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Autoimmune hemolytic anemia (AIHA) due to warm-acting IgA autoantibodies is rare. We explored the pathogenic mechanisms underlying destruction of red blood cells (RBCs) in a patient with severe AIHA mediated exclusively by polymeric immunoglobulin A (pIgA) anti-Band 3 autoantibodies. The follow-up period was 17 months. RBCs were not destroyed by complement activation as no deposition of complement was observed on the patient's RBCs. pIgA eluted from the patient's RBCs did not induce RBC destruction through phagocytosis by monocytes or antibody–dependent cell–mediated cytotoxicity by natural killer cells. Induction of eryptosis (ie, RBC apoptosis) due to direct alteration of the RBC membrane by pIgA autoantibodies was also excluded. By contrast, upon incubation with pIgA-opsonized RBCs, substantial RBC membrane transfers (ie, trogocytosis) to monocytes were observed that might contribute to RBC immune destruction. This effect was poorly inhibited by blockers of Fc receptors, excluding a major contribution of FcαRI to this process. Histologic analysis revealed a massive accumulation of agglutinated RBCs with little sign of erythrophagocytosis in the spleen. These results, together with the efficacy of splenectomy 17 months after AIHA onset, suggest that the trapping and subsequent sequestration of agglutinated RBCs in the spleen are the principal pathogenic mechanisms of pIgA-mediated AIHA.

EndoS from Streptococcus pyogenes is an immunomodulating enzyme that specifically hydrolyzes glycans from human immunoglobulin G and thereby affects antibody effector functions. Autoimmune hemolytic anemia is caused by antibody-mediated red blood cell (RBC) destruction and often resists treatment with corticosteroids that also cause frequent adverse effects. We show here that anti-RhD (anti-D) and rabbit anti–human-RBC antibodies (anti-RBC) mediated destruction of RBC, ie, phagocytosis, complement activation, and hemolysis in vitro and in vivo was inhibited by EndoS. Phagocytosis by monocytes in vitro was inhibited by pretreatment of anti-D with EndoS before sensitization of RBCs and abrogated by direct addition of EndoS to blood containing sensitized RBCs. The toxic effects of monocytes stimulated with anti-D–sensitized RBCs, as measured by interleukin-8 secretion and oxygen metabolite production, was restrained by EndoS. Agglutination of RBCs and complement-mediated hemolysis in vitro in whole human blood caused by rabbit anti-RBCs was inhibited by EndoS. Development of anemia in mice caused by a murine anti-RBC immunoglobulin G2a monoclonal autoantibody and complement activation and erythrophagocytosis by Kupffer cells in the liver were reduced by EndoS. Our data indicate that EndoS is a potential therapeutic agent that might be evaluated as an alternative to current treatment regimens against antibody-mediated destruction of RBCs.

Growth arrest-specific gene 6 (Gas6) is expressed in antigen-presenting cells and endothelial cells (ECs) but not in T cells. When wild-type (WT) or Gas6−/− mice received allogeneic non–T cell–depleted bone marrow cells, hepatic graft-versus-host disease (GVHD) was alleviated in Gas6−/− recipients regardless of donor genotype, but not in WT recipients. T-cell infiltration was more prominent and diffuse in WT than in Gas6−/− recipients' liver. When mice received 0.5 × 106 allogeneic T cells with T cell–depleted allogeneic bone marrow, clinical signs indicated that GVHD was less severe in Gas6−/− than in WT recipients, as shown by a significant improvement of the survival and reduced liver GVHD. These data demonstrate that donor cells were not involved in the protection mechanism. In addition, lack of Gas6 in antigen-presenting cells did not affect WT or Gas6−/− T-cell proliferation. We therefore assessed the response of WT or Gas6−/− ECs to tumor necrosis factor-α. Lymphocyte transmigration was less extensive through Gas6−/− than WT ECs and was not accompanied by increases in adhesion molecule levels. Thus, the lack of Gas6 in ECs impaired donor T-cell transmigration into the liver, providing a rationale for considering Gas6 pathway as a potential nonimmunosuppressive target to minimize GVHD in patients receiving allogeneic hematopoietic stem cell transplantation.

Across the animal kingdom, multivalency discriminates antibodies from all other immunoglobulin superfamily members. The evolutionary forces conserving multivalency above other structural hallmarks of antibodies remain, however, incompletely defined.

By generating IgM and IgA switch variants of the 34-3C IgG2a anti–red blood cell (RBC) autoantibody, we evaluated the pathogenic activity of these 2 isotypes in view of the Fc-associated effector functions (ie, complement activation and polyvalency-dependent agglutination). We found that polymeric forms of 34-3C IgM and IgA anti-RBC autoantibody were as pathogenic as IgG2a, which was the most pathogenic among 4 different IgG subclasses, whereas their monomeric variants completely lacked pathogenic effects. Histological examination showed that 34-3C IgM and IgA autoantibodies caused anemia as a result of multivalency-dependent hemaggultination and subsequent sequestration of RBC in the spleen, in contrast to Fc receptor– and complement receptor–mediated erythrophagocytosis by Kupffer cells with IgG isotypes. In addition, the development of anemia induced by IgM and IgA isotypes of 34-3C antibody and by 2 additional IgM anti-RBC monoclonal autoantibodies was not inhibited at all in C3-deficient mice, indicating the lack of involvement of complement activation in the pathogenesis of IgM- and IgA-induced anemia. Our data demonstrate a remarkably high pathogenic potential of polymeric forms of IgM and IgA anti-RBC autoantibodies due to their ability to induce hemagglutination but completely independent of complement activation.

CD22 functions primarily as a negative regulator of B-cell receptor signaling. The Cd22a allele has been proposed as a candidate allele for murine systemic lupus erythematosus. In this study, we explored the possible expression of aberrant forms of CD22, which differ in the N-terminal sequences constituting the ligand-binding site due to synthesis of abnormally processed Cd22 mRNA, in several Cd22a mouse strains, including C57BL/6 Cd22 congenic mice. The staining pattern of splenic B cells obtained with CY34 anti-CD22 mAb, which was expected to bind poorly to the aberrant CD22, was more heterogeneous in Cd22a mice than in Cd22b mice. Moreover, CD22 detected on B cells of Cd22a mice was expressed more weakly and as a smaller-sized protein, compared with Cd22b mice. Significantly, analysis with a synthetic CD22 ligand demonstrated that Cd22a mice carried a larger proportion of CD22 that was not bound by cis ligands on the B-cell surface than Cd22b mice. Finally, the study of C57BL/6 Cd22 congenic mice revealed that Cd22a B cells displayed a phenotype reminiscent of constitutively activated B cells (reduced surface IgM expression and augmented MHC class II expression), as reported for B cells expressing a mutant CD22 lacking the ligand-binding domain. Our demonstration that Cd22a B cells express aberrant forms of CD22, which can potentially deregulate B-cell signaling because of their decreased ligand-binding capacity, provides further support for Cd22a as a potential candidate allele for murine systemic lupus erythematosus.

Using a cohort of C57BL/6 (B6) × (NZB × B6)F1 backcross male mice bearing the Yaa (Y-linked autoimmune acceleration) mutation, we mapped and characterized the NZB-derived susceptibility loci predisposing to the development of autoimmune hemolytic anemia (AHA). Our analysis identified 2 major loci on NZB chromosome 7 and chromosome 1 linked with Coombs antierythrocyte autoantibody production, and their contributions were confirmed by the analysis of B6.Yaa mice (B6 mice bearing the Yaa mutation) congenic for each NZB-derived susceptibility interval. A newly identified Aia3 (autoimmune anemia 3) locus present on NZB chromosome 7 selectively regulated Coombs antibody responses, while the second locus, directly overlapping with Nba2 (NZB autoimmunity 2) on chromosome 1, promoted the development of AHA, likely as part of its effect on overall production of lupus autoantibodies. A higher incidence of Coombs antibody production in B6.Aia3 congenic mice (B6 mice bearing the NZB-Aia3 locus) than B6.Nba2 mice (B6 mice bearing the NZB-Nba2 locus) indicated a major role for Aia3 in AHA. Notably, lack of expansion of B1 cells in B6.Aia3 congenic mice argued against the involvement of this subset in AHA. Finally, our analysis of BC mice also demonstrated the presence of a B6-derived H2-linked locus on chromosome 17 that apparently regulated the production of Coombs antibodies as a result of its overall autoimmune promoting effect.

Autoimmune hemolytic anemia (AIHA) is a disease in which autoantibodies against red blood cells (RBCs) lead to their premature destruction. Most clinically significant autoantibodies are of the immunoglobulin G (IgG) type, which leads primarily to the uptake and destruction of RBCs by splenic and hepatic macrophages. Therapies such as corticosteroids and splenectomy are directed at interfering with this process. Liposomally encapsulated clodronate (dichloromethylene diphosphonate) has previously been found to be a potent antimacrophage agent. It selectively depletes animals of macrophages within 24 hours of administration by inducing apoptosis in these cells. Therefore, we hypothesized that liposomal clodronate would be a useful agent for treating AIHA. We tested this hypothesis in a mouse model of AIHA in which animals were given either anti-RBC antibodies or preopsonized RBCs. In either case, liposomal clodronate substantially decreased RBC destruction. This drug formulation was effective within hours by first blocking and then depleting phagocytic macrophages, and its action lasted for 1 to 2 weeks. Thus, in AIHA, liposomal clodronate therapy may act like a temporary, medicinal splenectomy. As such, it may prove useful in situations where rapid response to therapy is critical or other medical therapies are inadequate.

The glycoprotein CD47 (integrin-associated protein, IAP) is present on the surface of virtually all cells, including red blood cells (RBCs). CD47 acts like a marker of self by ligating the macrophage inhibitory receptor signal regulatory protein α (SIRPα). In this manner mild reactivity of wild-type RBCs with macrophage phagocytic receptors is tolerated, whereas otherwise identical CD47-deficient RBCs are rapidly eliminated. We show here that virtually all CD47-deficient nonobese diabetic (NOD) mice spontaneously develop severe lethal autoimmune hemolytic anemia (AIHA) at 180 to 280 days of age, whereas none of the control CD47+ NOD mice develop lethal AIHA at least during the first year of life. This phenotype is at least partially due to a markedly increased rate of elimination of opsonized CD47−/− compared to CD47+ RBCs. Similarly, CD47−/−C57BL/6 mice were much more sensitive than their wild-type counterparts to experimental passive AIHA induced by anti-RBC monoclonal antibodies. Thus, CD47-SIRPα signaling can have a profound influence on the severity of AIHA, making manipulation of this signaling pathway a theoretically appealing avenue in the treatment of the disease.

Autoantibodies of the cryoprecipitating IgG3 isotype have been shown to play a significant role in the development of murine lupus–like autoimmune syndrome. At present, the structural basis of IgG3 cryoprecipitation and its role in autoantibody pathogenicity remain to be defined. Using molecular variants of an IgG3 monoclonal rheumatoid factor, 6-19, derived from an autoimmune MRL-Faslpr mouse, we have investigated the implication of charged residues in the heavy-chain variable (VH) region, potential CH3-linked oligosaccharides, and galactosylation of CH2-linked oligosaccharides in its cryoglobulin activity. The cryoglobulin activity of the IgG3 6-19 mutant bearing more negatively charged residues at VH 6 and 23 was found to be reduced but still highly significant, whereas that of the mutant lacking a potential CH3 glycosylation site remained unchanged. In marked contrast, IgG3 6-19 variants obtained from 6-19 heavy-chain transgenic mice displayed barely detectable cryoglobulin activity associated with an increased level of galactosylation in the CH2 oligosaccharide side chains. Thus, our data strongly suggest that the cryoglobulin activity of IgG3 6-19 autoantibody is critically determined by levels of galactosylation in the CH2 oligosaccharide side chains, whereas VH residues play a secondary role in 6-19 IgG3 cryoglobulin activity.

Adhesive interactions with stromal cells and the extracellular matrix are essential for the differentiation and migration of hematopoietic progenitors. In the erythrocytic lineage, a number of adhesion molecules are expressed in the developing erythrocytes and are thought to play a role in the homing and maturation of erythrocytic progenitors. However, many of these molecules are lost during the final developmental stages leading to mature erythrocytes. One of the adhesion molecules that remains expressed in mature, circulating erythrocytes is CD147. This study shows that blockade of this molecule on the cell surface by treatment with F(ab′)2fragments of anti-CD147 monoclonal antibody disrupts the circulation of erythrocytes, leading to their selective trapping in the spleen. Consequently, mice develop an anemia, and de novo, erythropoietin-mediated erythropoiesis in the spleen. In contrast, these changes were not seen in mice similarly treated with another antierythrocyte monoclonal antibody with a different specificity. These results suggest that the CD147 expressed on erythrocytes likely plays a critical role in the recirculation of mature erythrocytes from the spleen into the general circulation.

Cryoglobulin activity associated with murine immunoglobulin G3 (IgG3) has been shown to play a significant role in the development of murine lupuslike glomerulonephritis. A fraction, but not all, IgG3 monoclonal antibodies are capable of inducing a severe acute lupuslike glomerulonephritis as a result of direct localization of IgG3 cryoglobulins, suggesting the importance of qualitative features of cryoglobulins in their nephritogenic activities. Here a remarkable difference is shown in the renal pathogenicity of 2 murine IgG3 monoclonal cryoglobulins, identical in the amino acid sequences of their heavy and light chains but different in galactosylation patterns of oligosaccharide side chains because of their synthesis in different myeloma cells. The antibody lacking the capacity to induce severe glomerulonephritis displayed an increased proportion of galactosylated heavy chains. Changes in conformation, as revealed by gel filtration analysis, reduced cryoglobulin activity, and accelerated clearance could account for the lack of the renal pathogenicity of the more galactosylated variant. This observation provides a direct demonstration for the role of IgG galactosylation in the pathogenic potential of cryoglobulins.