Michael P. Madaio

The central and multiple roles of B cells in lupus pathogenesis

Summary: A standard view of B cells in systemic autoimmunity is that they promote lupus by producing autoantibodies (autoAb). However, this view is incomplete because recent studies have revealed that autoimmune disease can be dissociated from autoAb deposition. Furthermore, the spontaneous T‐cell activation and organ infiltration in systemic lupus erythematosus patients and animal models are difficult to explain entirely via a direct autoAb‐mediated mechanism. In this review, we describe work addressing the B‐cell functions of autoantigen presentation and autoAb production in lupus pathogenesis. In the JHD‐MRL‐Faslpr strain (JHD/lpr), a B‐cell‐deficient version of the lupus‐prone MRL‐Faslpr (MRL/lpr) mouse, spontaneous nephritis and dermatitis is abrogated, demonstrating that B cells have a primary role in disease. B cells play a similar role in Fas‐intact, lupus‐prone MRL mice. To address the role of autoantigen presentation, we analyzed transgenic mice which have B cells that cannot secrete immunoglobulin (mIgM transgenic mice). The restoration of B cells without antibody caused substantial interstitial nephritis and vasculitis although less marked than the intact MRL/lpr controls. To address the role of autoAb, we infused serum from aged MRL/lpr mice into JHD/lpr mice. At most, mild to no nephritis was observed in the infused mice. These results indicate that B cells are promoting autoimmunity in mechanisms other than autoAb secretion, and we describe a model depicting these B‐cell roles in the context of other inflammatory events in lupus.

Pathology and protection in nephrotoxic nephritis is determined by selective engagement of specific Fc receptors

Introduction of heterologous anti–glomerular basement membrane antiserum (nephrotoxic serum, NTS) into presensitized mice triggers the production of IgG anti-NTS antibodies that are predominantly IgG2b and the glomerular deposition of pathogenic immune complexes, leading to accelerated renal disease. The pathology observed in this model is determined by the effector cell activation threshold that is established by the coexpression on infiltrating macrophages of the IgG2a/2b restricted activation receptor FcγRIV and its inhibitory receptor counterpart, FcγRIIB. Blocking FcγRIV with a specific monoclonal antibody thereby preventing IgG2b engagement or treatment with high dose intravenous γ-globulin (IVIG) to down-regulate FcγRIV while up-regulating FcγRIIB, protects mice from fatal disease. In the absence of FcγRIIB, IVIG is not protective; this indicates that reduced FcγRIV expression alone is insufficient to protect animals from pathogenic IgG2b immune complexes. These results establish the significance of specific IgG subclasses and their cognate FcγRs in renal disease.

Receptor-mediated cellular entry of nuclear localizing anti-DNA antibodies via myosin 1.

A unique subset of anti-DNA antibodies enters living cells, interacts with DNase 1, and inhibits endonuclease activity, before their nuclear localization and subsequent attenuation of apoptosis. We now report that endocytosis of these immunoglobulins is mediated by cell surface binding to brush border myosin (myosin 1). Cellular entry and internalization via this unique receptor provides initial contact for entry and sorting these immunoglobulins to translocate to the nuclear pore and enter the nucleus, interact with DNase 1 within the cytoplasm, or recycle back to the cell surface. This internalization pathway provides clues to the translocation of large proteins across cell membranes and the functional effects of intracellular antibodies on cytopathology. This is the first demonstration that brush border myosin functions as a specific cell surface receptor for internalization of large proteins.

IL-10 Regulates Murine Lupus

MRL/MpJ-Tnfrsf6lpr (MRL/MpJ-Faslpr; MRL-Faslpr) mice develop a spontaneous lupus syndrome closely resembling human systemic lupus erythematosus. To define the role of IL-10 in the regulation of murine lupus, IL-10 gene-deficient (IL-10−/−) MRL-Faslpr (MRL-Faslpr IL-10−/−) mice were generated and their disease phenotype was compared with littermates with one or two copies of an intact IL-10 locus (MRL-Faslpr IL-10+/− and MRL-Faslpr IL-10+/+ mice, respectively). MRL-Faslpr IL-10−/− mice developed severe lupus, with earlier appearance of skin lesions, increased lymphadenopathy, more severe glomerulonephritis, and higher mortality than their IL-10-intact littermate controls. The increased severity of lupus in MRL-Faslpr IL-10−/− mice was closely associated with enhanced IFN-γ production by both CD4+ and CD8+ cells and increased serum concentration of IgG2a anti-dsDNA autoantibodies. The protective effect of IL-10 in this lupus model was further supported by the observation that administration of rIL-10 reduced IgG2a anti-dsDNA autoantibody production in wild-type MRL-Faslpr animals. In summary, our results provide evidence that IL-10 can down-modulate murine lupus through inhibition of pathogenic Th1 cytokine responses. Modulation of the level of IL-10 may be of potential therapeutic benefit for human lupus.

ANTI-DNA ANTIBODY IDIOTYPES IN SYSTEMIC LUPUS ERYTHEMATOSUS

Monoclonal anti-DNA antibodies prepared by the hybridoma technique were used for an analysis of idiotypes of anti-DNA antibodies in systemic lupus erythematosus (SLE). Serum levels of one idiotypic marker, 16/6/R, were higher than normal in 40 of 74 patients (54%) with active SLE, compared with only 6 of 24 patients (25%) with inactive SLE, 9 of 38 patients (25%) with rheumatoid arthritis, and 4 of 96 normal subjects (4%). Levels of the 16/6/R idiotypic marker were determined in serially collected serum samples from 12 patients with SLE. Concordance was found between idiotype levels and clinical activity in 8 of the 12 patients. Levels of the 16/6/R idiotype tended to correlate with levels of antibodies to double-stranded DNA, but in some cases the clinical status was reflected better by the idiotype levels than by the levels of anti-double-stranded-DNA antibodies. Measurement of idiotypes of anti-DNA antibodies may provide information valuable in monitoring the clinical course of patients with SLE.

Short Term Administration of Costimulatory Blockade and Cyclophosphamide Induces Remission of Systemic Lupus Erythematosus Nephritis in NZB/W F1 Mice by a Mechanism Downstream of Renal Immune Complex Deposition

NZB/W F1 mice with established nephritis were treated with a single dose of cyclophosphamide with or without a 2-wk course of murine CTLA4Ig, either alone or in combination with anti-CD154. Sixty to 80% of treated mice entered remission, and remission could be reinduced following relapse. A decrease in the frequency of anti-DNA-producing B cells and activated T cells was observed in treated mice, but this effect lasted only 3–6 wk, while remissions were sustained for up to 20 wk. Light microscopy of the kidneys of mice in remission revealed less glomerular inflammation, less tubular damage, and less infiltration of inflammatory cells. By immunofluorescence, however, IgG and C3 staining of glomeruli was no different in treated mice vs controls. Since chemokines and their receptors play an important role in inflammatory cell infiltration of affected organs in autoimmune diseases, we examined chemokine expression in the kidneys. Decreases in the expression of inflammatory cytokines and chemokines were evident in mice in the early stages of remission, but these differences were no longer present in late remission. Increased expression of CXCL13 was detected in the inflammatory infiltrates of the control NZB/NZW mice. Strikingly, we could not detect any CXCL13 in the kidneys of the treated group even in late remission. These findings suggest that costimulatory blockade together with cyclophosphamide influence the activation state of renal CD11c-positive cells and therefore lead to less B and T cell infiltration and nephritis.

Mechanism of Action of Transmembrane Activator and Calcium Modulator Ligand Interactor-Ig in Murine Systemic Lupus Erythematosus

B cell-activating factor belonging to the TNF family (BAFF) blockade prevents the onset of disease in systemic lupus erythematosus (SLE)-prone NZB/NZW F1 mice. To determine the mechanism of this effect, we administered a short course of TACI-Ig with and without six doses of CTLA4-Ig to 18- to 20-wk-old NZB/NZW F1 mice and evaluated the effect on B and T cell subsets and on anti-dsDNA Ab-producing B cells. Even a brief exposure to TACI-Ig had a beneficial effect on murine SLE; CTLA4-Ig potentiated this effect. The combination of TACI-Ig and CTLA4-Ig resulted in a temporary decrease in serum IgG levels. However, after cessation of treatment, high titers of IgG anti-dsDNA Abs appeared in the serum and IgG Abs deposited in the kidneys. Despite the appearance of pathogenic autoantibodies, the onset of proteinuria was markedly delayed; this was associated with prolonged depletion of B cells past the T1 stage, a decrease in the size of the spleen and lymph nodes, and a decrease in the absolute number of activated and memory CD4+ T cells. TACI-Ig treatment normalized serum levels of IgM that are markedly elevated in NZB/W F1 mice; this appeared to be due to a prolonged effect on the ability of the splenic microenvironment to support short-lived IgM plasma cells. Finally, a short course of combination TACI-Ig and CTLA4-Ig prolonged life and even reversed proteinuria in aged NZB/W F1 mice, suggesting that BAFF blockade may be an effective therapeutic strategy for active SLE.

Pathogenic autoantibodies in lupus nephritis

Lupus nephritis is a major complication of systemic lupus erythematosus (SLE) and is associated with a high rate of morbidity and mortality. While many different immunologic and nonimmunologic factors contribute to disease expression in lupus nephritis, a large body of evidence suggests that the production of anti-DNA antibodies and the formation of glomerular immune deposits are important initial events in the pathogenesis of the disease. This review will summarize our current understanding of the differences between pathogenic and nonpathogenic autoantibodies, the mechanisms by which these autoantibodies induce renal injury and the effector mechanisms which are subsequently activated by the deposited autoantibodies that ultimately lead to the expression of the different lupus lesions.

FcR-bearing myeloid cells are responsible for triggering murine lupus nephritis.

Lupus glomerulonephritis is initiated by deposition of IgG-containing immune complexes in renal glomeruli. FcR engagement by immune complexes (IC) is crucial to disease development as uncoupling this pathway in FcRγ−/− abrogates inflammatory responses in (NZB × NZW)F1 mice. To define the roles of FcR-bearing hemopoietic cells and of kidney resident mesangial cells in pathogenesis, (NZB × NZW)F1 bone marrow chimeras were generated. Nephritis developed in (NZB × NZW)F1 mice expressing activating FcRs in hemopoietic cells. Conversely, recipients of FcRγ−/− bone marrow were protected from disease development despite persistent expression of FcRγ in mesangial cell populations. Thus, activating FcRs on circulating hemopoietic cells, rather than on mesangial cells, are required for IC-mediated pathogenesis in (NZB × NZW)F1. Transgenic FcRγ−/− mice expressing FcRγ limited to the CD11b+ monocyte/macrophage compartment developed glomerulonephritis in the anti-glomerular basement disease model, whereas nontransgenic FcRγ−/− mice were completely protected. Thus, direct activation of circulating FcR-bearing myeloid cells, including monocytes/macrophages, by glomerular IC deposits is sufficient to initiate inflammatory responses.

Deletion of decay-accelerating factor (CD55) exacerbates autoimmune disease development in MRL/lpr mice.

Decay-accelerating factor (DAF, CD55) is a glycosylphosphatidylinositol-anchored membrane protein that restricts complement activation on autologous cells. It is also a ligand for CD97, an activation-associated lymphocyte antigen with seven transmembrane domains. It is widely expressed on cells of both the hematopoietic and nonhematopoietic lineages. Although deficiency of DAF on human erythrocytes is associated with the hemolytic anemia syndrome paroxysmal nocturnal hemoglobinuria, the in vivo biology of DAF is still poorly understood. We addressed the in vivo function of DAF in a knockout mouse model and describe here that deletion of DAF exacerbates autoimmune disease development in MRL/lpr mice, a model for human systemic lupus erythematosus. Compared to DAF-sufficient littermate controls, DAF-deficient female MRL/lpr mice developed exacerbated lymphadenopathy and splenomegaly, higher serum anti-chromatin autoantibody levels, and aggravated dermatitis. Consistent with the phenotype of aggravated dermatitis in DAF-deficient mice, Northern and Western blots and immunofluorescence studies showed DAF to be expressed abundantly in the mouse skin, suggesting that it may play a particularly important role in this tissue. Histology and immunostaining demonstrated inflammatory infiltrate and focal C3 deposition in early skin lesions, mostly along the dermal-epidermal junction. These results reveal a protective function of DAF in the development of a systemic autoimmune syndrome and suggest that dysfunction or down-regulation of DAF may contribute to autoimmune disease pathogenesis and manifestation.