducted using two or three different preparations of purified CA IgM from two different patients; similar results were obtained in each case.

Electron microscopy studies have shown that small numbers of hexamers can be found in serum, but IgM pentamers constitute the vast majority of serum IgM (5, 6, 7, 8). Precise quantitation of the fraction of hexamers in serum has proven difficult due to the predominance of pentameric IgM, but we estimate that hexamers comprise much less than 5% of the serum IgM in normal, healthy mice (data not shown). The low frequency of hexamers in serum could indicate that very little of the IgM secreted under normal physiologic conditions is hexameric. Alternatively, IgM hexamers might be produced at higher rates in vivo than apparent from their distribution in normal serum. Either an intrinsic instability in these molecules and/or their enhanced capacity to activate complement might lead to accelerated removal from the circulation.

To help distinguish between these possibilities, we compared the half-life of IgM hexamers and pentamers in vivo. CH12 cells were used as a source of IgM for this purpose, since they secrete approximately equivalent amounts of IgM hexamers and pentamers in response to LPS (11) (Fig. 2). They therefore provide a clonal source of both types of polymers, allowing direct comparison between the clearance of IgM pentamers and hexamers. CH12 IgM binds with low affinity to a haptenic component, trimethylammonium, of the self Ag phosphatidylcholine (29, 30). Importantly, there is no measurable difference in the avidity of CH12 pentamers and hexamers for this Ag (11). In addition, CH12 IgM exhibits weak and limited polyreactivity with other Ags (24).

FIGURE 2.

Production of hexameric IgM by normal B cells. Murine spleen (S) and peritoneal (P) cells were stimulated in vitro with LPS. Secreted IgM in the culture supernatants was analyzed by nonreducing agarose/SDS-PAGE and Western blotting using a labeled anti-μ Ab. Culture supernatant from LPS-stimulated CH12 cells serves as a control for the identification of pentameric (lower band) and hexameric (upper band) IgM. Cells from both male (M) and female (F) mice of varying ages were examined.

FIGURE 2.

Production of hexameric IgM by normal B cells. Murine spleen (S) and peritoneal (P) cells were stimulated in vitro with LPS. Secreted IgM in the culture supernatants was analyzed by nonreducing agarose/SDS-PAGE and Western blotting using a labeled anti-μ Ab. Culture supernatant from LPS-stimulated CH12 cells serves as a control for the identification of pentameric (lower band) and hexameric (upper band) IgM. Cells from both male (M) and female (F) mice of varying ages were examined.

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Metabolically labeled secreted IgM from CH12 cells was affinity purified and injected into mice. The presence of the injected IgM was first followed by monitoring the serum for 35S-labeled IgM. In agreement with other studies (31), we found the half-life of polymeric IgM to be approximately 1 day (Fig. 1; t1/2 = 30 h). To distinguish the half-life of pentameric and hexameric IgM, an equivalent volume of serum from each time point was subjected to nondenaturing PAGE analysis, which allows IgM pentamers and hexamers in monoclonal samples to be resolved (11). Autoradiography of the gel revealed the labeled IgM pentamers and hexamers present in the circulation of the animals over the course of the experiment. The respective bands were excised from the gel and quantitated directly for radioactivity. As shown in Figure 1, the clearance of IgM hexamers in vivo was indistinguishable from that of pentameric IgM. Similar results were obtained in a second experiment. These results are consistent with the interpretation that the scarcity of circulating hexameric IgM in serum reflects the low level production of this polymer in vivo.

We next asked whether normal Ab-secreting B cells have the capacity to secrete hexameric IgM. Peritoneal and spleen cells from both individual female and male mice of various ages were cultured in vitro in the presence of LPS. Secreted IgM in the culture supernatants was analyzed by denaturing agarose/SDS-PAGE, which resolves IgM polymers based on size (23). IgM from CH12 cells served as a marker for IgM pentamers and hexamers on these gels (11). Both forms of polymeric IgM could be detected in most normal cell samples analyzed regardless of the anatomical source of the B cells or the age or sex of the animal, and these migrated as pentamers and hexamers (Fig. 2). Some samples, particularly those from the spleen, contained almost all pentamers, while other samples, especially those from peritoneal B cells, contained easily detectable hexamers. In this and other experiments the relative abundance of the total IgM that was secreted as IgM hexamers by LPS-stimulated spleen or peritoneal cells ranged from as little as 3% to as much as 50%, as estimated by densitometry of the Western blots. High abundance hexamers were only produced by peritoneal cells, and in all cases splenic B cells produced fewer hexamers than peritoneal cells from the same mouse. We conclude that IgM hexamers can be secreted by normal Ab-secreting B cells, at least in vitro.

Two lines of evidence prompted us to investigate whether hexameric IgM, with its enhanced ability to activate complement, might function deleteriously in vivo. First, our data suggest that IgM hexamer production is tightly regulated in vivo, since normal B cells are capable of secreting abundant IgM hexamers, but apparently do not normally do so at high levels in vivo. Second, peritoneal B cells, which are rich in CD5+ B cells (19, 32), are more apt to secrete IgM hexamers than splenic B cells (see Fig. 2), and CD5+ B cells have been implicated in the production of autoantibodies (33, 34). We therefore asked whether autoimmune Abs might contain hexameric IgM. We first screened Ig from a number of mouse strains with autoimmune syndromes, such as NZB and MRL/lpr, but found no convincing evidence for significant increases in the presence of hexameric IgM. However, the autoimmune conditions in these mice are predominantly mediated by IgG Abs (35), and we therefore searched for autoimmune diseases specifically known to be mediated by IgM.

CA syndrome is one such disease. It is a human autoimmune condition characterized by the presence of monoclonal IgM autoantibodies specific for the Ii Ags on red cells (27, 36, 37). Patients with this disease have variable outcomes, but in some patients these Abs can cause severe hemolytic anemia due to complement activation by the CA IgM (38, 39). We therefore purified CA IgM from seven patients with confirmed monoclonal CA syndrome by adsorption and elution from human red cells and analyzed these samples for the presence of different polymeric forms of IgM using nonreducing native (nondenaturing) PAGE and Western blotting. In monoclonal samples of IgM, the presence of different polymeric forms of IgM can be distinguished because they migrate to different positions on these gels, with IgM hexamers migrating more slowly than IgM pentamers (11). As shown in Figure 3 A, IgM from several patients, including RH, IL, and MR, exhibited at least two major bands of IgM, suggesting that their CA IgM might contain both IgM hexamers and pentamers. The CA IgM from other patients, such as AB and MS, exhibited only one form of IgM, suggesting that their autoantibodies probably consist only of IgM pentamers, a conclusion supported by analysis of J chain content (see below). It should be noted that on nondenaturing gels IgM migrates according to intrinsic charge. As a result, IgM from different clonal sources will migrate to different positions due to charge differences resulting from sequence diversity in the V regions and as a result of any charge differences that might be contributed by the sialic acid content on N-linked glycans of the μ heavy chain (our unpublished observations). The fact that IgM from different patients migrate differently is not surprising because there is known sequence diversity in CA IgM (40).

FIGURE 3.

CA IgM from some patients contain two forms of IgM polymers. A, Distribution of CA IgM on nondenaturing (native) gels. CA IgM from seven different patients was fractionated on a 4% PAGE gel, transferred, and probed with an 125I-labeled anti-human μ-chain-specific Ab as described in Materials and Methods. IgM from the murine B cell line, CH12, was included as a control. B, Fractionation of CA IgM from patient MR on sucrose density gradients. IgM from individual fractions were collected from the bottom of the gradient and analyzed on nonreducing SDS-PAGE gels. Polymers were revealed by Western blotting as in A. The slowest migrating band from the bottom of the gradient (fractions, left of gel) represent IgM hexamers, while the predominant band is from IgM pentamers. The rapidly migrating band from the top of the gradient, found in the last four fractions, have been shown to be IgM tetramers in other studies (11).

FIGURE 3.

CA IgM from some patients contain two forms of IgM polymers. A, Distribution of CA IgM on nondenaturing (native) gels. CA IgM from seven different patients was fractionated on a 4% PAGE gel, transferred, and probed with an 125I-labeled anti-human μ-chain-specific Ab as described in Materials and Methods. IgM from the murine B cell line, CH12, was included as a control. B, Fractionation of CA IgM from patient MR on sucrose density gradients. IgM from individual fractions were collected from the bottom of the gradient and analyzed on nonreducing SDS-PAGE gels. Polymers were revealed by Western blotting as in A. The slowest migrating band from the bottom of the gradient (fractions, left of gel) represent IgM hexamers, while the predominant band is from IgM pentamers. The rapidly migrating band from the top of the gradient, found in the last four fractions, have been shown to be IgM tetramers in other studies (11).

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To confirm that the autoantibodies from patients with CA syndrome that exhibit two forms of IgM on native gels (Fig. 3,A) do contain IgM hexamers and pentamers, we fractionated the CA IgM on sucrose gradients, which separates IgM polymers based on their molecular size. The IgM in individual sucrose gradient fractions from one such patient, MR, was then analyzed on nondenaturing gels to resolve the polymers present. As shown in Figure 3,B, Western blotting revealed two predominant forms of IgM that migrated on these gradients at the expected positions for IgM hexamers and pentamers (11). A small amount of IgM tetramers, not uncommon in IgM samples (11, 41), was also present in some of the fractions at the top of the gradient. Similar results were obtained using CA IgM from another patient, IL (see Fig. 4), which also migrated as two bands on native gels. Taken together, these data demonstrate that the presence of IgM hexamers is not uncommon in patients with monoclonal CA disease. This conclusion is not necessarily unexpected, based on early studies that revealed that two polymeric classes of this IgM could often be detected by analytic ultracentrifugation of IgM from CA patients (3, 4). The major peak sedimented as a 19S protein complex and was determined to be pentameric IgM, but a second, larger form of IgM was often observed. In light of our data, it is likely that these investigators had resolved both IgM hexamers and pentamers.

FIGURE 4.

Human CA IgM hexamers lack J chain. A, Individual sucrose gradient fractions were analyzed on native cells as described in Figure 3 B. B, Individual sucrose gradient fractions containing predominantly IgM hexamers (fraction 12) or pentamers (fraction 20) were reduced, fractionated on SDS-PAGE gels, transferred, and Western blotted for the presence of μ-chains or J chain.

FIGURE 4.

Human CA IgM hexamers lack J chain. A, Individual sucrose gradient fractions were analyzed on native cells as described in Figure 3 B. B, Individual sucrose gradient fractions containing predominantly IgM hexamers (fraction 12) or pentamers (fraction 20) were reduced, fractionated on SDS-PAGE gels, transferred, and Western blotted for the presence of μ-chains or J chain.

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To test which of the IgM polymers contained J chain, we fractionated CA IgM from patient IL on sucrose gradients and resolved the fractions on native gels to identify those that contained predominantly IgM hexamers (i.e., fraction 12) or pentamers (fraction 20; Fig. 4,A). IgM samples from these fractions was then separated on reducing SDS-PAGE and analyzed for the presence of μ-chains or J chain. As shown in Figure 4 B, only the fraction enriched in IgM pentamers contained detectable J chain. IgM from a fraction containing the slower migrating species of IgM, fraction 12, lacked detectable J chain, providing further evidence for the identity of this polymeric species as IgM hexamers. We conclude that the IgM autoantibodies from several patients with monoclonal CA disease contain both IgM hexamers and IgM pentamers.

We next compared the relative abundance of μ heavy chain and J chain proteins in the CA IgM from two patients that differ in hexamer content. The CA IgM from patient MR contains IgM hexamers, while the IgM from patient MS appears to be comprised only of IgM pentamers (Fig. 3). As shown in Figure 5, the CA IgM from patient MR contained much lower levels of J chain than equivalent amounts of IgM from patient MS. We compared the ratios of J chain to μ-chains in the CA IgM with normal human serum IgM, using densitometry for quantification of the bands shown in Figure 5. We found that the ratio of J:μ in the CA IgM from patient MS was very similar (75.7%) to that of normal serum IgM, while the J:μ ratio in the CA IgM from patient MR was only 5.3% of normal levels. Because more than half of the IgM in the CA samples from MR is pentameric, the data suggest that many of these pentamers also lack J chain. These data are consistent with those from other studies that have demonstrated that IgM pentamers can be assembled and secreted without J chain (9, 14). These data are also consistent with a model in which J chain is limiting in the CA IgM-secreting cells of patients such as MR, a condition that would favor IgM hexamer production (11, 13).

FIGURE 5.

CA IgM from patients containing IgM hexamers (patient MR) contain less J chain than normal human IgM or IgM from CA patients that is expressed predominantly as pentamers (patient MS). The presence of μ- and J chains was determined by Western blotting following fractionation of reduced samples on SDS-PAGE gels.

FIGURE 5.

CA IgM from patients containing IgM hexamers (patient MR) contain less J chain than normal human IgM or IgM from CA patients that is expressed predominantly as pentamers (patient MS). The presence of μ- and J chains was determined by Western blotting following fractionation of reduced samples on SDS-PAGE gels.

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We next determined whether there were differences in the ability of human IgM hexamers and pentamers to activate complement. Murine IgM hexamers have been shown to activate complement up to 20-fold more efficiently than IgM pentamers (10, 11), indicating that an increased presence of hexamers could significantly alter the lytic capacity of an IgM Ab. To compare the lytic capacity of human IgM polymers, we first fractionated IgM from patient MR on sucrose gradients. Defined amounts of IgM from individual fractions were analyzed for their ability to lyse erythrocytes in the presence of complement. As shown in Figure 6, the lytic capacity of fractions increased with increasing hexamer content, suggesting that human IgM hexamers activate complement more efficiently than IgM pentamers.

FIGURE 6.

Hemolytic activity of CA IgM hexamers and pentamers. A, Sucrose gradient fractionation of CA IgM from patient MR. Representative samples of individual sucrose gradient fractions were analyzed on native gels by Western blotting, as described in Figure 3. B, Hemolytic activity of IgM. IgM from individual fractions was tested for its ability to lyse rabbit erythrocytes (which express the I Ag) using guinea pig serum as a source of complement.

FIGURE 6.

Hemolytic activity of CA IgM hexamers and pentamers. A, Sucrose gradient fractionation of CA IgM from patient MR. Representative samples of individual sucrose gradient fractions were analyzed on native gels by Western blotting, as described in Figure 3. B, Hemolytic activity of IgM. IgM from individual fractions was tested for its ability to lyse rabbit erythrocytes (which express the I Ag) using guinea pig serum as a source of complement.

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To further explore the pathologic potential of CA IgM hexamers, we used an entirely homologous system (human red cells and human complement) rather than the heterologous system used above to compare the lytic activity of fractions enriched in CA IgM hexamers with that of those enriched in CA IgM pentamers. As shown in Figure 7, the hexamer-containing fraction was more potent in activating complement than the pentamer fraction regardless of the source of red cells or complement. The difference was at least sevenfold in this assay, but the data almost certainly underestimate the relative activity of the IgM hexamers, since the pentamer fractions contain some hexamers (∼13% by densitometry), and the hexamer fractions contain some IgM pentamers. These results demonstrate that human IgM hexamers are more efficient at complement activation than IgM pentamers of identical specificity and reveal the deleterious potential of hexameric IgM autoantibodies in vivo.

FIGURE 7.

Increased hemolytic activity of CA IgM hexamers against human erythrocytes using human complement. Top panel, CA IgM sucrose gradient fractions were tested for their ability to lyse rabbit erythrocytes in the presence of heterologous (guinea pig) complement. Lower panel, The same fractions were tested for their ability to lyse human erythrocytes in the presence of human serum as a source of complement. Fraction 8 contained 94% hexamers and 6% pentamers, as determined by quantitation using laser densitometry. In contrast, fraction 16 contained 13% hexamers (relative to pentamers). This fraction also contained some tetramers, which do not activate complement (our unpublished observation).

FIGURE 7.

Increased hemolytic activity of CA IgM hexamers against human erythrocytes using human complement. Top panel, CA IgM sucrose gradient fractions were tested for their ability to lyse rabbit erythrocytes in the presence of heterologous (guinea pig) complement. Lower panel, The same fractions were tested for their ability to lyse human erythrocytes in the presence of human serum as a source of complement. Fraction 8 contained 94% hexamers and 6% pentamers, as determined by quantitation using laser densitometry. In contrast, fraction 16 contained 13% hexamers (relative to pentamers). This fraction also contained some tetramers, which do not activate complement (our unpublished observation).

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A hallmark of the immune system is the employment of multimeric protein complexes that specifically and efficiently execute various effector functions. This is particularly evident in humoral immunity and is exemplified by IgM. IgM Abs are generally of low affinity, but they interact efficiently with the complement system and constitute a first line of defense in the adaptive immune system. While the majority of serum Ab is pentameric in form, a second polymeric form, hexameric IgM, has been characterized in recent years, primarily as a secreted product of IgM-secreting B cell lines cultured in vitro. While IgM hexamers do not exhibit significant increases in avidity compared with their pentameric counterparts, they are particularly effective in activating the complement pathway (10, 11), suggesting that these polymers could represent a form of IgM adapted to increased biologic activity (2). However, the role of hexameric IgM has remained elusive due to the lack of in vivo data and analysis of normal sources. In the current studies, we have shown that normal B cells are capable of producing hexameric IgM and, importantly, our data implicate these highly lytic IgM Abs in the pathogenicity of certain autoimmune conditions.

Our data demonstrate that normal B cells can produce IgM hexamers (Fig. 2), at least in vitro, thereby excluding the possibility that the secretion of hexameric IgM represents an artifact of transformed B cell lines, many of which have been shown to secrete abundant IgM hexamers (13, 14). On the other hand, numerous studies have indicated that IgM hexamers are not frequent in the serum (3, 4, 5, 6, 7, 8). This suggests either that IgM hexamers are produced but cleared more rapidly in situ, or that under most circumstances hexamer production does not occur in vivo at significant rates. We have found that IgM pentamers and hexamers exhibit similar half-lives in vivo (Fig. 1), suggesting that the scarcity of hexamers in serum cannot simply be attributed to an inherent instability or to an accelerated clearance of these molecules in vivo. We cannot rule out the possibility that IgM hexamers are normally produced in sequestered sites and do not enter the circulation, or that the IgM produced during a normal immune response might be cleared more rapidly due to their increased ability to activate complement. However, the IgM Ab we used in these studies was selected because it does bind the haptenic self Ag phosphatidylcholine (29, 30).

From these considerations, we suggest that the significant bias toward the presence of pentameric IgM must be due at least in part to the exertion of tight regulatory control on the type of polymer assembled in normal IgM-secreting cells in vivo. This would result in the increased secretion of pentamers relative to hexamers, but our data do not allow us to conclude which of these polymeric forms is being regulated. Using model B cell lines, J chain has been shown to play a role in regulating IgM assembly by promoting IgM pentamer assembly at the expense of hexamers (13, 14). J chain has this effect because late in the assembly process it is preferentially inserted into an assembling pentamer, excluding the incorporation of a sixth monomeric subunit required to produce an IgM hexamer (42). J chain might play an important role in the preferential assembly of IgM pentamers in primary B cells as well. J chain has been reported to be highly expressed in Ab-secreting cells in vivo (43, 44), and it therefore seems likely that most modes of B cell stimulation must lead to levels of J chain sufficient to ensure the predominant production of pentamers. In addition, other factors might complement the role that J chain plays in regulating IgM assembly in vivo.

Our results do not exclude a protective role for IgM hexamers in the immune response. For example, small amounts of hexameric IgM could be secreted during immune responses, and even a small percentage would be expected to have a significant effect on the overall ability of specific IgM to activate complement. While we have not detected changes in the abundance of hexamers in mice following immunization, our current methods of resolving IgM polymers would not be sufficiently sensitive to allow us to detect small changes in the polymeric form of polyclonal IgM produced in response to a given Ag. If IgM hexamers have a protective role in vivo, it might be expected to function during the T-independent phases of immune responses, such as during bacterial infections, in which B cells may be directly stimulated such that J chain synthesis may not be optimally activated (2).

While a normal physiologic role for IgM hexamers remains unclear, our results raise the intriguing possibility that hexameric IgM may play a pathogenic role in IgM-mediated autoimmunity. The rate of hemolysis in CA disease is dependent to some degree on the concentration and characteristics of the IgM Ab, and the rate of exposure to temperatures sufficiently low to permit interaction of Ab and Ag, followed by the activation of complement (26). Several characteristics have been identified that increase hemolysis by a given amount of Ab (45, 46). These include 1) the thermal amplitude, that is the highest temperature at which there is detectable interaction between the Ab and the Ag, 2) the degree of inhibition of Ab interaction by the presence of complement fragments on the membrane surface, and 3) the ability of the Ab molecule to fix the first component of complement. Clearly, CA IgM hexamers exhibit an enhanced ability (at least sevenfold) to facilitate complement-mediated hemolysis of red cells compared with their pentameric counterparts (Fig. 7). It seems likely that the presence of hexameric CA Abs could contribute to the hemolysis in CA disease, where chronic hemolytic anemia or episodic acute hemolysis is a major concern (27, 36, 37, 38, 39). To fully explore this possibility, it will be necessary to perform a longitudinal study of patients with CA disease to determine whether there is a relationship between the presence and the abundance of IgM hexamers and disease severity.

Given the fact that most IgM normally found in vivo is pentameric, the presence of hexamers in the IgM autoantibodies of many patients with CA disease is an anomaly. This conclusion is based not only on our current study, but also reflects earlier studies that showed that many patients with CA disease contained CA IgM that was larger than pentamers, presumably hexamers (3, 4). At this time, it is not clear why the cells that secrete CA IgM might be predisposed to secrete higher levels of IgM hexamers than normal B cells in vivo. Clearly, the CA condition itself does not automatically yield a high level of hexameric IgM, since at least some CA IgM samples apparently contain exclusively IgM pentamers (Fig. 3). There are at least three possible explanations for how CA B cells might become IgM hexamer producers. First, the mode of B cell stimulation may be critically important. CA Abs recognize cell surface oligosaccharides (the Ii Ags) (25, 37), precisely the type of Ag that might elicit IgM production without concomitantly stimulating optimal J chain expression (13). This possibility fits well with the demonstrated role of J chain in favoring pentamer production. Our results suggest that there may be significant variation in the J chain content in cells secreting CA Abs, as revealed by the relative levels of J chain in the secreted CA IgM (Fig. 5). Second, certain B cell subpopulations may be predisposed toward hexamer production. In this regard, the higher quantities of hexameric polymers in IgM secreted by murine peritoneal vs splenic B cells (Fig. 2) might reflect differences in the B cell subpopulations present in these anatomical sites and/or the previous antigenic experience of B cells in these locations. The peritoneal cavity is rich in CD5+ (B1) B cells (19, 32), a subset that primarily responds to T-independent Ags and that has been implicated in the production of autoantibodies (33, 34, 47, 48). Third, the rapid expansion of a B cell clone coupled with high rate IgM production might facilitate an increase in hexamer secretion. In this regard, it is important to note that monoclonal CA is often the initial manifestation in the emergence of B cell lymphomas (49), suggesting a relationship between the loss of control of B cell proliferation and disease. Interestingly, in another case in which IgM hexamers have been detected in vivo, the IgM was detected in a patient with Waldenström’s macroglobulinemia (8). Thus, there may be a relationship between the unusual expansion of a B cell clone and the breakdown in normal regulatory processes that control IgM polymer assembly.

We thank R. M. E. Parkhouse (Division of Immunology, Pirbright Laboratory, Surrey, U.K.) for providing the anti-J chain antiserum used in these studies, T. D. Randall for advice during the early stages of this work, and L. Kleinman (Boston University School of Medicine) and Sharron Hoffman (Duke Medical Center) for technical assistance.

1

This work was supported by a Biomedical Science Grant from the Arthritis Foundation, Grant AI31209 from the National Institutes of Health, and an Arthritis Foundation Fellowship (to A.D.C.).

7

Abbreviations used in this paper: CA, cold agglutinin; PNH, paroxysmal nocturnal hemoglobinuria.

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