Thomas Moll

Complement Activation Selectively Potentiates the Pathogenicity of the IgG2b and IgG3 Isotypes of a High Affinity Anti-Erythrocyte Autoantibody

By generating four IgG isotype-switch variants of the high affinity 34–3C anti-erythrocyte autoantibody, and comparing them to the IgG variants of the low affinity 4C8 anti-erythrocyte autoantibody that we have previously studied, we evaluated in this study how high affinity binding to erythrocytes influences the pathogenicity of each IgG isotype in relation to the respective contributions of Fcγ receptor (FcγR) and complement. The 34–3C autoantibody opsonizing extensively circulating erythrocytes efficiently activated complement in vivo (IgG2a = IgG2b > IgG3), except for the IgG1 isotype, while the 4C8 IgG autoantibody failed to activate complement. The pathogenicity of the 34–3C autoantibody of IgG2b and IgG3 isotypes was dramatically higher (>200-fold) than that of the corresponding isotypes of the 4C8 antibody. This enhanced activity was highly (IgG2b) or totally (IgG3) dependent on complement. In contrast, erythrocyte-binding affinities only played a minor role in in vivo hemolytic activities of the IgG1 and IgG2a isotypes of 34–3C and 4C8 antibodies, where complement was not or only partially involved, respectively. The remarkably different capacities of four different IgG isotypes of low and high affinity anti-erythrocyte autoantibodies to activate FcγR-bearing effector cells and complement in vivo demonstrate the role of autoantibody affinity maturation and of IgG isotype switching in autoantibody-mediated pathology.

Targeting IL-15 Receptor-Bearing Cells with an Antagonist Mutant IL-15/Fc Protein Prevents Disease Development and Progression in Murine Collagen-Induced Arthritis

It has been suggested that the inflammatory cytokine IL-15 plays an important role in the development of several autoimmune diseases, including rheumatoid arthritis. We have generated a unique lytic and antagonistic IL-15 mutant/Fcγ2a fusion protein (CRB-15) that targets the IL-15R. In the present study we examined the effects of targeting the IL-15R on the prevention and treatment of collagen-induced arthritis (CIA) in mice and probed the possible mechanisms of action of this IL-15 mutant/Fcγ2a protein. Upon immunization with type II collagen, DBA/1 mice develop severe articular inflammation and destruction. Treatment of DBA/1 mice with a brief course of CRB-15 at the time of type II collagen challenge markedly inhibited the incidence and severity of arthritis. Moreover, in animals with ongoing established arthritis, treatment with CRB-15 effectively blocked disease progression compared with that in control-treated animals. The therapeutic effect of CRB-15 on either disease development or disease progression is remarkably stable, because withdrawal of treatment did not lead to disease relapse. A detailed analysis revealed that treatment with CRB-15 decreased synovitis in the joints; reduced bone erosion and cartilage destruction; reduced in situ production of the proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-17; and decreased the responder frequency of autoreactive T cells. Our study suggests that the effective targeting of IL-15R-triggered events with CRB-15 can be of therapeutic importance in the treatment of rheumatoid arthritis.

The Yaa Mutation Promoting Murine Lupus Causes Defective Development of Marginal Zone B Cells

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of an as yet unidentified mutant gene, Yaa (Y-linked autoimmune acceleration). In view of a possible role of marginal zone (MZ) B cells in murine SLE, we have explored whether the expression of the Yaa mutation affects the differentiation of MZ and follicular B cells, thereby implicating the acceleration of the disease. In this study, we show that both BXSB and C57BL/6 Yaa mice, including two different substrains of BXSB Yaa males that are protected from SLE, displayed an impaired development of MZ B cells early in life. Studies in bone marrow chimeras revealed that the loss of MZ B cells resulted from a defect intrinsic to B cells expressing the Yaa mutation. The lack of selective expansion of MZ B cells in diseased BXSB Yaa males strongly argues against a major role of MZ B cells in the generation of pathogenic autoantibodies in the BXSB model of SLE. Furthermore, a comparative analysis with mice deficient in CD22 or expressing an IgM anti-trinitrophenyl/DNA transgene suggests that the hyperreactive phenotype of Yaa B cells, as judged by a markedly increased spontaneous IgM secretion, is likely to contribute to the enhanced maturation toward follicular B cells and the block in the MZ B cell generation.

P-selectin Glycoprotein Ligand-1 and E-selectin Ligand-1 Are Differentially Modified by Fucosyltransferases Fuc-TIV and Fuc-TVII in Mouse Neutrophils

P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) are the two major selectin ligands on mouse neutrophils. Transfection experiments demonstrate that each ligand requires α1,3-fucosylation for selectin-binding. However, the relative contributions made by the two known myeloid α1,3-fucosyltransferases Fuc-TVII or Fuc-TIV to this α1,3-fucosylation are not yet clear. To address this issue, we have used mice deficient in Fuc-TIV and/or Fuc-TVII to examine how these enzymes generate selectin-binding glycoforms of PSGL-1 and ESL-1 in mouse neutrophils. Selectin binding was analyzed by affinity isolation experiments using recombinant, antibody-like forms of the respective endothelial selectins. We observe essentially normal binding of E- or P-selectin to PSGL-1 expressed by Fuc-TIV-deficient neutrophils but find that PSGL-1 expressed by Fuc-TVII-deficient neutrophils is not bound by E- or P-selectin. By contrast, E-selectin binds with normal efficiency to ESL-1 on Fuc-TVII-deficient neutrophils but exhibits an 80% reduction in its ability to bind ESL-1 isolated from Fuc-TIV-deficient neutrophils. The same specificity with which Fuc-TVII and Fuc-TIV generate selectin-binding forms of PSGL-1 and ESL-1 was found in transfection experiments with CHO-Pro−5 cells. In contrast, each fucosyltransferase alone could generate selectin-binding glycoforms of each of the two ligands in CHO-DUKX-B1 cells. Our data imply that in mouse neutrophils and their precursors, Fuc-TVII exclusively directs expression of PSGL-1 glycoforms bound with high affinity by P-selectin. By contrast, Fuc-TIV preferentially directs expression of ESL-1 glycoforms that exhibit high affinity for E-selectin. This substrate specificity can be mimicked in CHO-Pro−5 cells.

Differential role of three major New Zealand Black-derived loci linked with Yaa-induced murine lupus nephritis

By assessing the development of Y-linked autoimmune acceleration (Yaa) gene-induced systemic lupus erythematosus in C57BL/6 (B6) × (New Zealand Black (NZB) × B6.Yaa)F1 backcross male mice, we mapped three major susceptibility loci derived from the NZB strain. These three quantitative trait loci (QTL) on NZB chromosomes 1, 7, and 13 differentially regulated three different autoimmune traits: anti-nuclear autoantibody production, gp70-anti-gp70 immune complex (gp70 IC) formation, and glomerulonephritis. Contributions to the disease traits were further confirmed by generating and analyzing three different B6.Yaa congenic mice, each carrying one individual NZB QTL. The chromosome 1 locus that overlapped with the previously identified Nba2 (NZB autoimmunity 2) locus regulated all three traits. A newly identified chromosome 7 locus, designated Nba5, selectively promoted anti-gp70 autoantibody production, hence the formation of gp70 IC and glomerulonephritis. B6.Yaa mice bearing the NZB chromosome 13 locus displayed increased serum gp70 production, but not gp70 IC formation and glomerulonephritis. This locus, called Sgp3 (serum gp70 production 3), selectively regulated the production of serum gp70, thereby contributing to the formation of nephritogenic gp70 IC and glomerulonephritis, in combination with Nba2 and Nba5 in NZB mice. Among these three loci, a major role of Nba2 was demonstrated, because B6.Yaa Nba2 congenic male mice developed the most severe disease. Finally, our analysis revealed the presence in B6 mice of an H2-linked QTL, which regulated autoantibody production. This locus had no apparent individual effect, but most likely modulated disease severity through interaction with NZB-derived susceptibility loci.

Differentially Regulated Expression and Function of CD22 in Activated B-1 and B-2 Lymphocytes

CD22 is a B cell-restricted transmembrane protein that apparently controls signal transduction thresholds initiated through the B cell Ag receptor (BCR) in response to Ag. However, it is still poorly understood how the expression of CD22 is regulated in B cells after their activation. Here we show that the expression levels of CD22 in conventional B-2 cells are markedly down-regulated after cross-linking of BCR with anti-IgM mAb but are up-regulated after stimulation with LPS, anti-CD40 mAb, or IL-4. In contrast, treatment with anti-IgM mAb barely modulated the expression levels of CD22 in CD5+ B-1 cells, consistent with a weak Ca2+ response in anti-IgM-treated CD5+ B-1 cells. Moreover, in CD22-deficient mice, anti-IgM treatment did not trigger enhanced Ca2+ influx in CD5+ B-1 cells, unlike CD22-deficient splenic B-2 cells, suggesting a relatively limited role of CD22 in BCR signaling in B-1 cells. In contrast, CD22 levels were markedly down-regulated on wild-type B-1 cells in response to LPS or unmethylated CpG-containing oligodeoxynucleotides. These data indicate that the expression and function of CD22 are differentially regulated in B-1 and conventional B-2 cells, which are apparently implicated in innate and adaptive immunity, respectively.

Contribution of NZB Autoimmunity 2 to Y-Linked Autoimmune Acceleration-Induced Monocytosis in Association with Murine Systemic Lupus

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of the Y-linked autoimmune acceleration (Yaa) mutation, which induces an age-dependent monocytosis. Using a cohort of C57BL/6 (B6) × (NZB × B6)F1 backcross male mice bearing the Yaa mutation, we defined the pathogenic role and genetic basis for Yaa-associated monocytosis. We observed a remarkable correlation of monocytosis with autoantibody production and subsequent development of lethal lupus nephritis, indicating that monocytosis is an additional useful indicator for severe SLE. In addition, we identified an NZB-derived locus on chromosome 1 predisposing to the development of monocytosis, which peaked at Fcgr2b encoding FcγRIIB and directly overlapped with the previously identified NZB autoimmunity 2 (Nba2) locus. The contribution of Nba2 to monocytosis was confirmed by the analysis of Yaa-bearing B6 mice congenic for the NZB-Nba2 locus. Finally, we observed a very low-level expression of FcγRIIB on macrophages bearing the NZB-type Fcgr2b allele, compared with those bearing the B6-type allele, and the development of monocytosis in FcγRIIB haploinsufficient B6 mice carrying the Yaa mutation. These data suggest that the Nba2 locus may play a supplementary role in the pathogenesis of SLE by promoting the development of monocytosis and the activation of effector cells bearing stimulatory FcγR, in addition to its implication in the dysregulated activation of autoreactive B cells.

An antagonist mutant IL-15/Fc promotes transplant tolerance.

Background. IL-15 is a proinflammatory and antiapoptotic T-cell growth factor that plays an important role in a variety of autoimmune disorders and transplant rejection. To inhibit IL-15 function and to target IL-15 receptor (IL-15R) bearing cells, we have generated a unique lytic antagonistic mutant IL-15/Fc fusion protein (mIL-15/Fc). Methods. In this study, we further examined the efficacy of mIL-15/Fc in preventing allograft rejection cross minor and major histocompatibility barriers. Results. A short-course treatment with mIL-15/Fc fusion protein is sufficient to prevent cardiac allograft rejection and induce antigen-specific tolerance in minor histocompatibility complex-mismatched recipients, and permit prolonged cardiac allograft survival in fully MHC mismatched recipients. In addition, mIL-15/Fc treatment, in combination with a suboptimal dose of anti-CD154 antibody, confers permanent cardiac allograft engraftment in a fully MHC-mismatched mouse strain combination. In a murine islet allograft model, mIL-15/Fc monotherapy is capable to permit permanent allograft survival in 50% fully MHC-mismatched recipients. Conclusion. Immunochemistry studies demonstrated that prolonged graft survival was accompanied by reduced intragraft mononuclear cell infiltration and pro-inflammatory cytokine gene expression in the mIL-15/Fc treated recipients. Moreover, parallel experiments employing a mutated nonlytic IgG2a Fc demonstrate that the Fc portion of mIL-15/Fc contributes to the overall efficacy of the molecule in vivo.

A critical role for Fc gamma RIIB in the induction of rheumatoid factors

Rheumatoid factors (RF) are autoantibodies with specificity for the Fc portion of IgG, and IgG-containing immune complexes are likely to be the major source of RF autoantigens. Therefore, the activation of RF-producing B cells could be controlled specifically through recognition of IgG immune complexes by the low-affinity IgG FcR, FcγRIIB, a potent negative regulator of the BCR. To test this possibility, we determined the development of RF in C57BL/6 (B6) mice lacking FcγRIIB, in relation to the H2 haplotype, complement C3, and the Y-linked autoimmune acceleration (Yaa) mutation. FcγRIIB-null B6 mice displayed substantial anti-IgG2a RF activities in their sera, in addition to anti-DNA autoantibodies. Their RF and anti-DNA responses were linked to the H2b haplotype, but were suppressed almost completely by the H2d haplotype. Strikingly, the absence of C3 failed to modulate RF production, but strongly inhibited anti-DNA production. Furthermore, we observed that partial FcγRIIB deficiency (i.e., heterozygous level of FcγRIIB expression) was sufficient to induce the production of RF and anti-DNA autoantibodies in the presence of the Yaa mutation. In contrast to FcγRIIB, the deficiency in another BCR negative regulator, CD22, was unable to promote RF and anti-DNA autoimmune responses in B6 mice. Our results indicate that RF autoimmune responses are critically controlled by FcγRIIB, together with the H2b and Yaa gene, while C3 regulates positively and specifically anti-DNA, but not RF autoimmune responses.

Differential Activation of Anti-Erythrocyte and Anti-DNA Autoreactive B Lymphocytes by the Yaa Mutation

An as-yet-unidentified mutation, Y-linked autoimmune acceleration (Yaa), is responsible for the accelerated development of lupus-like autoimmune syndrome in mice. In view of a possible role for Yaa as a positive regulator of BCR signaling, we have explored whether the expression of the Yaa mutation affects the development and activation of transgenic autoreactive B cells expressing either 4C8 IgM anti-RBC or Sp6 IgM anti-DNA. In this study, we show that the expression of the Yaa mutation induced a lethal form of autoimmune hemolytic anemia in 4C8 transgenic C57BL/6 mice, likely as a result of activation of 4C8 anti-RBC autoreactive B cells early in life. This was further supported, although indirectly, by increased T cell-independent IgM production in spleens of nontransgenic C57BL/6 mice bearing the Yaa mutation. In contrast, Yaa failed to induce activation of Sp6 anti-DNA autoreactive B cells, consistent with a lack of increased IgM anti-DNA production in nontransgenic C57BL/6 Yaa mice. Our results suggest that Yaa can activate autoreactive B cells in a BCR-dependent manner, related to differences in the form and nature of autoantigens.