Silke Winkler

Chlorpromazine induces apoptosis in activated human lymphoblasts: a mechanism supporting the induction of drug-induced lupus erythematosus?

OBJECTIVE Drug-induced lupus erythematosus is a serious side effect of certain medications, such as procainamide, quinidine, hydralazine, chlorpromazine, and isoniazid, the underlying pathogenesis of which is unresolved. In this study, we examined the influence of these drugs on the regulation of apoptosis, or programmed cell death, in quiescent and activated human lymphocytes. We also discuss the dysregulation of apoptosis as a pathogenetic factor in systemic lupus erythematosus. METHODS Peripheral blood mononuclear cells or activated lymphoblasts from normal donors were incubated with different concentrations of each of the above-mentioned drugs. RESULTS We did not find induction of apoptosis in quiescent cells over a broad concentration range. In contrast, lymphoblasts readily underwent apoptosis when cultured with chlorpromazine, but not any of the other drugs, after stimulation with interleukin-2 (IL-2) in a dose-, time- and cell cycle-dependent manner. By several lines of evidence, toxicity was ruled out. Characteristic features of apoptosis-like incorporation of propidium iodide (PI), such as increased annexin V binding, changes in mitochondrial membrane potential, and induction of DNA breaks (as evidenced by TUNEL techniques), could be induced in lymphoblasts after chlorpromazine treatment. Chlorpromazine did not cause apoptosis by inhibition of cytokine binding or blockade of early intracellular signaling. The protease inhibitor Z-VAD and the ceramide inhibitor sphingosine 1-phosphate effectively blocked chlorpromazine-induced apoptosis (by PI staining and by externalization of phosphatidylserine), in contrast to the caspase 3/CPP32 inhibitor DEVD, which had only minor effects. Western blot analysis revealed IL-2-mediated phosphorylation of extracellular signal-regulated kinase, which was sensitive to chlorpromazine. Using lymphoblasts from a patient with Canale-Smith syndrome, we found that chlorpromazine-mediated apoptosis is Fas/ APO-1 independent. CONCLUSION These data suggest that chlorpromazine mediates apoptosis in human lymphoblasts through specific activation of intracellular proapoptotic signaling cascades. This mechanism might lead to an unsynchronized inflow of apoptotic break-down products and thereby to the induction of (auto)immunity against nuclear components.

Autoantibodies against galectins are associated with antiphospholipid syndrome in patients with systemic lupus erythematosus

The presence of autoantibodies against immunoregulatory effectors can be relevant for onset and/or the progression of autoimmune disease. Emerging insights into an immunological activity profile including a role as opsonins give reason to systematically monitor sera of patients for immunoglobulin G (IgG) autoantibodies, preferably for several galectins at the same time. Here, we report on a study of chronic inflammatory rheumatic diseases, i.e. systemic lupus erythematosus (SLE; pilot cohort p, n = 40; confirmation cohort c, n = 109), rheumatoid arthritis (RA; p, n = 32; c, n = 25) and primary antiphospholipid syndrome (APS; c, n = 64). Enzyme-linked immunosorbent assay-based series using galectin-1, -2, -3, -4, -7, -8 and -9 and natural processing products, i.e. the truncated version of galectin-3 and the N-terminal domains of galectin-4, -8 and -9, were performed. Normal healthy donors (p, n = 20; c, n = 21) and patients with paraproteins (c, n = 19) served as controls. Highly significant optical density-value readings for IgG autoantibodies were consistently detected for the proto-type galectin-7 (SLE) and the tandem repeat-type galectin-8 and -9 (SLE and RA). Their presence was independent from the autoantibody status against double-stranded DNA (for patients with SLE) or a rheumatoid factor (for patients with RA), respectively. Importantly, anti-galectin-2 autoantibodies highly significantly correlated with the appearance of a secondary APS in patients with SLE so that this parameter may serve as an additional biomarker for APS. Equally of note, the presence of IgG autoantibodies against galectins capable to act as an opsonin may contribute to a sustained immune dysregulation in patients with chronic inflammatory rheumatic diseases.

CRP/anti-CRP antibodies assembly on the surfaces of cell remnants switches their phagocytic clearance toward inflammation

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease characterized by the production of autoantibodies, formation of immune complexes (IC), and activation of complement that ultimately fuel acute and/or chronic inflammation. Accumulation in blood and tissues of post-apoptotic remnants is considered of etiological and pathological importance for patients with SLE. Besides receptors directly recognizing apoptotic cells, soluble opsonins of the innate immune system bind apoptotic material dependent on the stage of apoptosis. We describe the binding to the surface of secondary necrotic cells (SNEC) of the serum opsonin CRP and further opsonins. We show that anti-dsDNA and anti-CRP autoantibodies bind and sensitize SNEC. Autoantibody-sensitized SNEC were cleared by macrophages in vitro and induced a pro-inflammatory cytokine response. In conclusion, anti-CRP, CRP, and SNEC form a ternary pyrogen endowed with strong pro-inflammatory capabilities which is able to maintain and perpetuate chronic inflammation.

CD45 Tyrosine Phosphatase Controls Common γ-Chain Cytokine-Mediated STAT and Extracellular Signal-Related Kinase Phosphorylation in Activated Human Lymphoblasts: Inhibition of Proliferation Without Induction of Apoptosis

The objective of this study was to test whether CD45 signals can influence signaling processes in activated human lymphoblasts. To this end, we generated lymphoblasts which proliferate in response to common γ-chain cytokines, but readily undergo apoptosis after cytokine withdrawal. In experiments with the CD45R0 mAb UCHL-1, but not control CD45 mAbs, we found significant inhibition of proliferation. Interestingly, the pan-CD45 mAb GAP8.3, which is most effective in inhibition of OKT-3-mediated proliferation in quiescent lymphocytes, was ineffective in lymphoblasts. Addition of CD3 mAb OKT-3 had no influence on IL-2-mediated proliferation (with or without UCHL-1). In contrast, after addition of OKT-3 to IL-4- and IL-7-stimulated proliferation assays, UCHL-1 signals could not significantly alter cellular proliferation. We did not find induction of apoptosis following CD45R0 signaling. In Western blots using mAbs detecting phosphorylated STAT-3, STAT-5, STAT-6, or extracellular signal-related kinase 1/2, we found that CD45R0 signaling could effectively diminish phosphorylation of these intracellular signaling components. Using RT-PCR, we found that CD45R0 signaling inhibited IL-2 mRNA production without major influence on IL-13, IL-5, or IFN-γ mRNA levels. Costimulation with OKT-3 and IL-2 optimally induced secretion of IFN-γ, TNF-α, and IL-5, which was not decreased by CD45 signals. In conclusion, we illustrate that CD45R0 signals control early cytokine receptor-associated signaling processes and mRNA and DNA synthesis in activated human lymphoblasts. Furthermore, we show the existence of CD45 epitopes (GAP8.3), which are active and critical for signaling in quiescent lymphocytes, but are nonfunctional in activated human lymphoblasts.

Interleukin‐4 supports interleukin‐12‐induced proliferation and interferon‐γ secretion in human activated lymphoblasts and T helper type 1 cells

Interleukin‐12 (IL‐12) and IL‐4 are known to differentially promote T helper (Th) cell differentiation. While IL‐12 induces interferon‐γ (IFN‐γ) production and maturation of Th1 cells, IL‐4 is thought to antagonize IL‐12 and to favour Th2 development. Here we studied the combined action of various concentrations of common γ‐chain (γc‐chain) cytokines, including IL‐4 and the Th1 cytokine IL‐12, in human activated lymphoblasts and Th1 cells. IL‐4 and IL‐7 potentiated IL‐12‐induced proliferation at every concentration tested (1–10 ng/ml) without increasing rescue from apoptosis, indicating that proliferation was directly affected by these cytokine combinations. With regards to cytokine secretion, IL‐2 together with IL‐12 initiated tumour necrosis factor‐α synthesis, enhanced IFN‐γ production, and shedding of soluble IL‐2 receptor α as expected. Importantly, combining IL‐4 with IL‐12 also enhanced IFN‐γ secretion in lymphoblasts and a Th1 cell line. Investigating signal transduction in lymphoblasts induced by these cytokines, we found that not only IL‐2 but also IL‐4 enhances signal transducer and activator of transcription 3 (STAT3) tyrosine phosphorylation by IL‐12. Tyrosine phosphorylations of janus kinase 2 (JAK‐2), tyrosine kinase 2 (TYK2), extracellular signal‐regulated kinase (ERK) and STAT4, STAT5 and STAT6 were not potentiated by combinations of these cytokines, suggesting specificity for increased STAT3 phosphorylation. In conclusion, two otherwise antagonizing cytokines co‐operate in activated human lymphoblasts and Th1 cells, possibly via STAT3 as a converging signal. These data demonstrate that IL‐4 can directly enhance human Th1 cell function independently of its known actions on antigen‐presenting cells. These findings should be of importance for the design of cytokine‐targeted therapies of human Th‐cell‐driven diseases.

Detection of Low Level Cryoglobulins by Flow Cytometry

Several patients with cryoglobulin (CG) associated symptoms are seronegative for CG and other potentially causative biomarkers. We analyzed whether it is possible to detect cryoprecipitates by flow cytometry and whether the sensitivity of their demonstration can be increased as compared to visual inspection. Sera from 91 patients with suspected CG associated symptoms and 33 healthy controls were examined for the presence of CG by conventional visual testing and by flow cytometry for small diffracting particles. For calibration purposes we tested lipid micelle dilutions (positive controls) by both methods. The minimum concentrations of lipid micelles to be detected by visual inspection and flow cytometry were 128.5 and 2.0 pg ml−1, respectively. Among the 91 patients and 33 controls, only 1 patient serum was positive for CG by conventional testing. This sample was also positive on flow cytometry. In the serum of a patient known to be positive for CG, laser diffracting particles were quantified by flow cytometry after keeping serum at 4°C for 3 days. Of the 91 patients, 14 additional samples displayed cold precipitates which redissolved after rewarming during flow cytometry. All 15 (1 + 14) patients positive for CG on flow cytometry suffered from symptoms usually associated with CG. Some precipitates were labeled with anti IgG and IgM antibodies confirming that the particles detected by flow cytometry contained immunoglobulins. No small diffracting particles were detected in the sera of the 33 healthy controls. Flow cytometry is equally specific but much more sensitive in the detection of CG than visual inspection.

Accumulation of histones in cell lysates precedes expression of apoptosis-related phagocytosis signals in human lymphoblasts.

Abstract: Systemic lupus erythematosus (SLE) is characterized by the production of autoantibodies directed against several nuclear components, such as DNA and histones. Apoptosis was induced in activated human lymphoblasts (n= 6) by UV‐B irradiation for 30 sec followed by continuous culturing. An extranuclear accumulation of the nucleosomal histones H2A, H2B, H3, and H4 in cell lysates was observed very early in the process of apoptosis, even before phosphatidylserine externalization occurred on the outer membrane surface of apoptotically dying lymphoblasts. We hypothesize that a dysregulation of apoptosis during these early phases may contribute to the induction of autoimmunity against nuclear autoantigens as seen in SLE.

Autoantibodies against galectin-2 peptides as biomarkers for the antiphospholipid syndrome.

Autoantibodies against opsonins of dying and dead cells mediate Fcγ receptor-dependent phagocytosis of autologous apoptotic and necrotic cells and hereby tend to elicit inflammation instead of silent clearance. We analysed sera of patients with chronic autoimmune diseases for the occurrence of IgG autoantibodies recognizing galectins. These pluripotent effectors can also bind to apoptotic or necrotic cells. Patients with antiphospholipid syndrome (APS; n = 104) and systemic lupus erythematosus (SLE; n = 62) were examined, healthy donors (n = 31) served as controls. Selected peptides of galectin (Gal)-2 were employed for peptide-based ELISAs. Levels of anti-Gal-2PEP-IgG were significantly increased in SLE and APS when compared with controls. In addition, patients with APS showed significantly higher levels of anti-Gal-2PEP-IgG compared with patients with SLE. Anti-Gal-2PEP-IgG may, therefore, be considered novel biomarkers for APS.