German Arthritis Research Center

Berlin, Germany

German Arthritis Research Center

Berlin, Germany
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Konnecke I.,Charité - Medical University of Berlin | Serra A.,German Arthritis Research Center | El Khassawna T.,Justus Liebig University | Schlundt C.,Charité - Medical University of Berlin | And 8 more authors.
Bone | Year: 2014

Fracture healing is a regenerative process in which bone is restored without scar tissue formation. The healing cascade initiates with a cycle of inflammation, cell migration, proliferation and differentiation. Immune cells invade the fracture site immediately upon bone damage and contribute to the initial phase of the healing process by recruiting accessory cells to the injury site. However, little is known about the role of the immune system in the later stages of fracture repair, in particular, whether lymphocytes participate in soft and hard callus formation. In order to answer this question, we analyzed femoral fracture healing in mice by confocal microscopy. Surprisingly, after the initial inflammatory phase, when soft callus developed, T and B cells withdrew from the fracture site and were detectable predominantly at the femoral neck and knee. Thereafter lymphocytes massively infiltrated the callus region (around day 14 after injury), during callus mineralization. Interestingly, lymphocytes were not found within cartilaginous areas of the callus but only nearby the newly forming bone. During healing B cell numbers seemed to exceed those of T cells and B cells progressively underwent effector maturation. Both, osteoblasts and osteoclasts were found to have direct cell-cell contact with lymphocytes, strongly suggesting a regulatory role of the immune cells specifically in the later stages of fracture healing. © 2014 Elsevier Inc.


Toben D.,Charité - Medical University of Berlin | Toben D.,Berlin Brandenburg Center for Regenerative Therapies | Schroeder I.,Charité - Medical University of Berlin | Schroeder I.,Berlin Brandenburg Center for Regenerative Therapies | And 18 more authors.
Journal of Bone and Mineral Research | Year: 2011

Fracture healing is a unique biologic process starting with an initial inflammatory response. As in other regenerative processes, bone and the immune system interact closely during fracture healing. This project was aimed at further elucidating how the host immune system participates in fracture healing. A standard closed femoral fracture was created in wild-type (WT) and recombination activating gene 1 knockout (RAG1-/-) mice lacking the adaptive immune system. Healing was investigated using micro-computed tomography (ÂμCT), biomechanical testing, and histologic and mRNA expression analyses. Biomechanical testing demonstrated a significantly higher torsional moment on days 14 and 21 in the RAG1-/- mice compared to the WT group. ÂμCT evaluation of RAG1-/- specimens showed earlier mineralization and remodeling. Histologically, endochondral ossification and remodeling were accelerated in the RAG1-/- compared with the WT mice. Histomorphometric analysis on day 7 showed a significantly higher fraction of bone and a significantly lower fraction of cartilage in the callus of the RAG1-/- mice than in the WT mice. Endochondral ossification was accelerated in the RAG1-/- mice. Lymphocytes were present during the physiologic repair process, with high numbers in the hematoma on day 3 and during formation of the hard callus on day 14 in the WT mice. Expression of inflammatory cytokines was reduced in the RAG1-/- mice. In contrast, expression of anti-inflammatory interleukin 10 (IL-10) was strongly upregulated in RAG1-/- mice, indicating protective effects. This study revealed an unexpected phenotype of enhanced fracture healing in RAG1-/- mice, suggesting detrimental functions of lymphocytes on fracture healing. The shift from proinflammatory to anti-inflammatory cytokines suggests that immunomodulatory intervention strategies that maximise the regenerative and minimize the destructive effects of inflammation may lead to enhanced fracture repair. © 2011 American Society for Bone and Mineral Research.


Haupl T.,Charité - Medical University of Berlin | Stuhlmuller B.,Charité - Medical University of Berlin | Grutzkau A.,German Arthritis Research Center | Radbruch A.,German Arthritis Research Center | Burmester G.-R.,Charité - Medical University of Berlin
Annals of the Rheumatic Diseases | Year: 2010

Transcription profiling has become a standard technology in research. It is mainly applied in the search for biomarkers to improve diagnostic and prognostic classification, to quantify disease activity and to predict or indicate response to therapy. This review will focus on rheumatoid arthritis and discuss considerations for sample selection, prerequisites for functional interpretation of data and the current status of information deduced in the field of biomarkers for the various clinical questions. In the next few years, prediction of response to treatment is the most important aim of biomarker research. With the growing number of new biological agents, there is increasing pressure to identify molecular parameters that will not only guide the therapeutic decision but also help to define the most important targets for which new biological agents should be tested in clinical studies.


Spies C.M.,Charité - Medical University of Berlin | Hoff P.,Charité - Medical University of Berlin | Hoff P.,German Arthritis Research Center | Mazuch J.,Charité - Medical University of Berlin | And 15 more authors.
Clinical and Experimental Rheumatology | Year: 2015

Objective: The circadian rhythm of clinical symptoms in rheumatoid arthritis (RA) has been primarily attributed to circadian variations in humoral factors and hormones. In this study, we investigated circadian rhythms of cellular immunity in RA (CiRA study). Methods: Peripheral blood of female postmenopausal patients with active RA (DAS 28 = 4.2) (n=5) and female postmenopausal non-RA controls (n=5) was collected every 2 hours for 24 hours and analysed by flow cytometry, cytokine multiplex suspension array and quantitative RT-PCR of clock gene expression in isolated CD14+ monocytes. Endogenous circadian rhythms of macrophages were investigated by BMAL1-luciferase bioluminescence. Significance of circadian rhythms was tested by Cosinor analysis. Results: We found (i) circadian rhythms in the relative frequency of peripheral blood cell populations that were present in postmenopausal non-RA controls but absent in patients with active RA, (ii) circadian rhythms that were absent in non-RA controls but present in patients with RA and (iii) circadian rhythms that were present in both groups but with differences in peak phase or amplitude or amplitude/magnitude. The circadian rhythm in expression of the clock genes PER2 and PER3 in CD14+ monocytes was lost in patients with RA. The amplitude of BMAL1-luciferase bioluminescence tended to be lower in patients with RA than in non-RA controls. Conclusion: We conclude that (i) in RA some immune cell populations lose their normal circadian rhythms whereas others establish new "inflammatory" circadian rhythms and (ii) these findings provide a good basis for further identifying pathophysiological aspects of RA chronobiology with potential therapeutic implications. © Clinical and Experimental Rheumatology 2015.


Wagegg M.,Charite University Hospital | Wagegg M.,German Arthritis Research Center | Gaber T.,Charite University Hospital | Gaber T.,German Arthritis Research Center | And 19 more authors.
PLoS ONE | Year: 2012

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. © 2012 Wagegg et al.


Kolar P.,Charite University Hospital | Kolar P.,German Arthritis Research Center | Gaber T.,Charite University Hospital | Gaber T.,German Arthritis Research Center | And 8 more authors.
Clinical Orthopaedics and Related Research | Year: 2011

Background: An effective immune system, especially during the inflammatory phase, putatively influences the quality and likelihood of bone healing. If and how this is reflected within the initial fracture hematoma is unclear. Questions/purposes: We therefore asked the following questions: (1) Does the local expression in fracture hematoma of genes involved in adaptation to hypoxia, migration, angiogenesis, and osteogenesis vary as compared to the peripheral blood? (2) Do these changes occur time dependently? (3) Is the gene expression during fracture hematoma formation altered by irradiation? Methods: Cells from fracture hematoma of 20 patients and hematomas formed in 40 patients after THA (20 without and 20 with preoperative radiation) were isolated and RNA was extracted to analyze the influence of oxygen deprivation during fracture healing on mRNA expression of genes (HIF1A, LDHA, and PGK1) involved in immunoregulation (IL6, IL8, CXCR4), angiogenesis (VEGF, IL8), and osteogenesis (SPP1, RUNX2) by quantitative PCR. Results: We observed locally increased LDHA gene expression in fracture hematoma cells (6-72 h post fracture) reflecting the adaptation to hypoxia. IL6, IL8, and VEGF upregulation indicated hypoxia-mediated inflammation and angiogenesis; increased CXCR4 expression reflected immigration of immune cells. Osteogenic differentiation was reflected in the increased expression of the SPP1 and RUNX2 genes. The increased expression of the LDHA, VEGF, IL8, SPP1 and RUNX2 genes occurred time dependently. Irradiation suppressed HIF1A, IL6, IL8, CXCR4, and RUNX2 gene expression. Conclusions: Our data suggest cells in the fracture hematoma (1) adapt to hypoxia and (2) promote inflammation in fracture healing at the mRNA level, indicating early involvement of the immune system. Clinical Relevance: The initial fracture hematoma is important for the onset of angiogenesis, chemotaxis, and osteogenesis. © 2011 The Association of Bone and Joint Surgeons®.


Hoff P.,Charite University Hospital | Hoff P.,German Arthritis Research Center | Maschmeyer P.,Charite University Hospital | Maschmeyer P.,German Arthritis Research Center | And 27 more authors.
Cellular and Molecular Immunology | Year: 2013

The initial inflammatory phase of bone fracture healing represents a critical step for the outcome of the healing process. However, both the mechanisms initiating this inflammatory phase and the function of immune cells present at the fracture site are poorly understood. In order to study the early events within a fracture hematoma, we established an in vitro fracture hematoma model: we cultured hematomas forming during an osteotomy (artificial bone fracture) of the femur during total hip arthroplasty (THA) in vitro under bioenergetically controlled conditions. This model allowed us to monitor immune cell populations, cell survival and cytokine expression during the early phase following a fracture. Moreover, this model enabled us to change the bioenergetical conditions in order to mimic the in vivo situation, which is assumed to be characterized by hypoxia and restricted amounts of nutrients. Using this model, we found that immune cells adapt to hypoxia via the expression of angiogenic factors, chemoattractants and pro-inflammatory molecules. In addition, combined restriction of oxygen and nutrient supply enhanced the selective survival of lymphocytes in comparison with that of myeloid derived cells (i.e., neutrophils). Of note, non-restricted bioenergetical conditions did not show any similar effects regarding cytokine expression and/or different survival rates of immune cell subsets. In conclusion, we found that the bioenergetical conditions are among the crucial factors inducing the initial inflammatory phase of fracture healing and are thus a critical step for influencing survival and function of immune cells in the early fracture hematoma. © 2013 CSI and USTC. All rights reserved.


Hoff P.,Charite University Hospital | Hoff P.,German Arthritis Research Center | Buttgereit F.,Charite University Hospital | Buttgereit F.,German Arthritis Research Center | And 15 more authors.
International Orthopaedics | Year: 2013

Purpose: Two 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. Methods: Primary 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. Results: Microscopic 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. Conclusions: Significantly 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. © 2012 Springer-Verlag Berlin Heidelberg.


Dziurla R.,Charite University Hospital | Gaber T.,Charite University Hospital | Gaber T.,German Arthritis Research Center | Fangradt M.,Charite University Hospital | And 9 more authors.
Immunology Letters | Year: 2010

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-1β, 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. © 2010 Elsevier B.V.


Spies C.M.,Charite University Hospital | Gaber T.,Charite University Hospital | Gaber T.,German Arthritis Research Center | Hahne M.,Charite University Hospital | And 10 more authors.
Immunology Letters | Year: 2010

Rimexolone is a lipophilic glucocorticoid drug used for local application. Only few data are available describing its effects on immune cell functions. In this study we investigated the effects of rimexolone on the proliferation of human CD4+ T-cells using dexamethasone as standard reference. Isolated CD4+ T-cells were pre-incubated with rimexolone or dexamethasone at different concentrations for 10min (10-11/10-8/10-5M) and stimulated with anti-CD3/anti-CD28 for 96h. Proliferation was determined by flow cytometry. The percentage of dividing cells was significantly reduced by 10-5M rimexolone and dexamethasone; however, the average number of cell divisions was unchanged. In addition, production of IL-2 and other cytokines was reduced by both glucocorticoids at 10-5M. Interestingly, we observed a rimexolone-induced down-regulation of CD4 expression in unstimulated and non-dividing cells. The inhibitory effects on proliferation and CD4 expression could be blocked by the glucocorticoid-antagonist RU486 and were not due to glucocorticoid-induced apoptosis. Rimexolone and dexamethasone showed a similar potential to induce IκBα gene expression. We demonstrate rimexolone and dexamethasone to impair T-cell signalling pathways by rapid non-genomic suppression of the phosphorylation of Akt, p38 and ERK.We conclude that rimexolone and dexamethasone inhibit T-cell proliferation as well as cytokine production of activated CD4+ T-cells in a similar manner. As these inhibitory effects predominantly occur at high concentrations, a relatively high occupation-rate of cytosolic glucocorticoid receptors is needed, but receptor-mediated non-genomic effects may also be involved. It is implied that these effects contribute to the well-known beneficial anti-inflammatory and immunomodulatory effects of glucocorticoid therapy. © 2010 Elsevier B.V.

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