Kirsten Neubert

The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis

Autoantibody-mediated diseases like myasthenia gravis, autoimmune hemolytic anemia and systemic lupus erythematosus represent a therapeutic challenge. In particular, long-lived plasma cells producing autoantibodies resist current therapeutic and experimental approaches. Recently, we showed that the sensitivity of myeloma cells toward proteasome inhibitors directly correlates with their immunoglobulin synthesis rates. Therefore, we hypothesized that normal plasma cells are also hypersensitive to proteasome inhibition owing to their extremely high amount of protein biosynthesis. Here we show that the proteasome inhibitor bortezomib, which is approved for the treatment of multiple myeloma, eliminates both short- and long-lived plasma cells by activation of the terminal unfolded protein response. Treatment with bortezomib depleted plasma cells producing antibodies to double-stranded DNA, eliminated autoantibody production, ameliorated glomerulonephritis and prolonged survival of two mouse strains with lupus-like disease, NZB/W F1 and MRL/lpr mice. Hence, the elimination of autoreactive plasma cells by proteasome inhibitors might represent a new treatment strategy for antibody-mediated diseases.

Extensive Immunoglobulin Production Sensitizes Myeloma Cells for Proteasome Inhibition

Multiple myeloma is an incurable plasma cell neoplasia characterized by the production of large amounts of monoclonal immunoglobulins. The proteasome inhibitor bortezomib (PS-341, Velcade) induces apoptosis in various malignant cells and has been approved for treatment of refractory multiple myeloma. Inhibition of the antiapoptotic transcription factor nuclear factor-kappaB (NF-kappaB) apparently contributes to the antitumor effects of bortezomib; however, this mechanism cannot fully explain the exceptional sensitivity of myeloma cells. Extensive protein synthesis as in myeloma cells is inherently accompanied by unfolded proteins, including defective ribosomal products (DRiPs), which need to be degraded by the ubiquitin-proteasome system. Therefore, we hypothesized that the proapoptotic effect of bortezomib in multiple myeloma is mainly due to the accumulation of unfolded proteins in cells with high protein biosynthesis. Using the IgG-secreting human myeloma cell line JK-6L and murine muH-chain-transfected Ag8.H myeloma cells, apoptosis induction upon proteasome inhibition was clearly correlated with the amount of immunoglobulin production. Preferentially in immunoglobulin-high myeloma cells, bortezomib triggered activation of caspases and induction of proapoptotic CHOP, a component of the terminal unfolded protein response induced by endoplasmic reticulum (ER) stress. In immunoglobulin-high cells, bortezomib increased the levels of proapoptotic Bax while reducing antiapoptotic Bcl-2. Finally, IgG-DRiPs were detected in proteasome inhibitor-treated cells. Hence, proteasome inhibitors induce apoptosis preferentially in cells with high synthesis rate of immunoglobulin associated with accumulation of unfolded proteins/DRiPs inducing ER stress. These findings further elucidate the antitumor activities of proteasome inhibitors and have important implications for optimizing clinical applications.

The Proteasome Inhibitor Bortezomib Prevents Lupus Nephritis in the NZB/W F1 Mouse Model by Preservation of Glomerular and Tubulointerstitial Architecture

Background/Aims: Crucial steps in the initiation of lupus nephritis are the deposition of (auto-)antibodies and consequent complement activation. In spite of aggressive treatment patients may develop terminal renal failure. Therefore, new treatment strategies are needed. In extension to our previously published data we here analyzed the potential renoprotective mechanisms of bortezomib (BZ) in experimental lupus nephritis by focusing on morphological changes. Methods: Female NZB×NZW F1 mice develop lupus-like disease with extensive nephritis that finally leads to lethal renal failure. Treatment with 0.75 mg/kg BZ i.v. or placebo (PBS) twice per week started at 18 or 24 weeks of age. Antibody production was measured with ELISA and kidney damage was determined by quantitative morphological and immunohistochemical methods. Results: BZ treatment completely inhibited antibody production in both BZ-treated groups and prevented the development of nephritis in comparison to PBS-treated animals. Glomerular and tubulointerstitial damage scores, collagen IV expression, mean glomerular volume as well as tubulointerstitial proliferation and apoptosis were significantly lower after BZ treatment. Glomerular ultrastructure and in particular podocyte damage and loss were prevented by BZ treatment. Conclusions: BZ effectively prevents the development of nephritis in the NZB/W F1 mouse model. Specific protection of podocyte ultrastructure may critically contribute to renoprotection by BZ, which may also represent a potential new treatment option in human lupus nephritis.

Antigen Delivery to CD11c+CD8− Dendritic Cells Induces Protective Immune Responses against Experimental Melanoma in Mice In Vivo

Dendritic cells (DCs) are central modulators of immune responses and, therefore, interesting target cells for the induction of antitumor immune responses. Ag delivery to select DC subpopulations via targeting Abs to DC inhibitory receptor 2 (DCIR2, clone 33D1) or to DEC205 was shown to direct Ags specifically to CD11c+CD8− or CD11c+CD8+ DCs, respectively, in vivo. In contrast to the increasing knowledge about the induction of immune responses by efficiently cross-presenting CD11c+CD8+ DCs, little is known about the functional role of Ag-presenting CD11c+CD8− DCs with regard to the initiation of protective immune responses. In this study, we demonstrate that Ag targeting to the CD11c+CD8− DC subpopulation in the presence of stimulating anti-CD40 Ab and TLR3 ligand polyinosinic-polycytidylic acid induces protective responses against rapidly growing tumor cells in naive animals under preventive and therapeutic treatment regimens in vivo. Of note, this immunization protocol induced a mixed Th1/Th2-driven immune response, irrespective of which DC subpopulation initially presented the Ag. Our results provide important information about the role of CD11c+CD8− DCs, which have been considered to be less efficient at cross-presenting Ags, in the induction of protective antitumor immune responses.

Apoptotic cells selectively suppress the Th1 cytokine interferon γ in stimulated human peripheral blood mononuclear cells and shift the Th1/Th2 balance towards Th2

Apoptotic cells are readily recognized and engulfed by phagocytes and usually do not induce inflammation or tissue damage. Furthermore, they can actively suppress a pro-inflammatory response in phagocytes: In the presence of apoptotic cells, activated monocytes/macrophages produce more of the anti-inflammatory and immunoregulatory cytokines IL-10 and TGF-β, but less of the pro-inflammatory cytokines TNFα, IL-1β and IL-12. This immunoregulatory effect is most likely mediated by several receptors on monocytes/macrophages including the thrombospondin receptor (CD36). In addition to the modulation of cytokine secretion, apoptotic cell material inhibited the expression of MHC class II molecules on the surface of monocytes/macrophages. Decreased MHC II expression appeared to be mediated predominantly by increased IL-10 secretion in a para-/autocrine manner. Here, we show that the functional modulation of antigen-presenting monocytes/macrophages by apoptotic cells also influences T cell activation and function. When human peripheral blood mononuclear cells were stimulated with recall antigens in the presence of apoptotic cells, interferonγ (IFNγ) secretion was markedly suppressed, whereas secretion of the Th2 cytokine IL-4 was not significantly altered. Hence, apoptotic cells shift the T cell cytokine secretion pattern towards a Th2-like response. This Th2 shift can largely be prevented by neutralizing IL-10, indicating an important role of this cytokine for modulating T cell cytokine secretion patterns.

Proteasome Inhibition Aggravates Tumor Necrosis Factor―Mediated Bone Resorption in a Mouse Model of Inflammatory Arthritis

OBJECTIVE The proteasome inhibitor bortezomib has potent anti-myeloma and bone-protective activity. Recently, bortezomib was shown to directly inhibit osteoclastogenesis. The aim of this study was to analyze the influence and therapeutic effect of bortezomib in a mouse model of inflammatory arthritis. METHODS Heterozygous human tumor necrosis factor α (hTNFα)-transgenic mice and their wild-type (WT) littermates were intravenously injected with 0.75 mg/kg of bortezomib or phosphate buffered saline twice weekly. The mice were assessed for clinical signs of arthritis. After 6 weeks of treatment, mice were analyzed for synovial inflammation, cartilage damage, bone erosions, and systemic bone changes. Osteoclast precursors from WT and hTNF-transgenic mice were isolated from bone marrow, treated with bortezomib, and analyzed for osteoclast differentiation, bone resorption, and expression of osteoclast-specific genes as well as apoptosis and ubiquitination. RESULTS Bortezomib-treated hTNF-transgenic mice showed moderately increased inflammatory activity and dramatically enhanced bone erosions associated with a significant increase in the number of synovial osteoclasts. Interestingly, bortezomib did not alter systemic bone turnover in either hTNF-transgenic mice or WT mice. In vitro, treatment with therapeutically relevant concentrations of bortezomib resulted in increased differentiation of monocytes into osteoclasts and more resorption pits. Molecularly, bortezomib increased the expression of TNF receptor-associated factor 6, c-Fos, and nuclear factor of activated T cells c1 in osteoclast precursors. CONCLUSION In TNF-mediated bone destruction, bortezomib treatment increased synovial osteoclastogenesis and bone destruction. Hence, proteasome inhibition may have a direct bone-resorptive effect via stimulation of osteoclastogenesis during chronic arthritis.

The Early Marginal Zone B Cell-Initiated T-Independent Type 2 Response Resists the Proteasome Inhibitor Bortezomib

The proteasome inhibitor bortezomib is approved for the treatment of multiple myeloma and mantle cell lymphoma. We recently demonstrated that bortezomib eliminates autoreactive plasma cells in systemic lupus erythematosus mouse models, thereby representing a promising novel treatment for Ab-mediated diseases. In this study, we investigated the effects of bortezomib on the just developing and pre-existing T-dependent Ab response toward dinitrophenyl-keyhole limpet hemocyanin and the T-independent type 2 response toward (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP)-Ficoll in BALB/c mice. Bortezomib treatment strongly reduced T-dependent Ab titers mainly due to depletion of plasma cells. In contrast, the early T-independent type 2 response against i.v. administered NIP-Ficoll, which is predominantly dependent on marginal zone (MZ) B cells, resisted bortezomib. Upon bortezomib treatment, immunoproteasome subunits and the antiapoptotic unfolded protein response including NF-κB were induced in NIP-Ficoll–stimulated MZ B cells, but not in plasma cells and follicular B cells. In summary, bortezomib treatment decreases Ab titers arising from T-dependent immune responses predominantly by eliminating plasma cells. In contrast, the early T-independent type 2 response protecting the organism against blood-borne pathogens remains largely intact due to a remarkable resistance of MZ B cells against proteasome inhibition.

Efficient generation of a monoclonal antibody against the human C-type lectin receptor DCIR by targeting murine dendritic cells

Dendritic cells (DCs) are very important for the generation of long lasting immune responses against pathogens or the induction of anti-tumor responses. Targeting antigen to dendritic cells via monoclonal antibodies specific for DC cell surface receptors such as DEC205 was shown to elicit potent cellular and humoral immune responses in vivo. Therefore, we investigated whether this novel strategy might also be useful for the generation of new monoclonal antibodies against molecules of choice. We show, that by targeting the extracellular domain of the human C-type lectin receptor ClecSF6/DCIR/LLIR (hDCIR) to DEC205 on DCs in vivo, we were able to generate highly specific monoclonal antibodies against hDCIR.

DC subset–specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

Dendritic cells (DCs) are efficient antigen-presenting cells equipped with various cell surface receptors for the direct or indirect recognition of pathogenic microorganisms. Interestingly, not much is known about the specific expression pattern and function of the individual activating and inhibitory Fc&ggr; receptors (Fc&ggr;Rs) on splenic DC subsets in vivo and how they contribute to the initiation of T cell responses. By targeting antigens to select activating and the inhibitory Fc&ggr;R in vivo, we show that antigen uptake under steady-state conditions results in a short-term expansion of antigen-specific T cells, whereas under inflammatory conditions especially, the activating Fc&ggr;RIV is able to induce superior CD4+ and CD8+ T cell responses. Of note, this effect was independent of Fc&ggr;R intrinsic activating signaling pathways. Moreover, despite the expression of Fc&ggr;RIV on both conventional splenic DC subsets, the induction of CD8+ T cell responses was largely dependent on CD11c+CD8+ DCs, whereas CD11c+CD8− DCs were critical for priming CD4+ T cell responses.

Novel treatment strategies for antibody-mediated diseases: targeting long-lived plasma cells

Reinhard E Voll†, Kirsten Neubert, Silke Meister, Eva Guckel & Joachim R Kalden †Author for correspondence University of ErlangenNuremberg, Interdisciplinary Center for Clinical Research, IZKF-N2, Nikolaus-FiebigerCenter of Molecular Medicine, Gluckstrasse 6, 91054 Erlangen, Germany Tel.: +49 (0)9131 8539301 Fax: +49 (0)9131 8539311 rvoll@molmed.unierlangen.deand, University of ErlangenNuremberg, Department of Internal Medicine 3 (Rheumatology and Clinical Immunology), Krankenhausstrasse 12, 91054 Erlangen, Germany ‘Whenever pathogenic antibodies are predominantly secreted by long-lived plasma cells, good treatment responses become unlikely.’