Stefan Krienke

Reduced CD4+,CD25− T cell sensitivity to the suppressive function of CD4+,CD25high,CD127−/low regulatory T cells in patients with active systemic lupus erythematosus

OBJECTIVE CD4+,CD25high regulatory T (Treg) cells play a crucial role in the maintenance of self tolerance and prevention of organ-specific autoimmunity. The presence of many in vivo-preactivated CD4+,CD25++ T cells in patients with systemic lupus erythematosus (SLE) poses a difficulty in discriminating CD25++ activated T cells from CD25high Treg cells. To overcome this problem, we analyzed the phenotype and function of CD4+,CD25high,CD127(-/low) natural Treg (nTreg) cells isolated from the peripheral blood of patients with SLE. METHODS CD4+,CD25high,CD127(-/low) nTreg cells and CD4+,CD25- responder T (Tresp) cells from patients with SLE and normal donors were separated by fluorescence-activated cell sorting. Cell proliferation was quantified by 3H-thymidine incorporation, and immunophenotyping of the cells was done using FACScan. RESULTS Comparable percentages of CD4+,CD25high,FoxP3+ T cells were observed in patients with SLE and normal donors. Proliferation of SLE nTreg cells sorted into the subset CD4+,CD25high,CD127(-/low) was significantly decreased compared with that of SLE nTreg cells sorted into the subset CD4+,CD25high (mean +/- SEM 2,223 +/- 351 counts per minute versus 9,104 +/- 1,720 cpm, respectively), while in normal donors, these values were 802 +/- 177 cpm and 2,028 +/- 548 cpm, respectively, confirming that effector cell contamination was reduced. Notably, the suppressive activity of nTreg cells was intact in all groups. However, CD4+,CD25- Tresp cells isolated from patients with active SLE were significantly less sensitive than those from patients with inactive SLE to the suppressive function of autologous or normal donor CD4+,CD25high,CD127(-/low) nTreg cells. Furthermore, a significant inverse correlation was observed between the extent of T cell regulation in suppressor assays and the level of lupus disease activity. CONCLUSION This study is the first to show that, in human SLE, impaired sensitivity of Tresp cells to the suppressive effects of a comparably functional, highly purified nTreg cell population leads to a defective suppression of T cell proliferation in active SLE. Studies aiming to define the mechanisms leading to Tresp cell resistance might help in the development of highly specific, alternative immunotherapeutic tools for the control of systemic autoimmune diseases such as SLE.

Decrease of sialic acid residues as an eat-me signal on the surface of apoptotic lymphocytes

The silent clearance of apoptotic cells is essential for cellular homeostasis in multicellular organisms, and several mediators of apoptotic cell recognition have been identified. However, the distinct mechanisms involved are not fully deciphered yet. We analyzed alterations of the glycocalyx on the surfaces of apoptotic cells and its impact for engulfment. After apoptosis induction of lymphocytes, a decrease of α2,6-terminal sialic acids and sialic acids in α2,3-linkage with galactose was observed. Similar changes were to be found on the surface of apoptotic membrane blebs released during early stages of apoptosis, whereas later released blebs showed no impaired, but rather an increased, exposure of sialic acids. We detected an exposure of fucose residues on the surface of apoptotic-cell-derived membrane blebs. Cleavage by neuraminidase of sialic acids, as well as lectin binding to sialic acids on the surfaces, enhanced the engulfment of apoptotic cells and blebs. Interestingly, even viable lymphoblasts were engulfed in an autologous cell system after neuraminidase treatment. Similarly, the engulfment of resting apoptotic lymphocytes was augmented after neuraminidase treatment. However, the engulfment of resting viable lymphocytes was not significantly enhanced after neuraminidase treatment. Our findings support the importance of the glycocalyx, notably the terminal sialic acids, in the regulation of apoptotic cell clearance. Thus, depending on cell type and activation status, changes in surface glycosylation can either directly mediate cellular engulfment or enhance phagocytosis by cooperation with further engulfment signals.

Cholera toxin binds to lipid rafts but has a limited specificity for ganglioside GM1.

Lipid rafts and the formation of an immunological synapse are crucial for T‐cell activation. Binding of cholera toxin B subunit (CTB) to ganglioside GM1 is a marker to identify lipid rafts. Primary human T cells were isolated from healthy donors and were stimulated with superantigen staphylococcus enterotoxin B (SEB) and stained with cholera toxin B‐fluorescein isothiocyanate (CTB‐FITC). An optimized staining procedure is required to stain lipid rafts exclusively on the cell surface. Unstimulated T cells show a few CTB binding spots on the cell surface. The size and number of CTB‐binding lipid rafts are strongly upregulated during T‐cell activation in SEB‐stimulated CD4+ T cells. However, our data show that the specificity of CTB for GM1 ganglioside is limited, because the binding capacity is partly resistant to inhibition of ganglioside synthesis and sensitive to trypsin digestion. Our results indicate that the binding of FITC‐labeled CTB can be divided into at least three different categories: a specific binding of CTB to ganglioside GM1, a nonspecific binding of CTB probably to glycosylated surface proteins and a nonspecific binding of FITC to the cell surface.

Atorvastatin Inhibits T Cell Activation through 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase without Decreasing Cholesterol Synthesis

The localization of the TCR and other signaling molecules in membrane rafts (MR) is essential for the activation of T lymphocytes. MR are stabilized by sphingolipids and cholesterol. Activation of T lymphocytes leads to the confluence of small MR and the formation of an immunological synapse that is essential for sustained activation and proliferation. In this study, we investigated the effect of statins on MR and T cell activation in superantigen-stimulated human PBMC. Atorvastatin significantly inhibited cellular activation and proliferation. The binding of cholera toxin B subunit to isolated MR and to whole cells was inhibited by low doses of statins. Statins reduce the association of critical signaling proteins such as Lck and linker of activation in T cells with MR in stimulated T cells. The expression of activation markers CD69 and CD25 was inhibited. Several statin-mediated mechanisms, such as a lower stimulation with MHC-II, an inhibition of costimulation by direct binding of statins to LFA-1, a reduced secretion of cytokines, or a depletion of cellular cholesterol pools, were excluded. Inhibition of protein prenylation had a similar effect on T cell proliferation, suggesting that a reduced protein prenylation might contribute to the statin-mediated inhibition of T cell activation. Statins induce both lower levels of low-density lipoprotein cholesterol and inhibition of T cell activation, which might contribute to an inhibition of atherosclerosis.

Clinical manifestations but not cytokine profiles differentiate adult-onset Still's disease and sepsis.

Objective. To analyze clinical manifestations, serum ferritin, and serum cytokine levels in patients with adult-onset Still’s disease (AOSD) or bacterial sepsis and to evaluate their potential use for differential diagnosis. Methods. Twenty-two consecutive patients with the first flare of AOSD and 6 patients with an established diagnosis of AOSD under immunosuppressive therapy were compared with 14 patients with bacterial sepsis. Clinical manifestations were scored in a Pouchot AOSD activity score including elevated serum ferritin levels to obtain a modified Pouchot score. Serum cytokine profiles were analyzed from each patient. Results. The scores of clinical manifestations using a modified Pouchot activity score were significantly higher in patients with active untreated AOSD (mean 5.60 ± 1.93) compared with patients with chronic AOSD (mean 1.16 ± 0.98; p < 0.001) and patients with sepsis (mean 2.38 ± 1.19; p < 0.001). A modified Pouchot score ≥ 4 shows a sensitivity of 92% and a specificity of 93% for active AOSD. Serum cytokine levels of interleukin 1ß (IL-1ß), IL-6, IL-8, IL-10, IL-12, IL-18, interferon-γ, tumor necrosis factor-α, and calprotectin were elevated in acute AOSD and sepsis. Significant differences were detected only in patients with sepsis who had higher levels of IL-6 and IL-8. The overlap of the 2 groups limits the use of cytokines for differential diagnosis in individual patients. Conclusion. A modified Pouchot AOSD activity score including elevated serum ferritin levels was more useful to confirm the diagnosis of AOSD compared to patients with sepsis. Elevated serum cytokines correlate with inflammation but are of limited use to differentiate between active AOSD and bacterial sepsis.

Lack of evidence for inhibition of angiogenesis as a central mechanism of the antiarthritic effect of methotrexate.

ObjectivesThe aim of this study was to investigate whether methotrexate (MTX) has an antiangiogenic effect and whether this property plays a role in the control of rheumatoid arthritis (RA).MethodsA human placenta angiogenesis assay was used to examine the antiangiogenic effects of MTX in vitro. In addition, DBA/1 mice were used to compare the antiarthritic effect of MTX in collagen-induced arthritis (CIA) and its antiangiogenic effect in a murine in vivo matrigel model for angiogenesis.ResultsThe spreading of microvessels from placental vessel fragments was not significantly inhibited by MTX. Treatment with MTX reduced significantly the incidence of CIA in DBA/1 mice in a dose-dependent manner. However, treatment with the same doses of MTX did not significantly reduce vessel growth in subcutaneous depots of bFGF-enriched matrigel.ConclusionThese data support the hypothesis that inhibition of angiogenesis does not significantly contribute to the antiarthritic effect of MTX seen in patients and animal models for RA. Therefore, the combination of MTX with antiangiogenic drugs appears to be a rational strategy in the treatment of RA.

Apoptotic-cell-derived membrane microparticles and IFN-α induce an inflammatory immune response.

ABSTRACT A dysregulation in the clearance of apoptotic material is considered a major pathogenetic factor for the emergence of autoimmune diseases. Apoptotic-cell-derived membrane microparticles (AdMPs), which are released from the cell surface during apoptosis, have been implicated in the pathogenesis of autoimmunity. Also of importance are cytokines, such as interferon-α (IFN-α), which is known to be a major player in patients with systemic lupus erythematosus (SLE). This study investigates the combined effect of AdMPs and IFN-α on professional phagocytes. In the presence of IFN-α, phagocytosis of AdMPs by human monocytes was significantly increased in a dose-dependent manner. The combination of AdMPs and raised IFN-α concentrations resulted in an increase in the secretion of pro-inflammatory cytokines and an upregulation of surface molecule expression involved in antigen uptake. In addition, macrophage polarisation was shifted towards a more inflammatory type of cell. The synergism between IFN-α and AdMPs seemed to be mediated by an upregulation of phosphorylated STAT1. Our results indicate that IFN-α, together with AdMPs, amplify the initiation and maintenance of inflammation. This mechanism might especially play a crucial role in disorders with a defective clearance of apoptotic material. Summary: High levels of IFN-α and apoptotic microparticles lead to a pro-inflammatory environment through the synergistic increase of phosphorylated STAT1 when acting upon professional phagocytes.

Extracellular vesicles mediate intercellular communication: Transfer of functionally active microRNAs by microvesicles into phagocytes

Cell activation and apoptosis lead to the formation of extracellular vesicles (EVs) such as exosomes or microvesicles (MVs). EVs have been shown to modulate immune responses; recently, MVs were described to carry microRNA (miRNA) and a role for MVs in the pathogenesis of autoimmune diseases has been discussed. Here we systematically characterized MVs and exosomes according to their release stimuli. The miRNA content of viable or apoptotic human T lymphocytes and the corresponding MVs was analyzed. miRNA, protein and surface marker expression, as well as cytokine release by human monocytes was measured after EV engulfment. Finally, miRNA expression in T lymphocytes and MVs of healthy individuals was compared with those of systemic lupus erythematosus (SLE) patients. We demonstrate that, depending on the stimuli, distinct subtypes of EVs are released, differing in size and carrying a specific RNA profile. We observed an accumulation of distinct miRNAs in MVs after induction of apoptosis and the transfer of functional miRNA by MVs into human monocytes. MVs released from apoptotic cells provoke less of an inflammatory response than those released from viable cells. MiR‐155*, miR‐34b and miR‐34a levels in T lymphocytes and corresponding MVs were deregulated in SLE when compared to healthy individuals.

Synovial Fibroblasts Selectively Suppress Th1 Cell Responses through IDO1-Mediated Tryptophan Catabolism

The development of rheumatoid arthritis (RA) is linked to functional changes in synovial fibroblasts (SF) and local infiltration of T lymphocytes. Fibroblasts possess the capacity to suppress T cell responses, although the molecular mechanisms of this suppression remain incompletely understood. In this study, we aimed to define the mechanisms by which noninflammatory SF modulate Th cell responses and to determine the immunosuppressive efficacy of RASF. Hence, the influence of SF from osteoarthritis or RA patients on total Th cells or different Th cell subsets of healthy donors was analyzed in vitro. We show that SF strongly suppressed the proliferation of Th cells and the secretion of IFN-γ in a cell contact–independent manner. In cocultures of SF and Th cells, tryptophan was completely depleted within a few days, resulting in eukaryotic initiation factor 2α phosphorylation, TCRζ-chain downregulation, and proliferation arrest. Blocking IDO1 activity completely restored Th cell proliferation, but not IFN-γ production. Interestingly, only the proliferation of Th1 cells, but not of Th2 or Th17 cells, was affected. Finally, RASF had a significantly lower IDO1 expression and a weaker Th cell suppressive capacity compared with osteoarthritis SF. We postulate that the suppression of Th cell growth by SF through tryptophan catabolism may play an important role in preventing inappropriate Th cell responses under normal conditions. However, expansion of Th17 cells that do not induce IDO1-mediated suppression and the reduced capacity of RASF to restrict Th cell proliferation through tryptophan metabolism may support the initiation and propagation of synovitis in RA patients.

Microvesicles released by apoptotic human neutrophils suppress proliferation and IL-2/IL-2 receptor expression of resting T helper cells

Membrane‐coated microvesicles (MVs) have been identified as important mediators in intercellular communication. During the process of apoptosis, dying cells dynamically release MVs. Neutrophils are the most abundant type of leukocytes in the circulation. Due to their very short lifespan, it is likely that they are the source of large amounts of apoptotic cell‐derived MVs. Here, we show that MVs released by apoptotic human polymorphonuclear neutrophils (apoPMN‐MVs), but not the apoptotic neutrophils themselves, selectively suppress the proliferation of CD25 (IL‐2Rα)neg CD127 (IL‐7Rα)pos Th cells in a dose‐dependent manner. In contrast, the proliferation of total T cells is not affected by MVs. Importantly, apoPMN‐MVs suppress the secretion of IL‐2 as well as the expression of and signaling via the IL‐2 receptor (IL‐2R) by CD25negCD127pos Th cells. Addition of IL‐7 strongly reduced the suppression of T‐cell proliferation by MVs and the addition of IL‐2 completely abrogated the suppressive effect. Thus, apoPMN‐MVs suppressed a subset of Th cells by downregulating IL‐2 and IL‐2R expression and signaling. This may represent an important mechanism to prevent the activation and expansion of resting T cells in the absence of sufficient cytokine stimulation, and thereby maintaining immune tolerance.