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1.
J Allergy Clin Immunol ; 152(1): 230-243, 2023 07.
Article in English | MEDLINE | ID: mdl-36822481

ABSTRACT

BACKGROUND: Familial Mediterranean fever (FMF), caused by mutations in the pyrin-encoding MEFV gene, is characterized by uncontrolled caspase-1 activation and IL-1ß secretion. A similar mechanism drives inflammation in cryopyrin-associated periodic fever syndrome (CAPS) caused by mutations in NLRP3. CAPS and FMF, however, result in largely different clinical manifestations, pointing to additional, autoinflammatory pathways involved in FMF. Another hallmark of FMF is extraordinarily high expression of S100A8 and S100A9. These alarmins are ligands of Toll-like receptor 4 and amplifiers of inflammation. However, the relevance of this inflammatory pathway for the pathogenesis of FMF is unknown. OBJECTIVE: This study investigated whether mutations in pyrin result in specific secretion of S100A8/A9 alarmins through gasdermin D pores' amplifying FMF pathology. METHODS: S100A8/A9 levels in FMF patients were quantified by enzyme-linked immunosorbent assay. In vitro models with knockout cell lines and specific protein inhibitors were used to unravel the S100A8/A9 secretion mechanism. The impact of S100A8/A9 to the pathophysiology of FMF was analyzed with FMF (MEFVV726A/V726A) and S100A9-/- mouse models. Pyrin-S100A8/A9 interaction was investigated by coimmunoprecipitation, immunofluorescence, and enzyme-linked immunosorbent assay studies. RESULTS: The S100A8/A9 complexes directly interacted with pyrin. Knocking out pyrin, caspase-1, or gasdermin D inhibited the secretion of these S100 alarmins. Inflammatory S100A8/A9 dimers were inactivated by tetramer formation. Blocking this inactivation by targeted S100A9 deletion in a murine FMF model demonstrated the relevance of this novel autoinflammatory pathway in FMF. CONCLUSION: This is the first proof that members of the S100 alarmin family are released in a pyrin/caspase-1/gasdermin D-dependent pathway and directly drive autoinflammation in vivo.


Subject(s)
Cryopyrin-Associated Periodic Syndromes , Familial Mediterranean Fever , Animals , Mice , Alarmins , Calgranulin A/genetics , Caspases/metabolism , Cryopyrin-Associated Periodic Syndromes/genetics , Familial Mediterranean Fever/genetics , Gasdermins , Inflammation , Pyrin/genetics
2.
Blood ; 134(2): 134-146, 2019 07 11.
Article in English | MEDLINE | ID: mdl-31076441

ABSTRACT

The inflammatory responsiveness of phagocytes to exogenous and endogenous stimuli is tightly regulated. This regulation plays an important role in systemic inflammatory response syndromes (SIRSs). In SIRSs, phagocytes initially develop a hyperinflammatory response, followed by a secondary state of hyporesponsiveness, a so-called "tolerance." This hyporesponsiveness can be induced by endotoxin stimulation of Toll-like receptor 4 (TLR4), resulting in an ameliorated response after subsequent restimulation. This modification of inflammatory response patterns has been described as innate immune memory. Interestingly, tolerance can also be triggered by endogenous TLR4 ligands, such as the alarmins myeloid-related protein 8 (MRP8, S100A8) and MRP14 (S100A9), under sterile conditions. However, signaling pathways that trigger hyporesponsiveness of phagocytes in clinically relevant diseases are only barely understood. Through our work, we have now identified 2 main signaling cascades that are activated during MRP-induced tolerance of phagocytes. We demonstrate that the phosphatidylinositol 3-kinase/AKT/GSK-3ß pathway interferes with NF-κB-driven gene expression and that inhibition of GSK-3ß mimics tolerance in vivo. Moreover, we identified interleukin-10-triggered activation of transcription factors STAT3 and BCL-3 as master regulators of MRP-induced tolerance. Accordingly, patients with dominant-negative STAT3 mutations show no tolerance development. In a clinically relevant condition of systemic sterile stress, cardiopulmonary bypass surgery, we confirmed the initial induction of MRP expression and the tolerance induction of monocytes associated with nuclear translocation of STAT3 and BCL-3 as relevant mechanisms. Our data indicate that the use of pharmacological JAK-STAT inhibitors may be promising targets for future therapeutic approaches to prevent complications associated with secondary hyporesponsiveness during SIRS.


Subject(s)
Phagocytes/metabolism , Signal Transduction/physiology , Systemic Inflammatory Response Syndrome/metabolism , Adult , Alarmins/immunology , Alarmins/metabolism , Animals , Cardiopulmonary Bypass/adverse effects , Female , Humans , Male , Mice , Mice, Inbred C57BL , Middle Aged , Phagocytes/immunology , Systemic Inflammatory Response Syndrome/immunology , Young Adult
3.
EMBO J ; 32(1): 100-11, 2013 Jan 09.
Article in English | MEDLINE | ID: mdl-23188082

ABSTRACT

Mrp8 and Mrp14 are endogenous alarmins amplifying inflammation via Toll-like receptor-4 (TLR-4) activation. Due to their pro-inflammatory properties, alarmins are supposed to enhance adaptive immunity via activation of dendritic cells (DCs). In contrast, analysing a model of allergic contact dermatitis (ACD) we observed a more severe disease outcome in Mrp8/14-deficient compared to wild-type mice. This unexpected phenotype was associated with an enhanced T-cell response due to an accelerated maturation of DCs in Mrp8/14-deficient mice. Accordingly, Mrp8, the active component of the heterocomplex, inhibits early DC maturation and antigen presentation in a TLR-4-dependent manner. Transfer of DCs purified from the local lymph nodes of sensitized Mrp8/14-deficient to wild-type mice determined the outcome of ACD. Our results link a pro-inflammatory role of the endogenous TLR-4 ligand Mrp8/14 to a regulatory function in adaptive immunity, which shows some similarities with the 'hygiene hypothesis' regarding continuous TLR-4 stimulation and decreased risk of allergy.


Subject(s)
Calgranulin A/metabolism , Calgranulin B/metabolism , Dermatitis, Allergic Contact/immunology , Leukocyte L1 Antigen Complex/immunology , Toll-Like Receptor 4/immunology , Adaptive Immunity , Animals , Antigen Presentation , Calgranulin A/blood , Calgranulin A/genetics , Calgranulin A/immunology , Calgranulin B/blood , Calgranulin B/genetics , Calgranulin B/immunology , Cell Communication , Cell Differentiation , Cell Proliferation , Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Dermatitis, Allergic Contact/metabolism , Ear/pathology , Leukocyte L1 Antigen Complex/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Time-Lapse Imaging , Toll-Like Receptor 4/metabolism
4.
J Immunol ; 194(2): 575-83, 2015 Jan 15.
Article in English | MEDLINE | ID: mdl-25505274

ABSTRACT

The alarmins myeloid-related protein (MRP)8 and MRP14 are the most prevalent cytoplasmic proteins in phagocytes. When released from activated or necrotic phagocytes, extracellular MRP8/MRP14 promote inflammation in many diseases, including infections, allergies, autoimmune diseases, rheumatoid arthritis, and inflammatory bowel disease. The involvement of TLR4 and the multiligand receptor for advanced glycation end products as receptors during MRP8-mediated effects on inflammation remains controversial. By comparative bioinformatic analysis of genome-wide response patterns of human monocytes to MRP8, endotoxins, and various cytokines, we have developed a model in which TLR4 is the dominant receptor for MRP8-mediated phagocyte activation. The relevance of the TLR4 signaling pathway was experimentally validated using human and murine models of TLR4- and receptor for advanced glycation end products-dependent signaling. Furthermore, our systems biology approach has uncovered an antiapoptotic role for MRP8 in monocytes, which was corroborated by independent functional experiments. Our data confirm the primary importance of the TLR4/MRP8 axis in the activation of human monocytes, representing a novel and attractive target for modulation of the overwhelming innate immune response.


Subject(s)
Calgranulin A/immunology , Immunity, Innate/physiology , Monocytes/immunology , Signal Transduction/immunology , Toll-Like Receptor 4/immunology , Animals , Calgranulin B/immunology , Female , Gene Expression Profiling , HEK293 Cells , Humans , Inflammation/immunology , Male , Mice , Monocytes/cytology , Receptor for Advanced Glycation End Products , Receptors, Immunologic/immunology
5.
J Allergy Clin Immunol ; 136(5): 1337-45, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26025129

ABSTRACT

BACKGROUND: Hyperzincemia and hypercalprotectinemia (Hz/Hc) is a distinct autoinflammatory entity involving extremely high serum concentrations of the proinflammatory alarmin myeloid-related protein (MRP) 8/14 (S100A8/S100A9 and calprotectin). OBJECTIVE: We sought to characterize the genetic cause and clinical spectrum of Hz/Hc. METHODS: Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene sequencing was performed in 14 patients with Hz/Hc, and their clinical phenotype was compared with that of 11 patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. PSTPIP1-pyrin interactions were analyzed by means of immunoprecipitation and Western blotting. A structural model of the PSTPIP1 dimer was generated. Cytokine profiles were analyzed by using the multiplex immunoassay, and MRP8/14 serum concentrations were analyzed by using an ELISA. RESULTS: Thirteen patients were heterozygous for a missense mutation in the PSTPIP1 gene, resulting in a p.E250K mutation, and 1 carried a mutation resulting in p.E257K. Both mutations substantially alter the electrostatic potential of the PSTPIP1 dimer model in a region critical for protein-protein interaction. Patients with Hz/Hc have extremely high MRP8/14 concentrations (2045 ± 1300 µg/mL) compared with those with PAPA syndrome (116 ± 74 µg/mL) and have a distinct clinical phenotype. A specific cytokine profile is associated with Hz/Hc. Hz/Hc mutations altered protein binding of PSTPIP1, increasing interaction with pyrin through phosphorylation of PSTPIP1. CONCLUSION: Mutations resulting in charge reversal in the y-domain of PSTPIP1 (E→K) and increased interaction with pyrin cause a distinct autoinflammatory disorder defined by clinical and biochemical features not found in patients with PAPA syndrome, indicating a unique genotype-phenotype correlation for mutations in the PSTPIP1 gene. This is the first inborn autoinflammatory syndrome in which inflammation is driven by uncontrolled release of members of the alarmin family.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cytoskeletal Proteins/metabolism , Leukocyte L1 Antigen Complex/metabolism , Metal Metabolism, Inborn Errors/immunology , Adaptor Proteins, Signal Transducing/genetics , Adolescent , Alarmins/genetics , Alarmins/metabolism , Calgranulin A/genetics , Calgranulin A/metabolism , Child , Cytokines/metabolism , Cytoskeletal Proteins/genetics , Female , Genotype , Humans , Leukocyte L1 Antigen Complex/genetics , Male , Metal Metabolism, Inborn Errors/genetics , Mutation, Missense/genetics , Phenotype , Phosphorylation , Protein Binding/genetics , Protein Interaction Maps/genetics , Protein Multimerization , Pyrin , Young Adult
6.
Biomedicines ; 11(3)2023 Mar 09.
Article in English | MEDLINE | ID: mdl-36979814

ABSTRACT

Immune reactions are characterized by the rapid immigration of phagocytes into sites of inflammation. Meticulous regulation of these migratory processes is crucial for preventing uncontrolled and harmful phagocyte extravasation. S100A8/S100A9 is the major calcium-binding protein complex expressed in phagocytes. After release, this complex acts as a proinflammatory alarmin in the extracellular space, but the intracellular functions of these highly abundant proteins are less clear. Results of this study reveal an important role of S100A8/S100A9 in coordinated cytoskeleton rearrangement during migration. We found that S100A8/S100A9 was able to cross-link F-actin and microtubules in a calcium- and phosphorylation-dependent manner. Cells deficient in S100A8/S100A9 showed abnormalities in cell adhesion and motility. Missing cytoskeletal interactions of S100A8/S100A9 caused differences in the surface expression and activation of ß1-integrins as well as in the regulation of Src/Syk kinase family members. Loss of S100A8/S100A9 led to dysregulated integrin-mediated adhesion and migration, resulting in an overall higher dynamic activity of non-activated S100A8/S100A9-deficient phagocytes. Our data suggest that intracellular S100A8/S100A9 is part of a novel regulatory mechanism that ensures the precise control necessary to facilitate the change between the quiescent and activated state of phagocytes.

7.
J Biol Chem ; 286(9): 7227-38, 2011 Mar 04.
Article in English | MEDLINE | ID: mdl-21177863

ABSTRACT

Ca(2+)-binding proteins of the S100 family participate in intracellular Ca(2+) signaling by binding to and regulating specific cellular targets in their Ca(2+)-loaded conformation. Because the information on specific cellular targets of different S100 proteins is still limited, we developed an affinity approach that selects for protein targets only binding to the physiologically active dimer of an S100 protein. Using this approach, we here identify IQGAP1 as a novel and dimer-specific target of S100P, a member of the S100 family enriched in the cortical cytoskeleton. The interaction between S100P and IQGAP1 is strictly Ca(2+)-dependent and characterized by a dissociation constant of 0.2 µM. Binding occurs primarily through the IQ domain of IQGAP1 and the first EF hand loop of S100P, thus representing a novel structural principle of S100-target protein interactions. Upon cell stimulation, S100P and IQGAP1 co-localize at or in close proximity to the plasma membrane, and complex formation can be linked to altered signal transduction properties of IQGAP1. Specifically, the EGF-induced tyrosine phosphorylation of IQGAP1 that is thought to function in assembling signaling intermediates at IQGAP1 scaffolds in the subplasmalemmal region is markedly reduced in cells overexpressing S100P but not in cells expressing an S100P mutant deficient in IQGAP1 binding. Furthermore, B-Raf binding to IQGAP1 and MEK1/2 activation occurring downstream of IQGAP1 in EGF-triggered signaling cascades are compromised at elevated S100P levels. Thus, S100P is a novel Ca(2+)-dependent regulator of IQGAP1 that can down-regulate the function of IQGAP1 as a signaling intermediate by direct interaction.


Subject(s)
Calcium-Binding Proteins/metabolism , MAP Kinase Signaling System/physiology , Neoplasm Proteins/metabolism , ras GTPase-Activating Proteins/metabolism , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Calmodulin/metabolism , Cell Membrane/metabolism , Cytoskeleton/metabolism , Dimerization , HeLa Cells , Humans , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Phosphorylation/physiology , Protein Interaction Domains and Motifs/physiology , Protein Structure, Tertiary , Proto-Oncogene Proteins B-raf/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , ras GTPase-Activating Proteins/chemistry , ras GTPase-Activating Proteins/genetics
8.
Cells ; 11(12)2022 06 20.
Article in English | MEDLINE | ID: mdl-35741108

ABSTRACT

Monocytes and macrophages are central players of the innate immune response and play a pivotal role in the regulation of inflammation. Thereby, they actively participate in all phases of the immune response, from initiating inflammation and triggering the adaptive immune response, through to the clearance of cell debris and resolution of inflammation. In this review, we described the mechanisms of monocyte and macrophage adaptation to rapidly changing microenvironmental conditions and discussed different forms of macrophage polarization depending on the environmental cues or pathophysiological condition. Therefore, special focus was placed on the tight regulation of the pro- and anti-inflammatory immune response, and the diverse functions of S100A8/S100A9 proteins and the scavenger receptor CD163 were highlighted, respectively. We paid special attention to the function of pro- and anti-inflammatory macrophages under pathological conditions.


Subject(s)
Macrophages , Monocytes , Anti-Inflammatory Agents/metabolism , Calgranulin A/metabolism , Humans , Inflammation/pathology , Macrophages/metabolism , Monocytes/metabolism
9.
Ann Rheum Dis ; 70(12): 2075-2081, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21908452

ABSTRACT

OBJECTIVES: To assess the sensitivity of the phagocyte-specific molecules myeloid-related protein (MRP) 8 and MRP14 (calprotectin) for monitoring disease activity during anti-interleukin (IL)-1 therapies in patients with cryopyrin-associated periodic syndromes (CAPS), including familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and chronic infantile neurological, cutaneous and articular (CINCA) syndrome. METHODS: A total of 39 patients with CAPS, including 5 FCAS, 16 MWS and 18 CINCA syndrome, received anti-IL-1 therapy. All patients with CINCA and 12 with MWS were treated with IL-1Ra (anakinra), 14 patients with MWS with a monoclonal anti-IL-1ß antibody (canakinumab) and patients with FCAS received IL-1 Trap (rilonacept). During serial clinical visits serum amyloid A, C-reactive protein, erythrocyte sedimentation rate and MRP8/14 serum levels were analysed. RESULTS: Untreated patients with CAPS had significantly elevated MRP8/14 values. In response to treatment there was a significant reduction of MRP8/14 levels in CINCA (2,830 (range 690 - 8,480) ng/ml to 670 ng/ml, p < 0.001) and MWS patients (anakinra-treated: 4,390 (1790 - 9780) ng/ml to 1,315 ng/ml (p = 0.003); canakinumab-treated: 3,000 (500 - 13060) ng/ml to 630 ng/ml (p=0.001)). However, in many patients with CAPS, MRP8/14 levels were still elevated compared with healthy individuals, reflecting residual disease activity. However, canakinumab-treated patients with CAPS showed normalised MRP8/14 levels, suggesting control of phagocyte activation. CONCLUSIONS: Monitoring of cellular systems involved in inflammatory cascades of the innate immunity was successfully applied to the IL-1-driven CAPS diseases. This is the first study illustrating different states of subclinical disease activity in all types of CAPS depending on the type of anti-IL-1 therapy. MRP8/14 is a sensitive biomarker for monitoring disease activity, status of inflammation and response to IL-1 blockade in patients with CAPS.


Subject(s)
ATP-Binding Cassette Transporters/blood , Calgranulin B/blood , Cryopyrin-Associated Periodic Syndromes/blood , Interleukin-1/antagonists & inhibitors , Adolescent , Adult , Aged , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized , Antirheumatic Agents/therapeutic use , Biomarkers/blood , C-Reactive Protein/metabolism , Child , Child, Preschool , Cryopyrin-Associated Periodic Syndromes/drug therapy , Cryopyrin-Associated Periodic Syndromes/immunology , Drug Monitoring/methods , Humans , Inflammation Mediators/blood , Interleukin 1 Receptor Antagonist Protein/therapeutic use , Interleukin-1beta/antagonists & inhibitors , Middle Aged , Prospective Studies , Recombinant Fusion Proteins/therapeutic use , Treatment Outcome , Young Adult
10.
Biochim Biophys Acta ; 1793(6): 1078-85, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19111582

ABSTRACT

S100 proteins function as Ca2+ signal transducers by regulating cellular targets in their Ca2+ bound conformation. S100P is a member of the S100 protein family that can activate the membrane and F-actin binding protein ezrin in a Ca2+ dependent manner at least in vitro. Here we generated a novel tool to elucidate directly the S100P-ezrin interaction in vivo. This was achieved by constructing a S100P derivative that contained mutations in the two EF hand loops predicted to lock the protein in a permanently active state. The resulting S100P mutant, termed here S100P pa, could be purified as a soluble protein and showed biochemical properties displayed by wild-type S100P only in the presence of Ca2+. Importantly, S100P pa bound to the N-terminal domain of ezrin in the absence of Ca2+ showing an affinity only slightly reduced as compared to that of Ca2+-bound WT S100P. In line with this permanent complex formation, S100P pa colocalized with ezrin to plasma membrane protrusions of epithelial cells even in the absence of intracellular Ca2+ transients. Thus, S100P pa is a novel type of S100 protein mutant locked in a permanently active state that shows an unregulated complex formation with its cellular target ezrin.


Subject(s)
Calcium-Binding Proteins , Calcium/metabolism , Mutation , Neoplasm Proteins , Amino Acid Sequence , Animals , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Surface Extensions/metabolism , Cytoskeletal Proteins/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment
11.
Sci Transl Med ; 11(490)2019 05 01.
Article in English | MEDLINE | ID: mdl-31043571

ABSTRACT

Interference with immune cell proliferation represents a successful treatment strategy in T cell-mediated autoimmune diseases such as rheumatoid arthritis and multiple sclerosis (MS). One prominent example is pharmacological inhibition of dihydroorotate dehydrogenase (DHODH), which mediates de novo pyrimidine synthesis in actively proliferating T and B lymphocytes. Within the TERIDYNAMIC clinical study, we observed that the DHODH inhibitor teriflunomide caused selective changes in T cell subset composition and T cell receptor repertoire diversity in patients with relapsing-remitting MS (RRMS). In a preclinical antigen-specific setup, DHODH inhibition preferentially suppressed the proliferation of high-affinity T cells. Mechanistically, DHODH inhibition interferes with oxidative phosphorylation (OXPHOS) and aerobic glycolysis in activated T cells via functional inhibition of complex III of the respiratory chain. The affinity-dependent effects of DHODH inhibition were closely linked to differences in T cell metabolism. High-affinity T cells preferentially use OXPHOS during early activation, which explains their increased susceptibility toward DHODH inhibition. In a mouse model of MS, DHODH inhibitory treatment resulted in preferential inhibition of high-affinity autoreactive T cell clones. Compared to T cells from healthy controls, T cells from patients with RRMS exhibited increased OXPHOS and glycolysis, which were reduced with teriflunomide treatment. Together, these data point to a mechanism of action where DHODH inhibition corrects metabolic disturbances in T cells, which primarily affects profoundly metabolically active high-affinity T cell clones. Hence, DHODH inhibition may promote recovery of an altered T cell receptor repertoire in autoimmunity.


Subject(s)
Crotonates/therapeutic use , Mitochondria/metabolism , Multiple Sclerosis/drug therapy , Multiple Sclerosis/immunology , T-Lymphocytes/immunology , Toluidines/therapeutic use , Aerobiosis/drug effects , Animals , Cell Proliferation/drug effects , Cell Respiration/drug effects , Crotonates/pharmacology , Dihydroorotate Dehydrogenase , Electron Transport Complex III/metabolism , Energy Metabolism/drug effects , Gene Expression Regulation/drug effects , Glycolysis/drug effects , Humans , Hydroxybutyrates , Lymphocyte Activation/drug effects , Lymphocyte Subsets/drug effects , Lymphocyte Subsets/immunology , Mitochondria/drug effects , Multiple Sclerosis/genetics , Multiple Sclerosis/pathology , Multiple Sclerosis, Relapsing-Remitting/immunology , Nitriles , Oxidative Phosphorylation/drug effects , Oxidoreductases Acting on CH-CH Group Donors/antagonists & inhibitors , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/drug effects , Toluidines/pharmacology
12.
Nat Rev Rheumatol ; 14(9): 528-541, 2018 09.
Article in English | MEDLINE | ID: mdl-30076385

ABSTRACT

Rheumatic diseases are characterized by sterile inflammation that causes severe long-term damage to various organ systems. A growing body of evidence supports a pivotal role for the pro-inflammatory calcium-binding S100 family of proteins in the pathogenesis of rheumatic diseases. Some S100 proteins are released at the site of inflammation and act as danger-associated molecular pattern molecules by activating pattern recognition receptors. Increased concentrations of S100 proteins in serum and synovial fluid closely correlate with disease activity in several rheumatic diseases and serve as useful biomarkers for monitoring disease activity. Some S100 proteins are also valid biomarkers for predicting response to treatment, systemic organ involvement or disease flares in rheumatic diseases. Analyses of knockout mouse models have confirmed a functional role for S100 proteins, particularly S100A8 and S100A9, in rheumatic diseases, indicating that blocking the expression, release or function of these proteins might be an innovative therapeutic strategy. Owing to their local pattern of expression, specific mechanism of release and autoregulatory effects, such therapeutic approaches would primarily target the local inflammatory process and present only minor risks of systemic adverse effects.


Subject(s)
Antirheumatic Agents/therapeutic use , Rheumatic Diseases/drug therapy , Rheumatic Diseases/metabolism , S100 Proteins/metabolism , Animals , Antirheumatic Agents/pharmacology , Biomarkers/blood , Biomarkers/metabolism , Clinical Trials as Topic , Gene Expression Regulation/drug effects , Humans , Rheumatic Diseases/blood , S100 Proteins/blood , Signal Transduction , Synovial Fluid/metabolism
13.
Expert Opin Ther Targets ; 21(7): 739-751, 2017 07.
Article in English | MEDLINE | ID: mdl-28494625

ABSTRACT

INTRODUCTION: In arthritis, inflammatory processes are triggered by numerous factors that are released from joint tissues, promoting joint destruction and pathological progression. During inflammation, a novel family of pro-inflammatory molecules called alarmins is released, amplifying inflammation and joint damage. Areas covered: With regard to the role of the alarmins S100A8 and S100A9 in the pathogenesis of arthritis, recent advances and the future prospects in terms of therapeutic implications are considered. Expert opinion: There is still an urgent need for novel treatment strategies addressing the local mechanisms of joint inflammation and tissue destruction, offering promising therapeutic alternatives. S100A8 and S100A9, which are the most up-regulated alarmins during arthritis, are endogenous triggers of inflammation, defining these proteins as promising targets for local suppression of arthritis. In murine models, the blockade of S100A8/S100A9 ameliorates inflammatory processes, including arthritis, and there are several lines of evidence that S100-alarmins may already be targeted in therapeutic approaches in man.


Subject(s)
Arthritis/drug therapy , Calgranulin A/metabolism , Calgranulin B/metabolism , Animals , Arthritis/physiopathology , Arthritis, Experimental/drug therapy , Arthritis, Experimental/physiopathology , Disease Progression , Drug Design , Humans , Inflammation/drug therapy , Inflammation/physiopathology , Mice , Molecular Targeted Therapy , Up-Regulation
14.
FEBS Lett ; 578(1-2): 95-8, 2004 Dec 03.
Article in English | MEDLINE | ID: mdl-15581623

ABSTRACT

Phosphorylation of the Ca2+ and membrane-binding protein annexin 1 by epidermal growth factor (EGF) receptor tyrosine kinase has been thought to be involved in regulation of the EGF receptor trafficking. To elucidate the interaction of annexin 1 during EGF receptor internalization, we followed the distribution of annexin 1-GFP fusion proteins at sites of internalizing EGF receptors. The observed association of annexin 1 with EGF receptors was confirmed by immunoprecipitation. We found that this interaction was independent of a functional phosphorylation site in the annexin 1 N-terminal domain but mediated through the Ca2+ binding core domain.


Subject(s)
Annexin A1/metabolism , Cell Membrane/metabolism , ErbB Receptors/metabolism , Recombinant Fusion Proteins/metabolism , Annexin A1/chemistry , Annexin A1/genetics , Calcium/metabolism , Endocytosis/physiology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Immunoprecipitation , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics
15.
Cell Rep ; 9(6): 2112-23, 2014 Dec 24.
Article in English | MEDLINE | ID: mdl-25497086

ABSTRACT

Hyporesponsiveness by phagocytes is a well-known phenomenon in sepsis that is frequently induced by low-dose endotoxin stimulation of Toll-like receptor 4 (TLR4) but can also be found under sterile inflammatory conditions. We now demonstrate that the endogenous alarmins MRP8 and MRP14 induce phagocyte hyporesponsiveness via chromatin modifications in a TLR4-dependent manner that results in enhanced survival to septic shock in mice. During sterile inflammation, polytrauma and burn trauma patients initially present with high serum concentrations of myeloid-related proteins (MRPs). Human neonatal phagocytes are primed for hyporesponsiveness by increased peripartal MRP concentrations, which was confirmed in murine neonatal endotoxinemia in wild-type and MRP14(-/-) mice. Our data therefore indicate that alarmin-triggered phagocyte tolerance represents a regulatory mechanism for the susceptibility of neonates during systemic infections and sterile inflammation.


Subject(s)
Calgranulin A/metabolism , Calgranulin B/metabolism , Immune Tolerance , Phagocytes/metabolism , Adult , Aged , Animals , Burns/immunology , Burns/metabolism , Calgranulin A/blood , Calgranulin A/genetics , Calgranulin B/blood , Calgranulin B/genetics , Cell Line , Cells, Cultured , Chromatin Assembly and Disassembly , Female , Humans , Inflammation/metabolism , Male , Mice , Middle Aged , NF-kappa B/metabolism , Phagocytes/immunology , Shock, Septic/immunology , Shock, Septic/metabolism , Stress, Physiological
16.
J Leukoc Biol ; 92(5): 1069-81, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22892107

ABSTRACT

The Staphylococcus aureus pore-forming toxin PVL is most likely causative for life-threatening necrotizing infections, which are characterized by massive tissue inflammation and necrosis. Whereas the cytotoxic action of PVL on human neutrophils is already well established, the PVL effects on other sensitive cell types, such as monocytes and macrophages, are less clear. In this study, we used different types of human leukocytes (neutrophils, monocytes, macrophages, lymphocytes) to investigate cell-specific binding of PVL subunits and subsequent proinflammatory and cytotoxic effects. In all PVL-sensitive cells, we identified the binding of the subunit LukS-PV as the critical factor for PVL-induced cytotoxicity, which was followed by binding of LukF-PV. LukS-PV binds to monocytes, macrophages, and neutrophils but not to lymphocytes. Additionally, we showed that PVL binding to monocytes and macrophages leads to release of caspase-1-dependent proinflammatory cytokines IL-1ß and IL-18. PVL activates the NLRP3 inflammasome, a signaling complex of myeloid cells that is involved in caspase-1-dependent IL-1ß processing in response to pathogens and endogenous danger signals. Specific inhibition of this pathway at several steps significantly reduced inflammasome activation and subsequent pyronecrosis. Furthermore, we found that PAMPs and DAMPs derived from dying neutrophils can dramatically enhance this response by up-regulating pro-IL-1ß in monocytes/macrophages. This study analyzes a specific host signaling pathway that mediates PVL-induced inflammation and cytotoxicity, which has high relevance for CA-MRSA-associated and PVL-mediated pathogenic processes, such as necrotizing infections.


Subject(s)
Bacterial Toxins/immunology , Carrier Proteins/immunology , Exotoxins/immunology , Inflammasomes/immunology , Inflammation/immunology , Leukocidins/immunology , Phagocytes/immunology , Animals , Bacterial Toxins/metabolism , Blotting, Western , Exotoxins/metabolism , Humans , Leukocidins/metabolism , Mice , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/immunology , Staphylococcal Infections/immunology , Staphylococcal Infections/metabolism , Staphylococcus aureus/immunology , Staphylococcus aureus/metabolism , Transfection
17.
J Biol Chem ; 283(43): 29331-40, 2008 Oct 24.
Article in English | MEDLINE | ID: mdl-18725408

ABSTRACT

Ezrin is a multidomain protein providing regulated membrane-cytoskeleton contacts that play a role in cell differentiation, adhesion, and migration. Within the cytosol of resting cells ezrin resides in an autoinhibited conformation in which the N- and C-terminal ezrin/radixin/moesin (ERM) association domains (ERMADs) interact with one another. Activation of the ezrin membrane-cytoskeleton linker function requires an opening of this interdomain association that can result from phosphatidylinositol 4,5-bisphosphate binding to the N-ERMAD and threonine 567 phosphorylation in the C-ERMAD. We have shown that ezrin can also be activated by Ca(2+)-dependent binding of the EF-hand protein S100P. We now provide a quantitative analysis of this interaction and map the respective binding sites to the F2 lobe in the ezrin N-ERMAD and a stretch of hydrophobic residues in the C-terminal extension of S100P. Phospholipid binding assays reveal that S100P and phosphatidylinositol 4,5-bisphosphate compete to some extent for at least partially overlapping binding sites in N-ERMAD. Using interaction-competent as well as interaction-incompetent S100P derivatives and permanently active ezrin mutants, we also show that the protein interaction and a resulting activation of ezrin promote the transendothelial migration of tumor cells. Thus, a prometastatic role of ezrin and S100P that had been proposed based on their overexpression in highly metastatic cancers is probably due to a direct interaction between the two proteins and the S100P-mediated activation of ezrin.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium/chemistry , Cytoskeletal Proteins/chemistry , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/chemistry , Neoplasms/metabolism , Cell Movement , Humans , Microcirculation , Models, Biological , Neoplasm Metastasis , Phospholipids/chemistry , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Surface Plasmon Resonance
18.
Biochemistry ; 47(12): 3762-9, 2008 Mar 25.
Article in English | MEDLINE | ID: mdl-18302339

ABSTRACT

In a quantitative manner, we investigated the mechanism of switching ezrin from the dormant to the active, F-actin binding state by recognition of PIP 2. For this purpose, we established a novel in vitro model mimicking ezrin-mediated membrane-cytoskeleton attachment and compared the F-actin binding capability of ezrin that either had been coupled via a His tag to a lipid bilayer displaying Ni-NTA or had been bound to supported membranes containing PIP 2. Epifluorescence and colloidal probe microscopy (CPM) were employed to demonstrate ezrin's conformational switch into an active conformation capable of binding F-actin. Epifluorescence images revealed attachment of fluorescently labeled F-actin solely to PIP 2-bound ezrin. For the first time, colloidal spheres equipped with an artificial cytoskeleton composed of firmly attached F-actin filaments were used to measure quantitatively the maximal adhesion forces and the work of adhesion of the ezrin-F-actin interface. We found that the work of adhesion between PIP 2-bound ezrin and F-actin is substantially larger than that measured between F-actin and ezrin bound to the membrane via the His tag. Collectively, these data indicate that activation of ezrin can occur as a consequence of PIP 2 binding and does not require additional cofactors.


Subject(s)
Actins/metabolism , Cytoskeletal Proteins/metabolism , Lipid Bilayers/metabolism , Phosphatidylinositol 4,5-Diphosphate/physiology , Colloids/chemistry , Lysine/analogs & derivatives , Lysine/chemistry , Microscopy, Atomic Force , Oleic Acids/chemistry , Protein Conformation , Succinates/chemistry
19.
Biochemistry ; 45(43): 13025-34, 2006 Oct 31.
Article in English | MEDLINE | ID: mdl-17059219

ABSTRACT

By means of the quartz crystal microbalance (QCM) and scanning force microscopy (SFM), the adsorption of ezrin, a member of the ezrin/radixin/moesin protein family, on l-alpha-phosphatidylinositol-4,5-bisphosphate (PIP(2)) containing solid-supported membranes was investigated. An increase in the PIP(2) content in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes resulted in an increased amount of bound ezrin strongly supporting the crucial role of PIP(2) for ezrin recruitment to membranes. No ezrin adsorption to membranes composed of pure POPC was detected. To characterize the binding process in more detail, the kinetics and reversibility of ezrin adsorption were investigated by the QCM technique, showing that the protein remains partly bound after rinsing with pure buffer, which we suspected to be a result of lateral interactions between the proteins. SFM images revealed the formation of two-dimensional ezrin clusters on PIP(2)-doped POPC membranes. Time-elapsed SFM images show that the growth of protein domains occurs from a few nucleation sites. The QCM data in conjunction with the results obtained by SFM led us to propose that the binding process of ezrin occurs in a positive cooperative manner. When lateral interactions of the proteins on the membrane were taken into account, we were able to simulate the kinetics obtained from time-resolved QCM readouts by employing a model developed by Minton. On the basis of the kinetic analysis, we were also able to reconstruct the adsorption isotherm.


Subject(s)
Cytoskeletal Proteins/metabolism , Membranes, Artificial , Phosphatidylinositol 4,5-Diphosphate/chemistry , Adsorption , Cytoskeletal Proteins/chemistry , Membrane Lipids/metabolism , Microscopy, Atomic Force/methods , Protein Binding , Time Factors
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