Autologous IgM Research Articles

Serologic Cross-Reactivity of Human IgM and IgG Antibodies to Five Species of Ebola Virus

Five species of Ebola virus (EBOV) have been identified, with nucleotide differences of 30–45% between species. Four of these species have been shown to cause Ebola hemorrhagic fever (EHF) in humans and a fifth species (Reston ebolavirus) is capable of causing a similar disease in non-human primates. While examining potential serologic cross-reactivity between EBOV species is important for diagnostic assays as well as putative vaccines, the nature of cross-reactive antibodies following EBOV infection has not been thoroughly characterized. In order to examine cross-reactivity of human serologic responses to EBOV, we developed antigen preparations for all five EBOV species, and compared serologic responses by IgM capture and IgG enzyme-linked immunosorbent assay (ELISA) in groups of convalescent diagnostic sera from outbreaks in Kikwit, Democratic Republic of Congo (n = 24), Gulu, Uganda (n = 20), Bundibugyo, Uganda (n = 33), and the Philippines (n = 18), which represent outbreaks due to four different EBOV species. For groups of samples from Kikwit, Gulu, and Bundibugyo, some limited IgM cross-reactivity was noted between heterologous sera-antigen pairs, however, IgM responses were largely stronger against autologous antigen. In some instances IgG responses were higher to autologous antigen than heterologous antigen, however, in contrast to IgM responses, we observed strong cross-reactive IgG antibody responses to heterologous antigens among all sets of samples. Finally, we examined autologous IgM and IgG antibody levels, relative to time following EHF onset, and observed early peaking and declining IgM antibody levels (by 80 days) and early development and persistence of IgG antibodies among all samples, implying a consistent pattern of antibody kinetics, regardless of EBOV species. Our findings demonstrate limited cross-reactivity of IgM antibodies to EBOV, however, the stronger tendency for cross-reactive IgG antibody responses can largely circumvent limitations in the utility of heterologous antigen for diagnostic assays and may assist in the development of antibody-mediated vaccines to EBOV.

Warm autoimmune hemolytic anemia is an IgM–IgG immune complex disease

Warm autoimmune hemolytic anemia (WAIHA) is characterized by polyclonal IgG autoantibodies binding to red blood cells (RBC). The characterization of the autoantigen in WAIHA has not yet led to definitive results, and the etiology of RBC autoantibodies remains unclear. An altered control of self-reactive IgG by autologous IgM has been proposed as the underlying mechanism of disease in WAIHA, suggesting that IgM–IgG immune complexes contribute to the pathophysiology of the disease. In the present study, we purified and characterized IgM from plasma of WAIHA patients and from healthy controls using FPLC-based protocols and optical biosensor technology, and investigated IgG present within the IgM fractions. We provide evidence that IgM–IgG immune complexes in plasma and associated with the RBC membrane are the characteristic feature of WAIHA, independent of the etiology of the disease. IgM–IgG immune complexes of WAIHA patients differ from IgM–IgG immune complexes of healthy individuals with regard to quantity and to structural composition. The data suggest that self-immunoglobulin is the original autoantigen underlying WAIHA. The molecular characterization of IgM–IgG immune complexes may define new targets for therapeutic intervention in WAIHA.

Regulation of the immune response by natural IgM: lessons from warm autoimmune hemolytic anemia.

Natural autoantibodies are immunoglobulins of isotypes IgM, IgG and IgA that are present under physiological conditions and that are directed toward self-antigens. Repertoires of self-reactive antibodies have been analysed intensively during the last decade and have been shown to be altered in a variety of autoimmune disorders, immunodeficiency syndromes and lymphoproliferative diseases. Immunoglobulin interactions via variable regions of antibody molecules account significantly for the functional integrity of natural self-reactive antibody repertoires. Recent data indicate that natural immunoglobulins of the isotype IgM might prove particularly useful for the control of antibody-mediated autoimmune diseases under certain conditions. Warm autoimmune hemolytic anemia (WAIHA), an IgG-mediated autoimmune disorder, turns out to be a clinically relevant in vivo model to analyse the impact of autologous IgM on the development of IgG-mediated autoimmunity in humans. We here summarize current knowledge on the role of autologous IgM for regulating self-reactivity. Since natural self-reactive antibodies are critical for the regulation not only of auto- but also of alloimmune responses, as they occur for example in the setting of organ transplantation, regulation of immune homeostasis by pools of human normal IgM might be an interesting therapeutic target of broad interest for clinical medicine.

Altered control of self-reactive IgG by autologous IgM in patients with warm autoimmune hemolytic anemia

Warm autoimmune hemolytic anemia (WAIHA) is characterized by an accelerated clearance of red blood cells (RBCs) associated with the presence of anti-RBC immunoglobulin (Ig)G autoantibodies. In the present study, we analyzed the self-reactive IgG and IgM antibody repertoires of patients with WAIHA using a technique of quantitative immunoblotting on a panel of whole tissue extracts as sources of self-antigens. Data were compared by means of multiparametric statistical analysis. We demonstrate that self-reactive antibody repertoires of IgG purified from plasma and of IgG purified from RBC eluates do not differ between healthy donors and patients with WAIHA, whereas autoreactive repertoires of IgM from patients exhibit broadly altered patterns of reactivity as compared with those of healthy controls. We further demonstrate that IgG purified from eluates of RBCs of healthy donors induces agglutination of RBCs in an indirect Coombs assay to a similar extent as IgG purified from eluates of RBCs of patients with WAIHA. The capability of IgG to induce agglutination of RBCs is suppressed in unfractionated eluates of healthy donors' cells, whereas it is readily found in unfractionated eluates of patients' RBCs. IgM is an essential factor in controlling the ability of IgG in unfractionated RBC eluates to induce agglutination of RBCs. These observations indicate that anti-RBC IgG autoantibodies of patients with WAIHA share extensive similarity with natural antiRBC autoantibodies of healthy donors and suggest that defective control of IgG autoreactivity by autologous IgM is an underlying mechanism for autoimmune hemolysis in WAIHA. (Blood. 2000;95:328-335)

Altered control of self-reactive IgG by autologous IgM in patients with warm autoimmune hemolytic anemia

AbstractWarm autoimmune hemolytic anemia (WAIHA) is characterized by an accelerated clearance of red blood cells (RBCs) associated with the presence of anti-RBC immunoglobulin (Ig)G autoantibodies. In the present study, we analyzed the self-reactive IgG and IgM antibody repertoires of patients with WAIHA using a technique of quantitative immunoblotting on a panel of whole tissue extracts as sources of self-antigens. Data were compared by means of multiparametric statistical analysis. We demonstrate that self-reactive antibody repertoires of IgG purified from plasma and of IgG purified from RBC eluates do not differ between healthy donors and patients with WAIHA, whereas autoreactive repertoires of IgM from patients exhibit broadly altered patterns of reactivity as compared with those of healthy controls. We further demonstrate that IgG purified from eluates of RBCs of healthy donors induces agglutination of RBCs in an indirect Coombs assay to a similar extent as IgG purified from eluates of RBCs of patients with WAIHA. The capability of IgG to induce agglutination of RBCs is suppressed in unfractionated eluates of healthy donors' cells, whereas it is readily found in unfractionated eluates of patients' RBCs. IgM is an essential factor in controlling the ability of IgG in unfractionated RBC eluates to induce agglutination of RBCs. These observations indicate that anti-RBC IgG autoantibodies of patients with WAIHA share extensive similarity with natural antiRBC autoantibodies of healthy donors and suggest that defective control of IgG autoreactivity by autologous IgM is an underlying mechanism for autoimmune hemolysis in WAIHA. (Blood. 2000;95:328-335)

Lack of Suppressive Antibody Activity in Sera from Patients with Active-Phase Autoimmune Diseases

To investigate the presence of a suppressive antibody activity in sera from patients with autoimmune diseases, the IgG autoreactivity in whole serum was compared to that of the IgG fraction purified by affinity chromatography on protein G-sepharose. Competitive inhibition assays on the binding to histone, dsDNA, RNP and thyroglobulin of the purified IgG fraction by the autologous IgM present in serum without IgG and depleted of < 100 kD components (named IgM fraction) were also performed. The IgG reactivity to the autoantigens tested was considerably increased in the IgG fraction than in the whole serum drawn from a healthy control and from three SLE patients in an inactive-phase of disease. Addition of the IgM fraction to the autologous purified IgG resulted in a dose-dependent inhibition of IgG binding to the autoantigens tested. However, no differences were observed between the autoreactivity of the IgG in whole serum and that of the purified IgG fraction from active-phase SLE patients, or from two patients with autoimmune thyroiditis, and the autologous IgM fractions did not inhibit significantly binding to the autoantigens under study of the purified IgG fraction. Our findings support the concept that the IgG autoreactivity in physiological conditions is regulated by idiotypic interactions between IgG and IgM, and suggest that this regulation is broken in the active phase of autoimmune diseases and that clinical remission from SLE could be associated with the restoration of this control mechanism. Additionally, qualitative differences, such as polyreactivity or change of idiotype in the autoreactive IgG fraction from active-phase disease might contribute to escape of regulation.

Autologous IgM, IgA, and Complement Binding to Sickle Erythrocytes In Vivo. Evidence for the Existence of Dense Sickle Cell Subsets

We have previously reported that sickle erythrocytes sedimenting at high specific density after gradient centrifugation exhibit increased IgG binding in vivo as compared with low-density paired samples. We have performed the present study to determine whether the opsonization of dense sickle cells in vivo could also involve autologous IgM, IgA, and complement. IgA, IgM, and complement binding in vivo to the surface of density-separated sickle erythrocytes was detected by flow cytometric analyses. IgM and complement C3 fragment binding was detected primarily on high-density sickle erythrocytes. With the exception outlined below, IgA binding was detected for all sickle cell fractions that sediment at densities > 1.085 g/mL. IgM, IgA, and complement C3 fragment binding was increased on high-density sickle erythrocytes as compared with low-density paired samples and exceeded that binding to normal erythrocytes by 30% +/- 10% (mean +/- range), 50% +/- 10%, and 41% +/- 5%, respectively. Two-color flow cytometry indicates that high-density sickle cell fractions contain at least two heterogeneous RBC subsets. One is an RBC subset that binds IgA in combination with IgM and C3, and the second subset is devoid of IgA yet binds IgM and C3. These findings indicate that high-density sickle cells exhibit a greater heterogeneity than has been reported in previous studies, which is based on autologous Ig binding in vivo; and suggest that RBC components of this most severely dehydrated sickle cell subpopulation could have heterogeneous origin and pathophysiologic significance. Although the functional role of IgA binding to human RBCs is unclear, our findings that IgM and complement bind to the same high-density sickle cell fractions suggest that both the IgM and the sickle erythrocyte-bound IgG determined in previous studies could mediate the deposition of complement on dense sickle cells in vivo. These findings support the hypotheses that irreversibly sickled cell-enriched high-density sickle RBC subpopulations could be removed from the circulation by erythrocyte phagocytosis that is enhanced by the presence of complement.

Autologous IgM, IgA, and complement binding to sickle erythrocytes in vivo. Evidence for the existence of dense sickle cell subsets

We have previously reported that sickle erythrocytes sedimenting at high specific density after gradient centrifugation exhibit increased IgG binding in vivo as compared with low-density paired samples. We have performed the present study to determine whether the opsonization of dense sickle cells in vivo could also involve autologous IgM, IgA, and complement. IgA, IgM, and complement binding in vivo to the surface of density-separated sickle erythrocytes was detected by flow cytometric analyses. IgM and complement C3 fragment binding was detected primarily on high-density sickle erythrocytes. With the exception outlined below, IgA binding was detected for all sickle cell fractions that sediment at densities &gt; 1.085 g/mL. IgM, IgA, and complement C3 fragment binding was increased on high-density sickle erythrocytes as compared with low-density paired samples and exceeded that binding to normal erythrocytes by 30% +/- 10% (mean +/- range), 50% +/- 10%, and 41% +/- 5%, respectively. Two-color flow cytometry indicates that high-density sickle cell fractions contain at least two heterogeneous RBC subsets. One is an RBC subset that binds IgA in combination with IgM and C3, and the second subset is devoid of IgA yet binds IgM and C3. These findings indicate that high-density sickle cells exhibit a greater heterogeneity than has been reported in previous studies, which is based on autologous Ig binding in vivo; and suggest that RBC components of this most severely dehydrated sickle cell subpopulation could have heterogeneous origin and pathophysiologic significance. Although the functional role of IgA binding to human RBCs is unclear, our findings that IgM and complement bind to the same high- density sickle cell fractions suggest that both the IgM and the sickle erythrocyte-bound IgG determined in previous studies could mediate the deposition of complement on dense sickle cells in vivo. These findings support the hypotheses that irreversibly sickled cell-enriched high- density sickle RBC subpopulations could be removed from the circulation by erythrocyte phagocytosis that is enhanced by the presence of complement.

Expression and control of the natural autoreactive IgG repertoire in normal human serum.

We have investigated the autoreactive repertoire expressed by serum IgG of healthy individuals of various age groups using a large panel of self antigens. Natural IgG autoantibodies against all self antigens of the panel were found in the purified IgG fraction of the serum of all donors that were tested. The mean binding activity to self antigens of IgG of pregnant women was higher than that of IgG purified from the serum of infants, young adults and aged individuals. No increase in IgG autoreactivity was observed with aging neither in the purified IgG fraction of serum nor in whole serum. Whereas autoantibody activity was easily detectable in purified IgG, it was low in serum. No difference was observed, however, between the binding activity of purified IgG and of IgG in serum in the case of foreign antigens nor in the case of anti-thyroglobulin autoantibodies of patients with hashimoto's thyroiditis. Purified IgM from normal serum bound to F(ab')2 fragments of autologous IgG in a dose-dependent fashion and inhibited the binding of autologous IgG to self antigens. Our results thus indicate that autologous IgM contributes to regulate expression of the natural IgG autoreactive repertoire through V region-dependent interactions, resulting in low levels of IgG autoreactivity in serum under physiological conditions.

Analysis of anti-idiotypic antibodies against anti-microsomal antibodies in patients with thyroid autoimmunity

Anti-microsomal antibody (AMA) activity was inhibited in 14 of 16 sera and in all 12 IgG preparations from patients with postpartum thyroiditis following incubation with F(ab′) 2 fragments from normal polyspecific immunoglobulin for therapeutic use (ivIg). Similar results were observed with sera from seven of seven patients with Graves' disease and five of six patients with autoimmune hypothyroidism. Results of these competitive binding assays and affinity chromatography of AMA IgG on Sepharose-bound F(ab′) 2 fragments from ivIg indicated that AMA antibodies reacted with ivIg through idiotypic-anti-idiotypic interactions. Eight out of 10 IgG preparations from patients with autoimmune thyroid disease also showed inhibition of AMA activity when coincubated with autologous IgM at various IgG:IgM molar ratios. These observations suggest that ivIg can inhibit anti-microsomal antibodies through idiotype-anti-idiotype interactions and that such interactions occur with IgM anti-idiotype antibodies in vivo, providing evidence of a role for idiotypic network regulation in the control of thyroid autoimmunity.

Movement of IgM antibody from blood to bile in rats

The movement from blood to bile of passively injected autologous IgM antibody against horse erythrocytes was studied in rats. Both native and neuraminidase-treated antibody entered bile intact, with the peak titers for both measured between 60 and 90 min after injection. A small part (0.38%) of the injected dose of native antibody and 1.05% of the asialo-IgM antibody appeared in bile over 24 hr. These recoveries represented only a small fraction of the activity, which apparently disappeared from serum over the period. The pathways used by IgM antibody to enter bile were partially assessed. Prior injection of fetuin was found to abrogate the biliary secretion of native antibody and to significantly reduce the recovery of asialo-antibody in bile. In contrast, the presence of asialofetuin reduced (but not significantly) the secretion of native antibody and markedly changed the kinetics of appearance of asialo-IgM activity in bile. Some candidate routes to bile can be excluded as unlikely. The parahepatocellular pathway to bile canaliculi appears uninvolved because the secretion of antibody is slow and the inhibitory effects of fetuin argue against secretory component-mediated transport. Instead, two secretory pathways appear to be present. The first appears to be available to both native and asialo-IgM antibody, and may involve the peribiliary capillary plexus. The second route to bile is available to asialo-IgM antibody alone and is probably initiated by binding to the hepatic asialoglycoprotein receptor.

Movement of IgM antibody from blood to bile in rats

The movement from blood to bile of passively injected autologous IgM antibody against horse erythrocytes was studied in rats. Both native and neuraminidase-treated antibody entered bile intact, with the peak titers for both measured between 60 and 90 min after injection. A small part (0.38%) of the injected dose of native antibody and 1.05% of the asialo-IgM antibody appeared in bile over 24 hr. These recoveries represented only a small fraction of the activity, which apparently disappeared from serum over the period. The pathways used by IgM antibody to enter bile were partially assessed. Prior injection of fetuin was found to abrogate the biliary secretion of native antibody and to significantly reduce the recovery of asialoantibody in bile. In contrast, the presence of asialofetuin reduced (but not significantly) the secretion of native antibody and markedly changed the kinetics of appearance of asialo-IgM activity in bile. Some candidate routes to bile can be excluded as unlikely. The parahepatocellular pathway to bile canaliculi appears uninvolved because the secretion of antibody is slow and the inhibitory effects of fetuin argue against secretory component-mediated transport. Instead, two secretory pathways appear to be present. The first appears to be available to both native and asialo-IgM antibody, and may involve the peribiliary capillary plexus. The second route to bile is available to asialo-IgM antibody alone and is probably initiated by binding to the hepatic asialoglycoprotein receptor.

IgG autoantibody activity in normal mouse serum is controlled by IgM.

In the serum of normal BALB/c mice, IgG antibody reactivity to mouse actin and tubulin, DNA, and TNP groups was very low compared to that of the IgM. This activity was considerably increased when IgG was separated, by affinity chromatography on protein A-Sepharose, whereas no difference in the IgM activity was observed. Addition of IgM to IgG isolated from the same serum resulted in the inhibition of IgG binding to these Ag. Isolation of IgG antibodies on actin, TNP, and tubulin immunoadsorbents has indicated that at least part of the IgG antibodies is polyreactive. In order to understand this inhibition better, experiments with F(ab')2 fragments of IgG were performed. IgM inhibited the binding of F(ab')2 to the antigens in a dose-dependent manner and reacted with immobilized F(ab')2. IgM isolated on F(ab')2 immunoadsorbent, as compared to the initial IgM preparation, were less active toward the Ag but more inhibitory for IgG binding to the Ag. In some pathologic situations, IgM failed to inhibit some IgG antibody activities. The anti-DNA IgG activity from (NZB x NZW)F1 mice was not affected by autologous IgM. Similarly the anti-tubulin IgG from mice infected with Trypanosoma cruzi were less inhibited by IgM from autologous serum than antitubulin IgG from normal mice. These results are compatible with the existence in normal mice of an idiotypic-like network, regulating via an IgM population in the serum, the binding of IgG autoantibodies to self Ag. Modifications of this idiotype-anti-idiotype system might lead to the expression and/or expansion of autoreactive IgG-producing clones.

Antigen-induced rheumatoid factors. Protein and carbohydrate antigens induce different rheumatoid factor responses.

Rheumatoid factors, autologous IgM anti-IgG, were produced after immunization with protein or carbohydrate antigens. After immunization with either type of antigen, the kinetics of the rheumatoid factor response reflected the kinetics of the dominant IgG isotype in the anti-antigen response. Secondary immunization with protein antigens induced an IgM rheumatoid factor response which was consistently greater than that seen after carbohydrate immunization, and almost exclusively specific for the IgG1 isotype. In contrast, primary or hyperimmunization with carbohydrate antigens gave rise to a more heterogeneous response dominated by IgM anti-IgG3, with lesser amounts of IgM anti-IgG2b and anti-IgG1. Direct immunization with immune complexes gave similar results, as complexes composed of IgG1 induced exclusively IgM anti-IgG1, whereas those complexes made up of IgG3 gave rise to IgM rheumatoid factors binding IgG3 and IgG2b. Rheumatoid factor production, with isotypic specificity defined by the immunizing antigen, appears to be a natural consequence of immunization with a variety of protein and carbohydrate antigens.