ABSTRACT
These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
Subject(s)
Allergy and Immunology/standards , Cell Separation/methods , Cell Separation/standards , Flow Cytometry/methods , Flow Cytometry/standards , Consensus , Humans , PhenotypeABSTRACT
During therapeutic application, mesenchymal stem cells (MSCs) may interact with their environment via their expressed toll-like-receptors (TLRs) leading to pro- or anti-inflammatory immune responses. The present study aimed to describe the gingival margin-derived stem/progenitor cells' (G-MSCs) TLR-induced immune regulatory response to specific TLR agonists. Gingival cells were obtained, immunomagnetically sorted via anti-STRO-1 antibodies and seeded out to achieve colony forming units (CFUs). G-MSCs were investigated for stem cell characteristics and TLR expression. Specific TLR agonists were applied and m-RNA expression of pro- and anti-inflammatory factors was analyzed via real-time polymerase chain reaction. G-MSCs showed all characteristics of stem/progenitor cells. All TLR agonists induced pro-inflammatory cytokines, except for the TLR3 agonist, which significantly promoted the anti-inflammatory response. (p⩽0.05, Wilcoxon-Signed-Ranks-Test). TLR-induced immunomodulation by G-MSCs could impact their therapeutic potential in vivo. Two distinctive pro-inflammatory and an anti-inflammatory TLR-induced phenotypes of G-MSCs become noticeable in this study.
Subject(s)
Cytokines/immunology , Immunomodulation/immunology , Mesenchymal Stem Cells/immunology , Toll-Like Receptors/immunology , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Differentiation/immunology , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Gene Expression/drug effects , Gene Expression/immunology , Gingiva/cytology , Humans , Lipopeptides/pharmacology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Poly I-C/pharmacology , Toll-Like Receptor 3/agonists , Toll-Like Receptor 3/immunology , Toll-Like Receptor 3/metabolism , Toll-Like Receptors/agonists , Toll-Like Receptors/metabolismABSTRACT
BACKGROUND/OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) still has a poor prognosis and current treatments including immunotherapy often fail. This might be due to the pronounced immunosuppressive milieu impairing infiltration and function of immune effector cells. This study aimed at a comprehensive analysis of immune cells in PDAC patients by determining absolute and relative peripheral blood cell numbers of immune cell subsets along with their functional capacity. METHODS: Whole blood cells or isolated peripheral blood mononuclear cells were characterized by flow cytometry. PDAC tissues were analyzed by immunohistochemistry. Anti-tumor activity of immune effector cells was determined by RTCA system. RESULTS: Our data demonstrate that relative CD4+ memory- and regulatory T cell numbers were enhanced, whereas determination of absolute cell numbers revealed generally lower immune cell numbers in PDAC patients compared to healthy controls. γδ T cells accumulated at higher numbers compared to αß T cells in the malignant ductal epithelium of PDAC tissues indicating that γδ T cells infiltrate into the tumor. Cytotoxicity against tumor cells of even small numbers of T- and NK cells could be induced by a bispecific antibody targeting CD3+ T cells to human epidermal growth factor receptor (HER)2 expressing PDAC cells or Trastuzumab. Importantly, a critical number of γδ T cells was required for significant tumor cell killing by a bispecific antibody engaging the γδ T cell receptor on γδ T cells and HER2 on tumor cells. CONCLUSION: Monitoring immune cells along with the determination of their functional capacity provides a comprehensive assessment as a prerequisite for a personalized immunotherapeutic PDAC treatment.
Subject(s)
Carcinoma, Pancreatic Ductal/immunology , Lymphocytes/immunology , Pancreatic Neoplasms/immunology , Antineoplastic Agents/therapeutic use , CD4-Positive T-Lymphocytes/immunology , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/pathology , Epithelium/pathology , Female , Flow Cytometry , Humans , Immunohistochemistry , Immunotherapy , Leukocyte Count , Male , Middle Aged , Monitoring, Physiologic , Pancreatic Ducts/pathology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , T-Lymphocytes, Regulatory/immunology , Trastuzumab/therapeutic useABSTRACT
BACKGROUND: Gingival margin-derived stem/progenitor cells (G-MSCs) show remarkable periodontal regenerative potential in vivo. During regeneration, G-MSCs may interact with their inflammatory environment via toll-like-receptors (TLRs). The present study aimed to depict the G-MSCs TLRs expression profile. MATERIAL AND METHODS: Cells were isolated from free gingival margins, STRO-1-immunomagnetically sorted and seeded to obtain single colony forming units (CFUs). G-MSCs were characterized for CD14, CD34, CD45, CD73, CD90, CD105, CD146 and STRO-1 expression, and for multilineage differentiation potential. Following G-MSCs' incubation in basic or inflammatory medium (IL-1ß, IFN-γ, IFN-α, TNF-α) a TLR expression profile was generated. RESULTS: G-MSCs showed all stem/progenitor cells' characteristics. In basic medium G-MSCs expressed TLRs 1, 2, 3, 4, 5, 6, 7, and 10. The inflammatory medium significantly up-regulated TLRs 1, 2, 4, 5, 7 and 10 and diminished TLR 6 (p≤0.05, Wilcoxon-Signed-Ranks-Test). CONCLUSIONS: The current study describes for the first time the distinctive TLRs expression profile of G-MSCs under uninflamed and inflamed conditions.
Subject(s)
Gingiva/cytology , Stem Cells/metabolism , Toll-Like Receptors/biosynthesis , Cells, Cultured , HumansABSTRACT
We previously demonstrated that tumour necrosis factor (TNF)-induced ceramide production by endosomal acid sphingomyelinase (A-SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase-9 and caspase-3 activation. The mechanism of TNF-mediated A-SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF-induced A-SMase activation depends on functional caspase-8 and caspase-7 expression. The active forms of all three enzymes, caspase-8, caspase-7 and A-SMase, but not caspase-3, colocalize in internalized TNF receptosomes. While caspase-8 and caspase-3 are unable to induce activation of purified pro-A-SMase, we found that caspase-7 mediates A-SMase activation by direct interaction resulting in proteolytic cleavage of the 72-kDa pro-A-SMase zymogen at the non-canonical cleavage site after aspartate 253, generating an active 57 kDa A-SMase molecule. Caspase-7 down modulation revealed the functional link between caspase-7 and A-SMase, confirming proteolytic cleavage as one further mode of A-SMase activation. Our data suggest a signalling cascade within TNF receptosomes involving sequential activation of caspase-8 and caspase-7 for induction of A-SMase activation by proteolytic cleavage of pro-A-SMase.
Subject(s)
Caspase 7/metabolism , Caspase 8/metabolism , Endosomes/metabolism , Enzyme Activation/physiology , Sphingomyelin Phosphodiesterase/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Apoptosis , Blotting, Western , Cell Line , Ceramides/metabolism , Chromatography, Thin Layer , Cloning, Molecular , Enzyme Activation/genetics , Flow Cytometry , Gene Knockdown Techniques , Humans , Jurkat Cells , Mice , Microscopy, ConfocalABSTRACT
BACKGROUND: 18:1/18:1-Dioleoyl-phosphatidylgycerol (DOPG) is a surfactant phospholipid that is nearly non-detectable in neonatal surfactant films. When alveolar macrophages are exposed to DOPG in vitro, secretory phospholipase A2 (sPLA2) production is blocked, resulting in suppressed macrophage activity and improved surfactant function. We investigated whether the addition of DOPG to a commercially available surfactant preparation would improve lung function in a neonatal piglet model of acute respiratory distress syndrome. MATERIALS AND METHODS: Respiratory failure was achieved by triple-hit lung injury (repeated broncho-alveolar lavage, injurious ventilation, tracheal lipopolysaccharide instillation, each intervention 24 h apart) in twenty-four domestic piglets aged 2-6 days and subject to mechanical ventilation. Following each lung injury protocol the piglets were treated with surfactant alone or with surfactant + DOPG. RESULTS: Within 72 h of mechanical ventilation, we observed significantly improved gas exchange (oxygenation and ventilation), lung mechanics (compliance and resistance of the respiratory system), and pulmonary oedema (extra-vascular lung water index) in the surfactant + DOPG group. This favourable clinical effect could be attributed to improved surfactant function, reduced sPLA2 secretion, inhibition of macrophage migration, reduced alveolar epithelial apoptosis, and suppression of amphiregulin and TGF-ß1 expression in pulmonary tissues as a prerequisite for fibrous lung repair. CONCLUSIONS: We conclude that surfactant fortified by DOPG preserves lung function, and prevents alveolar epithelial injury and fibrous stimulus by reduction of sPLA2 in a neonatal model of acute respiratory distress syndrome without any relevant discernable side effects. Hence, DOPG supplementation in a neonatal lung exerts important function protecting effects and seems to be justified in cases of overwhelming pulmonary inflammation.
Subject(s)
Apoptosis/drug effects , Phosphatidylglycerols/pharmacology , Pulmonary Surfactants/pharmacology , Respiratory Distress Syndrome, Newborn/prevention & control , Animals , Animals, Newborn , Disease Models, Animal , Epithelial Cells/drug effects , Epithelial Cells/pathology , Female , Macrophages, Alveolar/drug effects , Macrophages, Alveolar/metabolism , Male , Pulmonary Alveoli/cytology , Pulmonary Alveoli/drug effects , Pulmonary Alveoli/pathology , Pulmonary Edema/prevention & control , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/prevention & control , Respiration, Artificial , SwineABSTRACT
Previous studies have characterized phenotypic and functional alterations within T-cell receptor αß-expressing T cells in patients with granulomatosis with polyangiitis (GPA). We analyzed the frequency, subset composition and in vitro activation of blood γδ T cells in GPA patients. We observed a significant reduction of γδ T cell numbers, due to the selective depletion of the Vδ2 subset which remained consistent over time upon repeated analysis. The loss of Vδ2 T cells was not due to migration into the inflamed lesions as very few γδ T cells were detected in inflammatory infiltrates. The memory subset distribution did not differ among Vδ2 T cells from healthy donors and GPA patients. Importantly, the remaining Vδ2 T cells were capable of responding to phosphoantigen stimulation in vitro. The marked depletion of blood Vδ2 T cells in GPA suggests cellular exhaustion, possibly due to chronic exposure to and continuous overstimulation by microbial phosphoantigens.
Subject(s)
Granulomatosis with Polyangiitis/immunology , Receptors, Antigen, T-Cell, gamma-delta/deficiency , T-Lymphocyte Subsets/immunology , T-Lymphocytes/immunology , Adult , Aged , Female , Humans , Male , Middle Aged , Young AdultABSTRACT
BACKGROUND: Due to their restricted expression in male germ cells and certain tumors, cancer/testis (CT) antigens are regarded as promising targets for tumor therapy. CT45 is a recently identified nuclear CT antigen that was associated with a severe disease score in Hodgkin's lymphoma and poor prognosis in multiple myeloma. As for many CT antigens, the biological function of CT45 in developing germ cells and in tumor cells is largely unknown. METHODS: CT45 expression was down-regulated in CT45-positive Hodgkin's lymphoma (L428), fibrosarcoma (HT1080) and myeloma (U266B1) cells using RNA interference. An efficient CT45 knock-down was confirmed by immunofluorescence staining and/or Western blotting. These cellular systems allowed us to analyze the impact of CT45 down-regulation on proliferation, cell cycle progression, morphology, adhesion, migration and invasive capacity of tumor cells. RESULTS: Reduced levels of CT45 did not coincide with changes in cell cycle progression or proliferation. However, we observed alterations in cell adherence, morphology and migration/invasion after CT45 down-regulation. Significant changes in the distribution of cytoskeleton-associated proteins were detected by confocal imaging. Changes in cell adherence were recorded in real-time using the xCelligence system with control and siRNA-treated cells. Altered migratory and invasive capacity of CT45 siRNA-treated cells were visualized in 3D migration and invasion assays. Moreover, we found that CT45 down-regulation altered the level of the heterogeneous nuclear ribonucleoprotein syncrip (hnRNP-Q1) which is known to be involved in the control of focal adhesion formation and cell motility. CONCLUSIONS: Providing first evidence of a cell biological function of CT45, we suggest that this cancer/testis antigen is involved in the modulation of cell morphology, cell adherence and cell motility. Enhanced motility and/or invasiveness of CT45-positive cells could contribute to the more severe disease progression that is correlated to CT45-positivity in several malignancies.
ABSTRACT
The phospholipase neutral sphingomyelinase (N-SMase) has been recognized as a major mediator of processes such as inflammation, development and growth, differentiation and death of cells, as well as in diseases such as Alzheimer's, atherosclerosis, heart failure, ischemia/reperfusion damage, or combined pituitary hormone deficiency. Although activation of N-SMase by the proinflammatory cytokine TNF was described almost two decades ago, the underlying signaling pathway is unresolved. Here, we identify the Polycomb group protein EED (embryonic ectodermal development) as an interaction partner of nSMase2. In yeast, the N terminus of EED binds to the catalytic domain of nSMase2 as well as to RACK1, a protein that modulates the activation of nSMase2 by TNF in concert with the TNF receptor 1 (TNF-R1)-associated protein FAN. In mammalian cells, TNF causes endogenous EED to translocate from the nucleus and to colocalize and physically interact with both endogenous nSMase2 and RACK1. As a consequence, EED and nSMase2 are recruited to the TNF-R1.FAN.RACK1-complex in a timeframe concurrent with activation of nSMase2. After knockdown of EED by RNA interference, the TNF-dependent activation of nSMase2 is completely abrogated, identifying EED as a protein that both physically and functionally couples TNF-R1 to nSMase2, and which therefore represents the "missing link" that completes one of the last unresolved signaling pathways of TNF-R1.
Subject(s)
Receptors, Tumor Necrosis Factor/metabolism , Repressor Proteins/metabolism , Sphingomyelin Phosphodiesterase/metabolism , Enzyme Activation , HeLa Cells , Humans , Polycomb Repressive Complex 2ABSTRACT
PURPOSE: The treatment of ovarian tumors is carried out with platinum medicine which can lead to incompatibilities or resistances. Thus, it is of great interest to check new medicine suitability for its application. AZD1152 is an Aurora kinase inhibitor predominantly works against Aurora kinase B involved in the chromosome segregation. Cells become polyploidy and reduce the proliferation by this impairment. To investigate whether AZD1152, may play a role in the treatment of ovarian carcinoma we serving it to the cisplatinum-resistant cell line SKOV3 alone and in combination with platinum. METHODS: We look at the proliferation, the ploidy, the phases of cell cycle and the apoptosis activity of the cells. RESULTS AND CONCLUSION: We could show that the combination of both medicines in the preclinical experiment produces a working advantage.
Subject(s)
Antineoplastic Agents/pharmacology , Carboplatin/pharmacology , Carcinoma/drug therapy , Cisplatin/pharmacology , Organophosphates/pharmacology , Ovarian Neoplasms/drug therapy , Quinazolines/pharmacology , Apoptosis/drug effects , Aurora Kinase B/antagonists & inhibitors , Caspase 3/metabolism , Caspase 7/metabolism , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Synergism , Female , Humans , PolyploidyABSTRACT
D-myo-inositol-1,2,6-trisphosphate (IP3) is an isomer of the naturally occurring second messenger D-myo-inositol-1,4,5-trisphosphate, and exerts anti-inflammatory and antiedematous effects in the lung. Myo-inositol (Inos) is a component of IP3, and is thought to play an important role in the prevention of neonatal pulmonary diseases such as bronchopulmonary dysplasia and neonatal acute lung injury (nALI). Inflammatory lung diseases are characterized by augmented acid sphingomyelinase (aSMase) activity leading to ceramide production, a pathway that promotes increased vascular permeability, apoptosis, and surfactant alterations. A novel, clinically relevant triple-hit model of nALI was developed, consisting of repeated airway lavage, injurious ventilation, and lipopolysaccharide instillation into the airways, every 24 hours. Thirty-five piglets were randomized to one of four treatment protocols: control (no intervention), surfactant alone, surfactant + Inos, and surfactant + IP3. After 72 hours of mechanical ventilation, lungs were excised from the thorax for subsequent analyses. Clinically, oxygenation and ventilation improved, and extravascular lung water decreased significantly with the S + IP3 intervention. In pulmonary tissue, we observed decreased aSMase activity and ceramide concentrations, decreased caspase-8 concentrations, reduced alveolar epithelial apoptosis, the reduced expression of interleukin-6, transforming growth factor-ß1, and amphiregulin (an epithelial growth factor), reduced migration of blood-borne cells and particularly of CD14(+)/18(+) cells (macrophages) into the airspaces, and lower surfactant surface tensions in S + IP3-treated but not in S + Inos-treated piglets. We conclude that the admixture of IP3 to surfactant, but not of Inos, improves gas exchange and edema in our nALI model by the suppression of the governing enzyme aSMase, and that this treatment deserves clinical evaluation.
Subject(s)
Acute Lung Injury/drug therapy , Apoptosis/drug effects , Inositol Phosphates/pharmacology , Pulmonary Alveoli/drug effects , Pulmonary Edema/drug therapy , Acute Lung Injury/metabolism , Acute Lung Injury/pathology , Amphiregulin , Animals , Animals, Newborn , Bronchoalveolar Lavage Fluid , Caspase 8/metabolism , Ceramides/metabolism , Disease Models, Animal , Female , Glycoproteins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Interleukin-6/metabolism , Lung/drug effects , Lung/metabolism , Lung/pathology , Lymphotoxin-alpha/metabolism , Male , Pulmonary Alveoli/metabolism , Pulmonary Edema/metabolism , Pulmonary Edema/pathology , Pulmonary Gas Exchange/drug effects , Pulmonary Surfactants/metabolism , Pulmonary Surfactants/pharmacology , Respiration, Artificial/methods , Respiratory Mucosa/drug effects , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Sphingomyelin Phosphodiesterase/metabolism , Surface Tension/drug effects , SwineABSTRACT
Hypoxemic respiratory failure of the neonatal organism involves increased acid sphingomyelinase (aSMase) activity and production of ceramide, a second messenger of a pro-inflammatory pathway that promotes increased vascular permeability, surfactant alterations and alveolar epithelial apoptosis. We comparatively assessed the benefits of topical aSMase inhibition by either imipramine (Imi) or phosphatidylinositol-3,5-bisphosphate (PIP2) when administered into the airways together with surfactant (S) for fortification. In this translational study, a triple-hit acute lung injury model was used that entails repeated airway lavage, injurious ventilation and tracheal lipopolysaccharide instillation in newborn piglets subject to mechanical ventilation for 72 hrs. After randomization, we administered an air bolus (control), S, S+Imi, or S+PIP2. Only in the latter two groups we observed significantly improved oxygenation and ventilation, dynamic compliance and pulmonary oedema. S+Imi caused systemic aSMase suppression and ceramide reduction, whereas the S+PIP2 effect remained compartmentalized in the airways because of the molecule's bulky structure. The surfactant surface tensions improved by S+Imi and S+PIP2 interventions, but only to a minor extent by S alone. S+PIP2 inhibited the migration of monocyte-derived macrophages and granulocytes into airways by the reduction of CD14/CD18 expression on cell membranes and the expression of epidermal growth factors (amphiregulin and TGF-ß1) and interleukin-6 as pro-fibrotic factors. Finally we observed reduced alveolar epithelial apoptosis, which was most apparent in S+PIP2 lungs. Exogenous surfactant "fortified" by PIP2, a naturally occurring surfactant component, improves lung function by topical suppression of aSMase, providing a potential treatment concept for neonates with hypoxemic respiratory failure.
Subject(s)
Acute Lung Injury/drug therapy , Phosphatidylinositol Phosphates/administration & dosage , Acute Lung Injury/pathology , Administration, Topical , Amphiregulin , Animals , Animals, Newborn , Apoptosis/drug effects , Bronchoalveolar Lavage Fluid/cytology , CD18 Antigens/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Movement/drug effects , Ceramides/metabolism , Disease Models, Animal , Female , Glycoproteins/metabolism , Imipramine/administration & dosage , Intercellular Signaling Peptides and Proteins/metabolism , Interleukin-6/metabolism , Lipopolysaccharide Receptors/metabolism , Macrophages/drug effects , Macrophages/metabolism , Pulmonary Surfactants , Respiration, Artificial , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Swine , Transforming Growth Factor beta/metabolismABSTRACT
The inhibitor of κB kinase ε (IKKε) is pivotal for an efficient innate immune response to viral infections and has been recognized as breast cancer oncogene. The antiviral function of IKKε involves activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-κB, thus inducing the expression of type I IFN. Here, we have identified two novel splice variants of human IKKε, designated IKKε-sv1 and IKKε-sv2, respectively. Interestingly, RT-PCR revealed quantitatively different isoform expression in PBMC from different individuals. Moreover, we found cell type- and stimulus-specific protein expression of the various splice variants. Overexpression of full-length wt IKKε (IKKε-wt) leads to the activation of NF-κB- as well as IRF3-driven luciferase reporter genes. Although none of the splice variants activates IRF3, IKKε-sv1 still activates NF-κB, whereas IKKε-sv2 is also defective in NF-κB activation. Both splice variants form dimers with IKKε-wt and inhibit IKKε-wt-induced IRF3 signaling including the antiviral activity in a dominant-negative manner. The lack of IRF3 activation is likely caused by the failure of the splice variants to interact with the adapter proteins TANK, NAP1, and/or SINTBAD. Taken together, our data suggest alternative splicing as a novel regulatory mechanism suitable to shift the balance between different functions of IKKε.
Subject(s)
Alternative Splicing , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Interferon Regulatory Factor-3/metabolism , Leukocytes, Mononuclear/immunology , NF-kappa B/metabolism , Virus Diseases/immunology , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Cell Line , Down-Regulation , Humans , Interferon Regulatory Factor-3/antagonists & inhibitors , Interferon-beta/analysis , Molecular Sequence Data , Proteins/metabolism , tRNA MethyltransferasesABSTRACT
gammadelta T-cells are a numerically small subset of T-cells with distinct features. They recognise antigens that are not seen by other immune cells. At the functional level, gammadelta T-cells share some features with alphabeta T-cells but also exert functions that are otherwise performed by specialised subsets of alphabeta T-cells (e.g. IL-17 production, regulatory activity). We discuss the potential role of gammadelta T-cells in various clinical forms of vasculitis.
Subject(s)
Receptors, Antigen, T-Cell, gamma-delta/immunology , T-Lymphocytes, Regulatory/immunology , Vasculitis/immunology , Animals , HumansABSTRACT
Monomorphic MHC class II determinants are attractive targets for immunomodulation. HLA-DR ligation on antigen-presenting cells (APCs) can dramatically alter their function or induce cell death. In monocytes, HLA-DR triggering diminishes their capacity to stimulate T cell proliferation. To further investigate this monocyte-dependent T cell inhibition, we activated human T cells +/- HLA-DR triggering on APCs and tested whether this can induce T cell anergy. Only anti-HLA-DR, but not anti-proliferative control agent anti-CD45, could modulate monocytes in primary cultures with stimulated T cells, so that T cells were hyporesponsive during re-stimulation. Cell separation studies demonstrated that HLA-DR ligation on monocytes is sufficient for mediating T cell anergy. Secretion of monokines was severely reduced after primary culture. Monocytes anergized independently of soluble factors. Extracellular signal-regulated kinase (ERK) phosphorylation occurred early with anti-HLA-DR, but late with anti-CD45 antibody. However, ERK inhibition did not reverse the T cell-anergizing potential of HLA-DR-ligated monocytes implicating other signaling pathways involved in tolerance induction. When analyzing the anergized T cells, they were refractory to exogenous IL-2 and characterized by defective secretion of various cytokines. Expression of CD25, CD28, intracellular CD3zeta and CTLA-4 was reduced. The hyporesponsive T cells up-regulated cell-cycle inhibitors p27(kip1) and p21(cip1) in correlation with human T cell anergy. In contrast, caspase-3 and -8, known to contribute to T cell proliferation, were equally decreased in anti-HLA-DR- and anti-CD45-inhibited cultures. In summary, anti-HLA-DR treatment can generate tolerogenic monocytes transmitting T cell anergy that may be exploited for future immunomodulatory strategies to treat immune-mediated disease states.
Subject(s)
Antibodies, Monoclonal/immunology , Clonal Anergy/immunology , HLA-DR Antigens/immunology , Leukocytes, Mononuclear/immunology , T-Lymphocytes/immunology , Antigen-Antibody Reactions , Apoptosis , Cell Proliferation , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/immunology , Cyclin-Dependent Kinase Inhibitor p27/immunology , Cytokines/biosynthesis , Cytokines/metabolism , Extracellular Signal-Regulated MAP Kinases/immunology , Humans , Leukocytes, Mononuclear/cytology , Phosphorylation , Reference ValuesABSTRACT
PURPOSE: Physical activity can impact the immune system in different ways, e.g. by alteration of the humoral and cellular immune response. Physical activity at medium intensity enhances numbers of cytotoxic T cells, NK cells and macrophages in healthy people. The aim of this study was to compare the effects of endurance and resistance training on the immune system in breast cancer patients during adjuvant chemotherapy. METHODS: In a prospective, controlled and randomized intervention exploratory trial, 12-week supervised endurance or resistance training were compared with usual care twice a week. Endpoints were the absolute numbers of the immune cells such as CD3+ T lymphocytes including CD4+- and CD8+, αß T cells, γδT cells, CD3-/CD16+/56+ NK cells and CD19+ B cells, before and after 12 weeks of treatment. Cell numbers were analyzed using fluorescence-activated cell sorting. RESULTS: Despite different physical interventions in all groups immune cell count decreased in CD3 T cells including TCR αß and CD4 T cells, NK cells and CD19 B cells 12 weeks after initiation of chemotherapy and start of the physical intervention program, while the reduction of γδ T cells and CD8 T cells is less prominent in the RT and UC group. CONCLUSION: Chemotherapy led to a decrease in nearly all measured immune cells. In this study, physical intervention with endurance or resistance training did not suppress cellular immunity any further. Larger multicenter trials are needed to evaluate the exact impact of sports intervention on immune cell subpopulations.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/therapy , Exercise Therapy/methods , Exercise/physiology , Immunity, Cellular/physiology , Adult , Aged , B-Lymphocytes/pathology , Breast Neoplasms/drug therapy , Breast Neoplasms/immunology , Chemotherapy, Adjuvant , Combined Modality Therapy , Female , Humans , Killer Cells, Natural/pathology , Lymphocyte Count , Middle Aged , Physical Endurance , Resistance Training , T-Lymphocytes, Cytotoxic/pathologyABSTRACT
AIMS: Down syndrome-associated dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A) is a ubiquitously expressed protein kinase. Up to date a variety of targets have been identified, establishing a key role for Dyrk1a in selected signalling pathways. In cardiomyocytes, Dyrk1a acts as a negative regulator of hypertrophy by phosphorylating transcription factors of the NFAT family, but its mechanistic function in the heart remains poorly understood. This study was designed to investigate a potential protective role of Dyrk1a in cardiac hypertrophy in vivo. METHODS AND RESULTS: We generated transgenic mice with cardiac-specific overexpression of Dyrk1a. Counterintuitively, these mice developed severe dilated cardiomyopathy associated with congestive heart failure and premature death. In search for the cause of this unexpected phenotype, we found that Dyrk1a interacts with all members of the D-cyclin family and represses their protein levels in vitro and in vivo. Particularly, forced expression of Dyrk1a leads to increased phosphorylation of Ccnd2 on Thr280 and promotes its subsequent proteasomal degradation. Accordingly, cardiomyocytes overexpressing Dyrk1a display hypo-phosphorylated Rb1, suppression of Rb/E2f-signalling, and reduced expression of E2f-target genes, which ultimately results in impaired cell cycle progression. CONCLUSIONS: We identified Dyrk1a as a novel negative regulator of D-cyclin-mediated Rb/E2f-signalling. As dysregulation of this pathway with impaired cardiomyocyte proliferation leads to cardiomyopathy, dose-specific Dyrk1a expression and activity appears to be critical for the hyperplastic and hypertrophic growth of the developing heart.
Subject(s)
Cardiomegaly/enzymology , Cardiomyopathy, Dilated/enzymology , Cyclin D/metabolism , E2F Transcription Factors/metabolism , Myocytes, Cardiac/enzymology , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Retinoblastoma/metabolism , Signal Transduction , Animals , Cardiomegaly/genetics , Cardiomegaly/pathology , Cardiomegaly/physiopathology , Cardiomyopathy, Dilated/genetics , Cardiomyopathy, Dilated/pathology , Cardiomyopathy, Dilated/physiopathology , Cell Cycle , Cell Proliferation , Cyclin D/genetics , Disease Models, Animal , Gene Expression Regulation , HEK293 Cells , Heart Failure/enzymology , Heart Failure/genetics , Heart Failure/pathology , Heart Failure/physiopathology , Humans , Mice, Inbred C57BL , Mice, Transgenic , Myocytes, Cardiac/pathology , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Rats, Wistar , Time Factors , Transfection , Dyrk KinasesABSTRACT
The role of death receptors and caspases as mediators of programmed cell death is well established. This review focuses on new insights into alternative functions of these molecules in activation and proliferation of lymphocytes and other hematopoietic cell types. The involvement of the death receptor Fas and caspases in immunodeficiency and autoimmunity is discussed. Elucidation of the mechanisms that control the decision whether death receptors and caspases drive activation/proliferation or apoptosis may broaden our knowledge about the pathogenesis of numerous diseases and facilitate the development of novel therapeutic strategies.
Subject(s)
Autoimmunity/immunology , Caspases/metabolism , Lymphocytes/cytology , Lymphocytes/immunology , Receptors, Tumor Necrosis Factor/metabolism , Animals , Cell Death , Cell Proliferation , Humans , Lymphocytes/metabolismABSTRACT
Human repp86 becomes detectable in the nucleoplasm of cycling cells at the G(1)-S boundary, condenses at the centrosomes with the onset of mitosis, during which it progressively locates to the mitotic spindle and to the midbody, and vanishes at the completion of cytokinesis. The repp86 cDNA was cloned and sequenced. Full-length repp86 and its COOH-terminal domain cosediment with polymerized microtubules, linking repp86 to the family of microtubule-associated proteins. During prophase and metaphase, repp86 interacts on the mitotic spindle with the putative motor protein Hklp2. Thus, repp86 may function in targeting Hklp2 to the microtubule minus ends, its activity being regulated by phosphorylation of serine/threonine residues. Exogenous overexpression of repp86 provokes accumulation of cells in G(2)-M phase and subsequent polyploidization, suggesting that excess repp86 may interfere with correct nuclear division.
Subject(s)
Mitosis , Nuclear Proteins/metabolism , Amino Acid Sequence , Animals , COS Cells , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cloning, Molecular , Endonucleases , G2 Phase , Humans , Kinesins/metabolism , Microtubules/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phosphoproteins/metabolism , Protein Binding , Spindle Apparatus/metabolismABSTRACT
A major part of the proinflammatory activity of tumor necrosis factor (TNF) is brought about by cytosolic phospholipase A(2) (cPLA(2)) that generates arachidonic acid, the precursor for the production of leukotrienes and prostaglandins. The activation of cPLA(2) and induction of proinflammatory lipid mediators is in striking contrast to the teleologic meaning of apoptosis, which is to avoid an inflammatory reaction. In this review we highlight the evidence for a caspase-mediated cleavage and inactivation of cPLA(2), which seems to be an important mechanism by which TNF downregulates cPLA(2) activity in cells undergoing apoptosis.