Silke Meister

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.

Induction of inflammatory and immune responses by HMGB1–nucleosome complexes: implications for the pathogenesis of SLE

Autoantibodies against double-stranded DNA (dsDNA) and nucleosomes represent a hallmark of systemic lupus erythematosus (SLE). However, the mechanisms involved in breaking the immunological tolerance against these poorly immunogenic nuclear components are not fully understood. Impaired phagocytosis of apoptotic cells with consecutive release of nuclear antigens may contribute to the immune pathogenesis. The architectural chromosomal protein and proinflammatory mediator high mobility group box protein 1 (HMGB1) is tightly attached to the chromatin of apoptotic cells. We demonstrate that HMGB1 remains bound to nucleosomes released from late apoptotic cells in vitro. HMGB1–nucleosome complexes were also detected in plasma from SLE patients. HMGB1-containing nucleosomes from apoptotic cells induced secretion of interleukin (IL) 1β, IL-6, IL-10, and tumor necrosis factor (TNF) α and expression of costimulatory molecules in macrophages and dendritic cells (DC), respectively. Neither HMGB1-free nucleosomes from viable cells nor nucleosomes from apoptotic cells lacking HMGB1 induced cytokine production or DC activation. HMGB1-containing nucleosomes from apoptotic cells induced anti-dsDNA and antihistone IgG responses in a Toll-like receptor (TLR) 2–dependent manner, whereas nucleosomes from living cells did not. In conclusion, HMGB1–nucleosome complexes activate antigen presenting cells and, thereby, may crucially contribute to the pathogenesis of SLE via breaking the immunological tolerance against nucleosomes/dsDNA.

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.

Cutting Edge: Signaling and Cell Surface Expression of a μH Chain in the Absence of λ5: A Paradigm Revisited

Pre-B cell receptor (pre-BCR) signals are essential for pro-B cells to mature efficiently into pre-B cells. The pre-BCR is an Ig-like transmembrane complex that is assembled from two μH chains (μHC) and two surrogate L chains consisting of the non-covalently associated polypeptides VpreB and λ5. In λ5−/− mice, pro-B cell maturation is impaired, but not completely blocked, implying that a μHC induces differentiation signals in the absence of λ5. Using a mouse model, in which transgenic μHC expression can be controlled by tetracycline, we show that in the absence of λ5, the transgenic μHC promotes in vivo differentiation of pro-B cells, induces IL-7-dependent cell growth, and is expressed on the surface of pre-B cells. Our findings not only show that an incomplete pre-BCR can initiate signals, but also challenge the paradigm that an IgHC must associate with an IgLC or a SLC to gain transport and signaling competency.

Oxidation of the alarmin high-mobility group box 1 protein (HMGB1) during apoptosis.

The architectural chromosomal protein high-mobility group box 1 protein (HMGB1) acts as an alarmin when released from cells. It is involved in the pathogenesis of inflammatory and autoimmune diseases. HMGB1 can undergo post-translational modifications including oxidation. However, the mechanisms and functional relevance of HMGB1 oxidation are not yet understood. Increased concentrations of reactive oxygen species (ROS) have been reported during apoptosis and necrosis. Hence, we investigated the oxidative status of HMGB1 in dead cells. Immunoblot analyses under reducing and non-reducing conditions revealed that HMGB1 is oxidized in dead cells. Moreover, tagging of oxidized cysteine residues by a maleimide moiety linked to polyethylene glycol showed that HMGB1 passively released from primary and secondary necrotic cells was predominantly oxidized. Also HMGB1 in plasma of patients with systemic lupus was reversibly oxidized. In conclusion, HMGB1 undergoes reversible oxidative modifications at cysteine residues during cell death, which may modulate its biological properties.

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.

Sensitive detection of plasma/serum DNA in patients with systemic lupus erythematosus

Objective: To optimize the use of the fluorochromic PicoGreen assay for the sensitive detection and quantification of double stranded (ds) DNA in plasma/serum samples of patients with systemic lupus erythematosus (SLE). Methods: Plasma/serum samples were obtained from SLE patients and normal healthy donors (NHD). Plasma/serum proteins were digested with proteinase K. DNA was subsequently purified using silica-based ion exchange micro columns and detected using the PicoGreen assay. Results: Sensitive detection of plasma/serum DNA is impaired by proteins: (1) Proteins caused background fluorescence in the PicoGreen assay. (2) Packaging of dsDNA in nucleosomes markedly reduced PicoGreen fluorescence. Therefore, we digested proteins using proteinase K and purified DNA before detection by PicoGreen assay. This procedure resulted in a detection limit for plasma/serum dsDNA of less than 1 ng/ml, and is therefore markedly more sensitive than previously described methods. We found that DNA concentrations are higher in serum than in plasma of healthy donors, suggesting artifactual DNA release during coagulation. In addition, we found higher levels of DNA in plasma and serum of a group of SLE patients compared to NHD. Conclusions: We have optimized the use of the PicoGreen assay for the ultrasensitive and reliable quantification of DNA in plasma/serum samples. This new method can be used in future studies to explore a possible correlation between circulating DNA levels and disease activity in patients with SLE.

Simian Immunodeficiency Virus Utilizes Human and Sooty Mangabey but Not Rhesus Macaque STRL33 for Efficient Entry

ABSTRACT It has been established that many simian immunodeficiency virus (SIV) isolates utilize the orphan receptors GPR15 and STRL33 about as efficiently as the chemokine receptor CCR5 for entry into target cells. Most studies were performed, however, with coreceptors of human origin. We found that SIV from captive rhesus macaques (SIVmac) can utilize both human and simian CCR5 and GPR15 with comparable efficiencies. Strikingly, however, only human STRL33 (huSTRL33), not rhesus macaque STRL33 (rhSTRL33), functioned efficiently as an entry cofactor for a variety of isolates of SIVmac and SIV from sooty mangabeys. A single amino acid substitution of S30R in huSTRL33 impaired coreceptor activity, and the reverse change in rhSTRL33 greatly increased coreceptor activity. In comparison, species-specific sequence variations in N-terminal tyrosines in STRL33 had only moderate effects on SIV entry. These results show that a serine residue located just outside of the cellular membrane in the N terminus of STRL33 is critical for SIV coreceptor function. Interestingly, STRL33 derived from sooty mangabeys, a natural host of SIV, also contained a serine at the corresponding position and was used efficiently as an entry cofactor. These results suggest that STRL33 is not a relevant coreceptor in the SIV/macaque model but may play a role in SIV replication and transmission in naturally infected sooty mangabeys.

Amino Acid 324 in the Simian Immunodeficiency Virus SIVmac V3 Loop Can Confer CD4 Independence and Modulate the Interaction with CCR5 and Alternative Coreceptors

ABSTRACT The V3 loop of the simian immunodeficiency virus (SIV) envelope protein (Env) largely determines interactions with viral coreceptors. To define amino acids in V3 that are critical for coreceptor engagement, we functionally characterized Env variants with amino acid substitutions at position 324 in V3, which has previously been shown to impact SIV cell tropism. These changes modulated CCR5 engagement and, in some cases, allowed the efficient usage of CCR5 in the absence of CD4. The tested amino acid substitutions had highly differential effects on viral infectivity. Eleven of sixteen substitutions disrupted entry via CCR5 or the alternative coreceptor GPR15. Nevertheless, most of these variants replicated in the macaque T-cell line 221-89 and some also replicated in rhesus macaque peripheral blood monocytes, suggesting that efficient usage of CCR5 and GPR15 on cell lines is not a prerequisite for SIV replication in primary cells. Four variants showed enhanced entry into the macaque sMagi reporter cell line. However, sMagi cells did not express appreciable amounts of CCR5 and GPR15 mRNA, and entry into these cells was not efficiently blocked by a small-molecule CCR5 antagonist, suggesting that sMagi cells express as-yet-unidentified entry cofactors. In summary, we found that a single amino acid at position 324 in the SIV Env V3 loop can modulate both the efficiency and the types of coreceptors engaged by Env and allow for CD4-independent fusion in some cases.

Cells under pressure - treatment of eukaryotic cells with high hydrostatic pressure, from physiologic aspects to pressure induced cell death.

The research on high hydrostatic pressure in medicine and life sciences is multifaceted. According to the used pressure head the research has to be divided into two different parts. To study physiological aspects of pressure on eukaryotic cells physiological pressure (pHHP; < 100 MPa) is used. pHHP induces morphological alterations in the cellular organelles and evokes a reversible stress response similar to the well known heat shock response. pHHP induces highly reversible alterations and normally does not affect cellular viability. The treatment of eukaryotic cells with non-physiological pressure (HHP; > or = 100 MPa) reveals different outcomes. Treatment with HHP < 150 MPa does not markedly affect viability of human cells, but induces apoptosis in murine cells. In human cells apoptosis is observed after treatment with > or = 200 MPa. Moreover, HHP treatment with > 300 MPa leads to necrosis. Therefore, HHP plays a role for the sterilisation of human transplants, of food stuff, and pharmaceuticals. Human tumour cells subjected to HHP > 300 MPa display a necrotic phenotype along with a gelificated cytoplasm, preserve their shape, and retain their immunogenicity. These observations favour the use of HHP to produce whole cell based tumour vaccines. Further experiments revealed that the increment of pressure as well as the pressure holding time influences the cell death of tumour cells. We conclude that high hydrostatic pressure offers both, an economic, easy to apply, clean, and fast technique for the generation of vaccines, and a promising tool to study physiological aspects.