Timo Gaber

Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up-regulated after in vitro stimulation and in patients with rheumatoid arthritis

Glucocorticoids mediate their therapeutic actions mostly by genomic effects via cytosolic receptors, but some effects are too rapid to be mediated by changes at the genomic level. The detailed mechanisms of these nongenomic actions are still unclear. Membrane‐bound glucocorticoid receptors (mGCR) have been suggested to be involved, although their physiological existence in humans so far is hypothetical. For the first time we demonstrate the existence of mGCR on monocytes and B cells obtained from healthy blood donors using high‐sensitivity immunofluorescent staining. Immunostimulation with lipopolysaccharide increases the percentage of mGCR‐positive monocytes, which can be prevented by inhibiting the secretory pathway. Overexpression of the human glucocorticoid receptor α alone is not sufficient to enhance mGCR expression. These in vitro findings are consistent with our clinical observation that in patients with rheumatoid arthritis the frequency of mGCR positive monocytes is increased and positively correlated with disease activity. We conclude that mGCR are 1) indeed physiologically present in healthy blood donors, but remained unidentified by conventional techniques due to their small number per cell and 2) actively up‐regulated and transported through the cell after immunostimulation. These receptors may reflect a feedback mechanism of the organism upon immunostimulation and/or play a role in pathogenesis.—Bartholome, B., Spies, C. M., Gaber, T., Schuchmann, S., Berki, T., Kunkel, D., Bienert, M., Radbruch, A., Burmester, G.‐R., Lauster, R., Scheffold, A., Buttgereit, F. Membrane glucocorticoid receptors (mGCR) are expressed in normal human peripheral blood mononuclear cells and up‐regulated after in vitro stimulation and in patients with rheumatoid arthritis. FASEB J. 18, 70–80 (2004)

Mesenchymal Stem Cells Regulate Angiogenesis According to Their Mechanical Environment

In fracture and bone defect healing, MSCs largely drive tissue regeneration. MSCs have been shown to promote angiogenesis both in vivo and in vitro. Angiogenesis is a prerequisite to large tissue reconstitution. The present study investigated how mechanical loading of MSCs influences their proangiogenic capacity. The results show a significant enhancement of angiogenesis by conditioned media from mechanically stimulated compared with unstimulated MSCs in two‐dimensional tube formation and three‐dimensional spheroid sprouting assays. In particular, proliferation but not migration or adhesion of endothelial cells was elevated. Promotion of angiogenesis was dependent upon fibroblast growth factor receptor 1 (FGFR1) signaling. Moreover, stimulation of tube formation was inhibited by vascular endothelial growth factor receptor (VEGFR) tyrosine kinase blocking. Screening for the expression levels of different soluble regulators of angiogenesis revealed an enrichment of matrix metalloprotease 2, transforming growth factor β1, and basic fibroblast growth factor but not of vascular endothelial growth factor in response to mechanical stimulation. In conclusion, mechanical loading of MSCs seems to result in a paracrine stimulation of angiogenesis, most likely by the regulation of a network of several angiogenic molecules. The underlying mechanism appears to be dependent on the FGFR and VEGFR signaling cascades and might be mediated by an additional cross‐talk with other pathways.

Homeostatic imbalance of regulatory and effector T cells due to IL-2 deprivation amplifies murine lupus.

The origins and consequences of a regulatory T cell (Treg) disorder in systemic lupus erythematosus (SLE) are poorly understood. In the (NZBxNZW) F1 mouse model of lupus, we found that CD4+Foxp3+ Treg failed to maintain a competitive pool size in the peripheral lymphoid organs resulting in a progressive homeostatic imbalance of CD4+Foxp3+ Treg and CD4+Foxp3− conventional T cells (Tcon). In addition, Treg acquired phenotypic changes that are reminiscent of IL-2 deficiency concomitantly to a progressive decline in IL-2-producing Tcon and an increase in activated, IFN-γ-producing effector Tcon. Nonetheless, Treg from lupus-prone mice were functionally intact and capable to influence the course of disease. Systemic reduction of IL-2 levels early in disease promoted Tcon hyperactivity, induced the imbalance of Treg and effector Tcon, and strongly accelerated disease progression. In contrast, administration of IL-2 partially restored the balance of Treg and effector Tcon by promoting the homeostatic proliferation of endogenous Treg and impeded the progression of established disease. Thus, an acquired and self-amplifying disruption of the Treg-IL-2 axis contributed essentially to Tcon hyperactivity and the development of murine lupus. The reversibility of this homeostatic Treg disorder provides promising approaches for the treatment of SLE.

Hypoxia Promotes Osteogenesis but Suppresses Adipogenesis of Human Mesenchymal Stromal Cells in a Hypoxia-Inducible Factor-1 Dependent Manner

Background Bone fracture initiates a series of cellular and molecular events including the expression of hypoxia-inducible factor (HIF)-1. HIF-1 is known to facilitate recruitment and differentiation of multipotent human mesenchymal stromal cells (hMSC). Therefore, we analyzed the impact of hypoxia and HIF-1 on the competitive differentiation potential of hMSCs towards adipogenic and osteogenic lineages. Methodology/Principal Findings Bone marrow derived primary hMSCs cultured for 2 weeks either under normoxic (app. 18% O2) or hypoxic (less than 2% O2) conditions were analyzed for the expression of MSC surface markers and for expression of the genes HIF1A, VEGFA, LDHA, PGK1, and GLUT1. Using conditioned medium, adipogenic or osteogenic differentiation as verified by Oil-Red-O or von-Kossa staining was induced in hMSCs under either normoxic or hypoxic conditions. The expression of HIF1A and VEGFA was measured by qPCR. A knockdown of HIF-1α by lentiviral transduction was performed, and the ability of the transduced hMSCs to differentiate into adipogenic and osteogenic lineages was analyzed. Hypoxia induced HIF-1α and HIF-1 target gene expression, but did not alter MSC phenotype or surface marker expression. Hypoxia (i) suppressed adipogenesis and associated HIF1A and PPARG gene expression in hMSCs and (ii) enhanced osteogenesis and associated HIF1A and RUNX2 gene expression. shRNA-mediated knockdown of HIF-1α enhanced adipogenesis under both normoxia and hypoxia, and suppressed hypoxia-induced osteogenesis. Conclusions/Significance Hypoxia promotes osteogenesis but suppresses adipogenesis of human MSCs in a competitive and HIF-1-dependent manner. We therefore conclude that the effects of hypoxia are crucial for effective bone healing, which may potentially lead to the development of novel therapeutic approaches.

Origin and Functional Activity of the Membrane-Bound Glucocorticoid Receptor

OBJECTIVE Glucocorticoids (GCs) exert their antiinflammatory and immunosuppressive effects in humans primarily via the cytosolic GC receptor (cGR) but also via rapid, nongenomic mechanisms. Most likely, membrane-bound GRs (mGR) are involved in nongenomic GC signaling. The aim of this study was to investigate the origin and functional activity of mGR. METHODS We analyzed the origin of mGR using mGR-expressing HEK 293T cells, by transient and stable RNA interference-mediated GR reduction. GR messenger RNA (mRNA) and cGR and mGR protein levels were analyzed by real-time quantitative polymerase chain reaction, immunoblotting, and high-sensitivity immunofluorescence staining. Furthermore, we analyzed the functional activity of mGR, using membrane-impermeable bovine serum albumin (BSA)-bound dexamethasone (DEX-BSA) in human monocytes. Membrane-bound GR-expressing monocytes were treated with DEX, DEX-BSA, or BSA. Cell lysates were analyzed using PepChip arrays in order to identify kinases triggered by DEX-BSA, with validation using Bio-Plex assays and immunoblotting. RESULTS Our data showed that transient reduction of GR mRNA in HEK 293T cells decreased cGR protein levels but not mGR protein levels. However, stably transfected cells showed reduced cGR protein expression and significantly reduced mGR protein expression. Furthermore, 51 kinase substrates were identified for which phosphorylation was either reduced or increased. We observed p38 MAP kinase (MAPK) as one possible upstream kinase. Validation of these data by Bio-Plex phosphoprotein assay and immunoblotting showed increased phosphorylation of p38 MAPK after treatment with DEX-BSA. CONCLUSION Our data demonstrate that the human GR gene encodes for both cGR and mGR. Membrane-bound GR retains functional activity, as indicated by induced phosphorylation of p38 MAPK due to DEX-BSA treatment. Membrane-bound GR-mediated cellular signaling needs to be investigated further in order to clarify its therapeutic potential.

Osteoarthritis synovial fluid activates pro-inflammatory cytokines in primary human chondrocytes.

PurposeTwo of the most common joint diseases are rheumatoid arthritis (RA) and osteoarthritis (OA). Cartilage degradation and erosions are important pathogenetic mechanisms in both joint diseases and have presently gained increasing interest. The aim of the present study was to investigate the effects of the synovial fluid environment of OA patients in comparison with synovial fluids of RA patients on human chondrocytes in vitro.MethodsPrimary human chondrocytes were incubated in synovial fluids gained from patients with OA or RA. The detection of vital cell numbers was determined by histology and by using the Casy Cell Counter System. Cytokine and chemokine secretion was determined by a multiplex suspension array.ResultsMicroscopic analysis showed altered cell morphology and cell shrinkage following incubation with synovial fluid of RA patients. Detection of vital cells showed a highly significant decrease of vital chondrocyte when treated with RA synovial fluids in comparison with OA synovial fluids. An active secretion of cytokines such as vascular endothelial growth factor (VEGF) of chondrocytes treated with OA synovial fluids was observed.ConclusionsSignificantly increased levels of various cytokines in synovial fluids of RA, and surprisingly of OA, patients were shown. Activation of pro-inflammatory cytokines of human chondrocytes by synovial fluids of OA patient supports a pro-inflammatory process in the pathogenesis of OA.

Transgenic disruption of glucocorticoid signaling in mature osteoblasts and osteocytes attenuates K/BxN mouse serum–induced arthritis in vivo

OBJECTIVE Endogenous glucocorticoids (GCs) modulate numerous biologic systems involved in the initiation and maintenance of arthritis. Bone cells play a critical role in the progression of arthritis, and some of the effects of GCs on inflammation may be mediated via these cells. The aim of this study was to investigate the impact of osteoblast-targeted disruption of GC signaling on joint inflammation, cartilage damage, and bone metabolism in the K/BxN mouse serum transfer model of autoimmune arthritis. METHODS Intracellular GC signaling was disrupted in osteoblasts through transgenic overexpression of 11beta-hydroxysteroid dehydrogenase type 2 under the control of a type I collagen promoter. Arthritis was induced in 5-week-old male transgenic mice and their wild-type (WT) littermates, and paw swelling was assessed daily until the mice were killed. The mice were examined by histology, histomorphometry, and microfocal computed tomography, and serum was analyzed for cytokines, adrenocorticotropic hormone, and corticosterone. RESULTS Acute arthritis developed in both transgenic and WT mice treated with K/BxN mouse serum. However, the arthritis and local inflammatory activity were significantly attenuated in transgenic mice, as judged by clinical and histologic indices of inflammation and cartilage damage. Bone turnover and bone volume remained unchanged in arthritic transgenic mice, while WT mice exhibited stimulated bone resorption, suppressed osteoblast activity, and significantly reduced bone volume, compatible with the known effects of active inflammation on bone. Circulating levels of proinflammatory cytokines tended to be lower in arthritic transgenic mice than in control transgenic mice. CONCLUSION Disruption of GC signaling in osteoblasts significantly attenuates K/BxN mouse serum-induced autoimmune arthritis in mice. These data suggest that osteoblasts modulate the immune-mediated inflammatory response via a GC-dependent pathway.

Human CD4+ T cells maintain specific functions even under conditions of extremely restricted ATP production

We investigated the energy‐adaptive potential of human CD4+ T cells under conditions of impaired oxidative phosphorylation (OXPHOS) and/or low glucose (inhibiting glycolysis). These cells often encounter these conditions when executing their functions in injured/inflamed tissues, even though T cells themselves require constant and adequate energy supply via ATP. We assessed two specific functions, cytokine synthesis and proliferation, and addressed whether adaptive characteristics also emerged in vivo. In glucose‐containing medium, both cytokine production and proliferation were unaffected, even under complete OXPHOS suppression. Only when glucose was also absent were these functions significantly decreased. Partial recovery of OXPHOS and induced glycolysis were crucial for the maintenance of cellular energy supply. Adaptive regulatory mechanisms are clinically relevant because hypoxia up‐regulates glycolytic genes but down‐regulates OXPHOS genes in vivo. Our data demonstrate an unexpectedly high, clinically relevant adaptive potential of human CD4+ T cells to maintain specific functions even under severely impaired bioenergetic conditions.

Macrophage Migration Inhibitory Factor Counterregulates Dexamethasone-Mediated Suppression of Hypoxia-Inducible Factor-1α Function and Differentially Influences Human CD4+ T Cell Proliferation under Hypoxia

Hypoxia, a feature of inflammation and tumors, is a potent inducer of the proinflammatory cytokine macrophage migration inhibitory factor (MIF). In transformed cells, MIF was shown to modulate and to be modulated via the oxygen-sensitive transcription factor hypoxia-inducible factor (HIF)-1. Furthermore, anti-inflammatory glucocorticoids (GCs) were described to regulate MIF action. However, in-depth studies of the interaction between MIF and HIF-1 and GC action in nontransformed primary human CD4+ T cells under hypoxia are missing. Therefore, we investigated the functional relationship between MIF and HIF and the impact of the GC dexamethasone (DEX) on these key players of inflammation in human CD4+ T cells. In this article, we show that hypoxia, and specifically HIF-1, is a potent and rapid inducer of MIF expression in primary human CD4+ T cells, as well as in Jurkat T cells. MIF signaling via CD74, in turn, is essential for hypoxia-mediated HIF-1α expression and HIF-1 target gene induction involving ERK/mammalian target of rapamycin activity complemented by PI3K activation upon mitogen stimulation. Furthermore, MIF signaling enhances T cell proliferation under normoxia but not hypoxia. MIF also counterregulates DEX-mediated suppression of MIF and HIF-1α expression. Based on these data, we suggest that hypoxia significantly affects the expression of HIF-1α in a MIF-dependent manner leading to a positive-feedback loop in primary human CD4+ T cells, thus influencing the lymphoproliferative response and DEX action via the GC receptor. Therefore, we suggest that HIF and/or MIF could be useful targets to optimize GC therapy when treating inflammation.

Effects of hypoxia and/or lack of glucose on cellular energy metabolism and cytokine production in stimulated human CD4+ T lymphocytes.

Oxidative phosphorylation and/or glycolysis provide energy, mainly in the form of ATP, which ensures proper functioning of immune cells such as CD4(+) T lymphocytes. However, the main substrates, namely oxygen and glucose, are known to remain for a relatively short time in the inflamed tissue and in other clinical situations where immune cells need to function properly. Therefore, we examined the effect of hypoxia and/or lack of glucose on cellular energy metabolism and on cytokine secretion in stimulated human CD4(+) T lymphocytes. Human CD4(+) T cells were MACS-isolated using peripheral blood obtained from healthy donors. Stimulated cells were incubated in medium with or without glucose for 6h in a sealed chamber which led to cumulative hypoxia. During this incubation period, (i) oxygen saturation was measured continuously using a Clark-type electrode, and (ii) samples were taken at different time points in order to quantify for each the viability of cells, intracellular reactive oxygen species (iROS), ATP levels, glycolytic enzyme activity, mRNA expression of hexokinase-1 and superoxide dismutase-1, and concentrations of several different cytokines. Stimulated CD4(+) T cells which were incubated under normoxic conditions served as controls. Under hypoxic conditions, lack of glucose exerted a biphasic effect on cellular oxygen consumption: initially higher but later lower respiration rates were measured when compared to conditions where glucose was available. Lack of glucose strongly increased the number of dead cells and the formation of iROS under normoxia but not under hypoxia. Under both normoxic and hypoxic conditions, intracellular ATP levels remained almost unchanged during the incubation period if glucose was present, but decreased significantly in the absence of glucose, despite the enhanced glycolytic enzyme activity. Measurements of stimulated cytokine production demonstrated (i) that cumulative hypoxia stimulates especially the secretion of IL-1beta, IL-10 and IL-8, and (ii) that lack of glucose results in lower cytokine concentrations. We demonstrate that CD4(+) T cells are highly adaptive in bioenergetic terms which ensure their proper function under extreme conditions of glucose and/or oxygen availability as found under physiological and pathophysiological conditions. Hypoxia seems to facilitate inflammatory reactions and angiogenesis.