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1.
Annu Rev Immunol ; 28: 367-88, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20192808

RESUMO

The immune system has evolved to respond not only to pathogens, but also to signals released from dying cells. Cell death through necrosis induces inflammation, whereas apoptotic cell death provides an important signal for tolerance induction. High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein, released actively following cytokine stimulation as well as passively during cell death; it is the prototypic damage-associated molecular pattern (DAMP) molecule and has been implicated in several inflammatory disorders. HMGB1 can associate with other molecules, including TLR ligands and cytokines, and activates cells through the differential engagement of multiple surface receptors including TLR2, TLR4, and RAGE. RAGE is a multiligand receptor that binds structurally diverse molecules, including not only HMGB1, but also S100 family members and amyloid-beta. RAGE activation has been implicated in sterile inflammation as well as in cancer, diabetes, and Alzheimer's disease. While HMGB1 through interactions with TLRs may also be important, this review focuses on the role of the HMGB1-RAGE axis in inflammation and cancer.


Assuntos
Proteína HMGB1/imunologia , Inflamação/imunologia , Neoplasias/imunologia , Receptores Imunológicos/imunologia , Animais , Proteína HMGB1/química , Humanos , Inflamação/metabolismo , Ligantes , Neoplasias/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Transdução de Sinais
2.
Eur Respir J ; 62(3)2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37442582

RESUMO

BACKGROUND: Epithelial damage, repair and remodelling are critical features of chronic airway diseases including chronic obstructive pulmonary disease (COPD). Interleukin (IL)-33 released from damaged airway epithelia causes inflammation via its receptor, serum stimulation-2 (ST2). Oxidation of IL-33 to a non-ST2-binding form (IL-33ox) is thought to limit its activity. We investigated whether IL-33ox has functional activities that are independent of ST2 in the airway epithelium. METHODS: In vitro epithelial damage assays and three-dimensional, air-liquid interface (ALI) cell culture models of healthy and COPD epithelia were used to elucidate the functional role of IL-33ox. Transcriptomic changes occurring in healthy ALI cultures treated with IL-33ox and COPD ALI cultures treated with an IL-33-neutralising antibody were assessed with bulk and single-cell RNA sequencing analysis. RESULTS: We demonstrate that IL-33ox forms a complex with receptor for advanced glycation end products (RAGE) and epidermal growth factor receptor (EGFR) expressed on airway epithelium. Activation of this alternative, ST2-independent pathway impaired epithelial wound closure and induced airway epithelial remodelling in vitro. IL-33ox increased the proportion of mucus-producing cells and reduced epithelial defence functions, mimicking pathogenic traits of COPD. Neutralisation of the IL-33ox pathway reversed these deleterious traits in COPD epithelia. Gene signatures defining the pathogenic effects of IL-33ox were enriched in airway epithelia from patients with severe COPD. CONCLUSIONS: Our study reveals for the first time that IL-33, RAGE and EGFR act together in an ST2-independent pathway in the airway epithelium and govern abnormal epithelial remodelling and muco-obstructive features in COPD.


Assuntos
Interleucina-33 , Doença Pulmonar Obstrutiva Crônica , Humanos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Receptores ErbB , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33/genética , Interleucina-33/metabolismo , Oxirredução , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo
3.
J Immunol ; 205(1): 102-112, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32434940

RESUMO

To maintain homeostasis, macrophages must be capable of assuming either an inflammatory or an anti-inflammatory phenotype. To better understand the latter, we stimulated human macrophages in vitro with TLR ligands in the presence of high-density immune complexes (IC). This combination of stimuli resulted in a broad suppression of inflammatory mediators and an upregulation of molecules involved in tissue remodeling and angiogenesis. Transcriptomic analysis of TLR stimulation in the presence of IC predicted the downstream activation of AKT and the inhibition of GSK3. Consequently, we pretreated LPS-stimulated human macrophages with small molecule inhibitors of GSK3 to partially phenocopy the regulatory effects of stimulation in the presence of IC. The upregulation of DC-STAMP and matrix metalloproteases was observed on these cells and may represent potential biomarkers for this regulatory activation state. To demonstrate the presence of these anti-inflammatory, growth-promoting macrophages in a human infectious disease, biopsies from patients with leprosy (Hanseniasis) were analyzed. The lepromatous form of this disease is characterized by hypergammaglobulinemia and defective cell-mediated immunity. Lesions in lepromatous leprosy contained macrophages with a regulatory phenotype expressing higher levels of DC-STAMP and lower levels of IL-12, relative to macrophages in tuberculoid leprosy lesions. Therefore, we propose that increased signaling by FcγR cross-linking on TLR-stimulated macrophages can paradoxically promote the resolution of inflammation and initiate processes critical to tissue growth and repair. It can also contribute to infectious disease progression.


Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Hanseníase Virchowiana/imunologia , Hanseníase Tuberculoide/imunologia , Macrófagos/imunologia , Biópsia , Diferenciação Celular/imunologia , Linhagem Celular , Progressão da Doença , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Hanseníase Virchowiana/patologia , Hanseníase Tuberculoide/patologia , Ativação de Macrófagos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Neovascularização Fisiológica/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA-Seq , Receptores de IgG/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Pele/citologia , Pele/imunologia , Pele/patologia , Receptores Toll-Like/metabolismo , Adulto Jovem
4.
J Proteome Res ; 20(6): 3150-3164, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34008986

RESUMO

Citrullination is an important post-translational modification implicated in many diseases including rheumatoid arthritis (RA), Alzheimer's disease, and cancer. Neutrophil and mast cells have different expression profiles for protein-arginine deiminases (PADs), and ionomycin-induced activation makes them an ideal cellular model to study proteins susceptible to citrullination. We performed high-resolution mass spectrometry and stringent data filtration to identify citrullination sites in neutrophil and mast cells treated with and without ionomycin. We identified a total of 833 validated citrullination sites on 395 proteins. Several of these citrullinated proteins are important components of pathways involved in innate immune responses. Using this benchmark primary sequence data set, we developed machine learning models to predict citrullination in neutrophil and mast cell proteins. We show that our models predict citrullination likelihood with 0.735 and 0.766 AUCs (area under the receiver operating characteristic curves), respectively, on independent validation sets. In summary, this study provides the largest number of validated citrullination sites in neutrophil and mast cell proteins. The use of our novel motif analysis approach to predict citrullination sites will facilitate the discovery of novel protein substrates of protein-arginine deiminases (PADs), which may be key to understanding immunopathologies of various diseases.


Assuntos
Citrulinação , Mastócitos , Citrulina/metabolismo , Ionomicina/farmacologia , Aprendizado de Máquina , Espectrometria de Massas , Mastócitos/metabolismo , Neutrófilos/metabolismo , Desiminases de Arginina em Proteínas/genética
5.
Ann Rheum Dis ; 78(2): 228-237, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30459279

RESUMO

OBJECTIVE: Immune complexes (ICs) play a critical role in the pathology of autoimmune diseases. The aim of this study was to generate and characterise a first-in-class anti-FcγRIIA antibody (Ab) VIB9600 (previously known as MEDI9600) that blocks IgG immune complex-mediated cellular activation for clinical development. METHODS: VIB9600 was humanised and optimised from the IV.3 Ab. Binding affinity and specificity were determined by Biacore and ELISA. Confocal microscopy, Flow Cytometry-based assays and binding competition assays were used to assess the mode of action of the antibody. In vitro cell-based assays were used to demonstrate suppression of IC-mediated inflammatory responses. In vivo target suppression and efficacy was demonstrated in FcγRIIA-transgenic mice. Single-dose pharmacokinetic (PK)/pharmacodynamic study multiple dose Good Laboratory Practice (GLP) toxicity studies were conducted in non-human primates. RESULTS: We generated a humanised effector-deficient anti-FcγRIIA antibody (VIB9600) that potently blocks autoantibody and IC-mediated proinflammatory responses. VIB9600 suppresses FcγRIIA activation by blocking ligand engagement and by internalising FcγRIIA from the cell surface. VIB9600 inhibits IC-induced type I interferons from plasmacytoid dendritic cells (involved in SLE), antineutrophil cytoplasmic antibody (ANCA)-induced production of reactive oxygen species by neutrophils (involved in ANCA-associated vasculitis) and IC-induced tumour necrosis factor α and interleukin-6 production (involved in rheumatoid arthritis). In FcγRIIA transgenic mice, VIB9600 suppressed antiplatelet antibody-induced thrombocytopaenia, acute anti-GBM Ab-induced nephritis and anticollagen Ab-induced arthritis. VIB9600 also exhibited favourable PK and safety profiles in cynomolgus monkey studies. CONCLUSIONS: VIB9600 is a specific humanised antibody antagonist of FcγRIIA with null effector function that warrants further clinical development for the treatment of IC-mediated diseases.


Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Complexo Antígeno-Anticorpo/efeitos dos fármacos , Doenças Autoimunes/tratamento farmacológico , Fatores Imunológicos/farmacologia , Receptores de IgG/imunologia , Animais , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Complexo Antígeno-Anticorpo/imunologia , Doenças Autoimunes/imunologia , Células Dendríticas/imunologia , Humanos , Imunoglobulina G/imunologia , Interleucina-6/imunologia , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/imunologia , Fator de Necrose Tumoral alfa/imunologia
6.
Ann Rheum Dis ; 77(1): 141-148, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29070531

RESUMO

OBJECTIVES: The citrullinating enzyme peptidylarginine deiminase type 4 (PAD4) is the target of a polyclonal group of autoantibodies in patients with rheumatoid arthritis (RA). A subgroup of such antibodies, initially identified by cross-reactivity with peptidylarginine deiminase type 3 (PAD3), is strongly associated with progression of radiographic joint damage and interstitial lung disease and has the unique ability to activate PAD4. The features of these antibodies in terms of their T cell-dependent origin, genetic characteristics and effect of individual antibody specificities on PAD4 function remain to be defined. METHODS: We used PAD4 tagged with the monomeric fluorescent protein mWasabi to isolate PAD4-specific memory B cells from anti-PAD4 positive patients with RA and applied single cell cloning technologies to obtain monoclonal antibodies. RESULTS: Among 44 single B cells, we cloned five antibodies with PAD4-activating properties. Sequence analysis, germline reversion experiments and antigen specificity assays suggested that autoantibodies to PAD4 are not polyreactive and arise from PAD4-reactive precursors. Somatic mutations increase the agonistic activity of these antibodies at low calcium concentrations by facilitating their interaction with structural epitopes that modulate calcium-binding site 5 in PAD4. CONCLUSIONS: PAD4-activating antibodies directly amplify a key process in disease pathogenesis, making them unique among other autoantibodies in RA. Understanding the molecular basis for their functionality may inform the design of future PAD4 inhibitors.


Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Linfócitos B/imunologia , Desiminases de Arginina em Proteínas/imunologia , Afinidade de Anticorpos , Artrite Reumatoide/sangue , Autoanticorpos/sangue , Reações Cruzadas , Progressão da Doença , Humanos , Proteína-Arginina Desiminase do Tipo 3 , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas/sangue
7.
Mediators Inflamm ; 2015: 236451, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26078491

RESUMO

Autoantibodies against citrullinated proteins are diagnostic for rheumatoid arthritis. However, the molecular mechanisms driving protein citrullination in patients with rheumatoid arthritis remain poorly understood. Using two independent western blotting methods, we report that agents that trigger a sufficiently large influx of extracellular calcium ions induced a marked citrullination of multiple proteins in human neutrophils, monocytes, and, to a lesser extent, T lymphocytes and natural killer cells, but not B lymphocytes or dendritic cells. This response required 250-1,000 µM extracellular calcium and was prevented by EDTA. Other neutrophil activating stimuli, such as formyl-peptides, GM-CSF, IL-6, IL8, TNFα, or phorbol ester, did not induce any detectable increase in protein citrullination, suggesting that receptor-induced calcium mobilization is insufficient to trigger hypercitrullination. We conclude that loss of membrane integrity and subsequent influx of high levels of calcium, which can be triggered by perforin released from cytotoxic cells or complement mediated formation of membrane attack complexes in the joints of rheumatoid arthritis patients, are sufficient to induce extensive protein citrullination in immune cells, notably neutrophils. This mechanism may provide the citrullinated autoantigens that drive autoimmunity in this devastating disease.


Assuntos
Citrulina/metabolismo , Leucócitos/metabolismo , Neutrófilos/metabolismo , Artrite Reumatoide/metabolismo , Western Blotting , Células Cultivadas , Humanos , Ionomicina/farmacologia , Leucócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Perforina/farmacologia
8.
J Gen Virol ; 94(Pt 8): 1691-1700, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23559480

RESUMO

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection. Infection is critically dependent on the RSV fusion (F) protein, which mediates fusion between the viral envelope and airway epithelial cells. The F protein is also expressed on infected cells and is responsible for fusion of infected cells with adjacent cells, resulting in the formation of multinucleate syncytia. The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that is constitutively highly expressed by type I alveolar epithelial cells. Here, we report that RAGE protected HEK cells from RSV-induced cell death and reduced viral titres in vitro. RAGE appeared to interact directly with the F protein, but, rather than inhibiting RSV entry into host cells, virus replication and budding, membrane-expressed RAGE or soluble RAGE blocked F-protein-mediated syncytium formation and sloughing. These data indicate that RAGE may contribute to protecting the lower airways from RSV by inhibiting the formation of syncytia, viral spread, epithelial damage and airway obstruction.


Assuntos
Células Epiteliais/virologia , Células Gigantes/virologia , Interações Hospedeiro-Patógeno , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Vírus Sincicial Respiratório Humano/patogenicidade , Proteínas Virais de Fusão/metabolismo , Células Cultivadas , Humanos
9.
J Infect Dis ; 205(8): 1311-20, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22262795

RESUMO

Respiratory syncytial virus (RSV), a common respiratory pathogen in infants and the older population, causes pulmonary inflammation and airway occlusion that leads to impairment of lung function. Here, we have established a role for receptor for advanced glycation end products (RAGE) in RSV infection. RAGE-deficient (ager(-/-)) mice were protected from RSV-induced weight loss and inflammation. This protection correlated with an early increase in type I interferons, later decreases in proinflammatory cytokines, and a reduction in viral load. To assess the contribution of soluble RAGE (sRAGE) to RSV-induced disease, wild-type and ager(-/-) mice were given doses of sRAGE following RSV infection. Of interest, sRAGE treatment prevented RSV-induced weight loss and neutrophilic inflammation to a degree similar to that observed in ager(-/-) mice. Our work further elucidates the roles of RAGE in the pathogenesis of respiratory infections and highlights the opposing roles of membrane and sRAGE in modulating the host response to RSV infection.


Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Receptores Imunológicos/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Animais , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Nariz , Isoformas de Proteínas , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Carga Viral
10.
Br J Haematol ; 155(4): 426-37, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21902688

RESUMO

Human cluster of differentiation (CD) antigen 19 is a B cell-specific surface antigen and an attractive target for therapeutic monoclonal antibody (mAb) approaches to treat malignancies of B cell origin. MEDI-551 is an affinity-optimized and afucosylated CD19 mAb with enhanced antibody-dependent cellular cytotoxicity (ADCC). The results from in vitro ADCC assays with Natural Killer cells as effector cells, demonstrate that MEDI-551 is effective at lower mAb doses than rituximab with multiple cell lines as well as primary chronic lymphocytic leukaemia and acute lymphoblastic leukaemia samples. Targeting CD19 with MEDI-551 was also effective in several severe combined immunodeficiency lymphoma models. Furthermore, the combination of MEDI-551 with rituximab resulted in prolonged suppression of tumour growth, demonstrating that therapeutic mAbs with overlapping effector function can be combined for greater tumour growth inhibition. Together, the data demonstrate that MEDI-551 has potent antitumour activity in preclinical models of B cell malignancies. The results also suggest that the combination of the ADCC-enhanced CD19 mAb with an anti-CD20 mAb could be a novel approach for the treatment of B cell lymphomas.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD19/imunologia , Leucemia de Células B/imunologia , Leucemia de Células B/terapia , Linfoma de Células B/imunologia , Linfoma de Células B/terapia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Murinos/administração & dosagem , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos SCID , Engenharia de Proteínas/métodos , Receptores Fc/imunologia , Rituximab , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Arthritis Rheum ; 62(3): 742-52, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20131230

RESUMO

OBJECTIVE: To define the intrinsic capacity of fibroblast-like synoviocytes (FLS) to establish a 3-dimensional (3-D) complex synovial lining architecture characterized by the multicellular organization of the compacted synovial lining and the elaboration of synovial fluid constituents. METHODS: FLS were cultured in spherical extracellular matrix (ECM) micromasses for 3 weeks. The FLS micromass architecture was assessed histologically and compared with that of dermal fibroblast controls. Lubricin synthesis was measured via immunodetection. Basement membrane matrix and reticular fiber stains were performed to examine ECM organization. Primary human and mouse monocytes were prepared and cocultured with FLS in micromass to investigate cocompaction in the lining architecture. Cytokine stimuli were applied to determine the capacity for inflammatory architecture rearrangement. RESULTS: FLS, but not dermal fibroblasts, spontaneously formed a compacted lining architecture over 3 weeks in the 3-D ECM micromass organ cultures. These lining cells produced lubricin. FLS rearranged their surrounding ECM into a complex architecture resembling the synovial lining and supported the survival and cocompaction of monocyte/macrophages in the neo-lining structure. Furthermore, when stimulated by cytokines, FLS lining structures displayed features of the hyperplastic rheumatoid arthritis synovial lining. CONCLUSION: This 3-D micromass organ culture method demonstrates that many of the phenotypic characteristics of the normal and the hyperplastic synovial lining in vivo are intrinsic functions of FLS. Moreover, FLS promote survival and cocompaction of primary monocytes in a manner remarkably similar to that of synovial lining macrophages. These findings provide new insight into inherent functions of the FLS lineage and establish a powerful in vitro method for further investigation of this lineage.


Assuntos
Fibroblastos/fisiologia , Líquido Sinovial/química , Membrana Sinovial/citologia , Animais , Matriz Extracelular/ultraestrutura , Glicoproteínas/biossíntese , Humanos , Inflamação/fisiopatologia , Macrófagos/citologia , Camundongos , Técnicas de Cultura de Órgãos , Membrana Sinovial/anatomia & histologia
12.
Am J Respir Crit Care Med ; 181(9): 917-27, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20133931

RESUMO

RATIONALE: Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation and remodeling. High-mobility group box 1 (HMGB1), a nuclear protein that is released during inflammation and repair, interacts with proinflammatory cytokines and with the receptor for advanced glycation end products (RAGE), which is highly expressed in the lung. OBJECTIVES: To determine whether HMGB1 is augmented in COPD and is associated with IL-1beta and RAGE. METHODS: HMGB1 was assessed in the bronchoalveolar lavage (BAL) of 20 never-smokers, 20 smokers, and 30 smokers with COPD and it was correlated with inflammatory and clinical parameters. In parallel, HMGB1 and RAGE immunolocalization was determined in bronchial and lung tissues. Last, binding of HMGB1 to IL-1beta in human macrophages and in BAL fluid was examined. MEASUREMENTS AND MAIN RESULTS: BAL levels of HMGB1 were higher in smokers with COPD than in smokers and never-smokers (P < 0.0001 for both comparisons), and similar differences were observed in epithelial cells and alveolar macrophages. BAL HMGB1 correlated positively with IL-1beta (r(s) = 0.438; P = 0.0006) and negatively with FEV(1) (r(s) = -0.570; P < 0.0001) and transfer factor of the lung for carbon monoxide (r(s) = -0.382; P = 0.0026). HMGB1-IL-1beta complexes were found in BAL supernatant and alveolar macrophages from smokers and patients with COPD, as well as in the human macrophage cell line, THP-1, where they enhanced the synthesis of tumor-necrosis factor-alpha. RAGE was overexpressed in the airway epithelium and smooth muscle of patients with COPD and it colocalized with HMGB1. CONCLUSIONS: Elevated HMGB1 expression in COPD airways may sustain inflammation and remodeling through its interaction with IL-1beta and RAGE.


Assuntos
Proteína HMGB1/metabolismo , Interleucina-1beta/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Receptores Imunológicos/metabolismo , Remodelação das Vias Aéreas/fisiologia , Brônquios/metabolismo , Líquido da Lavagem Broncoalveolar/química , Linhagem Celular , Feminino , Imunofluorescência , Fluxo Expiratório Forçado , Humanos , Imuno-Histoquímica , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Masculino , Pessoa de Meia-Idade , Receptor para Produtos Finais de Glicação Avançada , Fumar/metabolismo
13.
J Pharmacol Exp Ther ; 335(1): 213-22, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20605905

RESUMO

The pan B-cell surface antigen CD19 is an attractive target for therapeutic monoclonal antibody (mAb) approaches. We have generated a new afucosylated anti-human (hu)CD19 mAb, MEDI-551, with increased affinity to human FcγRIIIA and mouse FcγRIV and enhanced antibody-dependent cellular cytotoxicity (ADCC). During in vitro ADCC assays with B-cell lines, MEDI-551 is effective at much lower mAb concentrations than the fucosylated parental mAb anti-CD19-2. Furthermore, the afucosylated CD19 mAb MEDI-551 depleted B cells from normal donor peripheral blood mononuclear cell samples in an autologous ADCC assay, as well as blood and tissue B cells in human CD19/CD20 double transgenic (Tg) mice at lower concentrations than that of the positive control mAb rituximab. In huCD19/CD20 Tg mice, both macrophage-mediated phagocytosis and complement-dependent cytotoxicity contribute to depletion with rituximab; MEDI-551 did not require complement for maximal B-cell depletion. Furthermore, extended B-cell depletion from the blood and spleen was achieved with MEDI-551, which is probably explained by bone marrow B-cell depletion in huCD19/CD20 Tg mice relative to the control mAb rituximab. In summary, MEDI-551 has potent B-cell-depleting activity in vitro and in vivo and may be a promising new approach for the treatment of B-cell malignancies and autoimmune diseases.


Assuntos
Antígenos CD19/imunologia , Linfócitos B/fisiologia , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD19/genética , Proliferação de Células/efeitos dos fármacos , Fucose/química , Humanos , Imunoglobulina G/imunologia , Camundongos , Camundongos Transgênicos , Engenharia de Proteínas , Rituximab
14.
PLoS One ; 15(3): e0229184, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32182251

RESUMO

Traditional cardiovascular disease (CVD) risk factors, such as hypertension, dyslipidemia and diabetes do not explain the increased CVD burden in systemic lupus erythematosus (SLE). The oxidized-LDL receptor, LOX-1, is an inflammation-induced receptor implicated in atherosclerotic plaque formation in acute coronary syndrome, and here we evaluated its role in SLE-associated CVD. SLE patients have increased sLOX-1 levels which were associated with elevated proinflammatory HDL, oxLDL and hsCRP. Interestingly, increased sLOX-1 levels were associated with patients with early disease onset, low disease activity, increased IL-8, and normal complement and hematological measures. LOX-1 was increased on patient-derived monocytes and low-density granulocytes, and activation with oxLDL and immune-complexes increased membrane LOX-1, TACE activity, sLOX-1 release, proinflammatory cytokine production by monocytes, and triggered the formation of neutrophil extracellular traps which can promote vascular injury. In conclusion, perturbations in the lipid content in SLE patients' blood activate LOX-1 and promote inflammatory responses. Increased sLOX-1 levels may be an indicator of high CVD risk, and blockade of LOX-1 may provide a therapeutic opportunity for ameliorating atherosclerosis in SLE patients.


Assuntos
Doenças Cardiovasculares/etiologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/complicações , Receptores Depuradores Classe E/fisiologia , Adulto , Aterosclerose/sangue , Aterosclerose/complicações , Doenças Cardiovasculares/sangue , Estudos de Casos e Controles , Progressão da Doença , Feminino , Humanos , Inflamação/sangue , Inflamação/complicações , Lúpus Eritematoso Sistêmico/patologia , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Receptores Depuradores Classe E/sangue , Adulto Jovem
15.
Sci Adv ; 6(26): eaba4353, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32637608

RESUMO

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here, we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1&2), a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS, for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1&2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1&2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore, PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway, and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1&2 reversed arthritis in mice, providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies.


Assuntos
Antirreumáticos , Artrite Reumatoide , Sinoviócitos , Animais , Antirreumáticos/uso terapêutico , Células Cultivadas , Fibroblastos/metabolismo , Camundongos , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Fator de Necrose Tumoral alfa/metabolismo
16.
Front Immunol ; 9: 34, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29403504

RESUMO

Protein citrullination catalyzed by peptidyl arginine deiminase (PADs) is involved in autoimmune disease pathogenesis, especially in rheumatoid arthritis. Calcium is a key regulator of PAD activity, but under normal physiological conditions it remains uncertain how intracellular calcium levels can be raised to sufficiently high levels to activate these enzymes. In pursuit of trying to identify other factors that influence PAD activity, we identified bicarbonate as a potential regulator of PAD activity. We demonstrate that physiological levels of bicarbonate upregulate citrullination by recombinant PAD2/4 and endogenous PADs in neutrophils. The impact of bicarbonate is independent of calcium and pH. Adding bicarbonate to commercial PAD activity kits could increase assay performance and biological relevance. These results suggest that citrullination activity is regulated by multiple factors including calcium and bicarbonate. We also provide commentary on the current understanding of PAD regulation and future perspective of research in this area.


Assuntos
Artrite Reumatoide/patologia , Bicarbonatos/metabolismo , Cálcio/metabolismo , Citrulinação/fisiologia , Desiminases de Arginina em Proteínas/metabolismo , Artrite Reumatoide/enzimologia , Citrulina/metabolismo , Humanos , Neutrófilos/enzimologia , Proteína-Arginina Desiminase do Tipo 2 , Proteína-Arginina Desiminase do Tipo 4
17.
Sci Rep ; 8(1): 15228, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30323221

RESUMO

Neutrophils are critical for the defense against pathogens, in part through the extrusion of extracellular DNA traps, phagocytosis, and the production of reactive oxygen species. Neutrophils may also play an important role in the pathogenesis of rheumatoid arthritis (RA) through the activation of protein arginine deiminases (PADs) that citrullinate proteins that subsequently act as autoantigens. We report that PAD4 is physically associated with the cytosolic subunits of the oxidative burst machinery, p47phox (also known as neutrophil cytosol factor 1, NCF1) and p67phox (NCF2). Activation of PAD4 by membranolytic insults that result in high levels of intracellular calcium (higher than physiological neutrophil activation) leads to rapid citrullination of p47phox/NCF1 and p67phox/NCF2, as well as their dissociation from PAD4. This dissociation prevents the assembly of an active NADPH oxidase complex and an oxidative burst in neutrophils stimulated by phorbol-ester or immune complexes. In further support of a substrate-to-inactive enzyme interaction, small-molecule PAD inhibitors also disrupt the PAD4-NCF complex and reduce oxidase activation and phagocytic killing of Staphylococcus aureus. This novel role of PAD4 in the regulation of neutrophil physiology suggests that targeting PAD4 with active site inhibitors for the treatment of RA may have a broader impact on neutrophil biology than just inhibition of citrullination.


Assuntos
Artrite Reumatoide/genética , NADPH Oxidases/genética , Desiminases de Arginina em Proteínas/genética , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Membrana Celular/genética , Citrulinação/genética , Citosol/metabolismo , Humanos , Neutrófilos/enzimologia , Neutrófilos/patologia , Fagócitos/metabolismo , Fagocitose/genética , Proteína-Arginina Desiminase do Tipo 4 , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade
18.
Lupus Sci Med ; 5(1): e000261, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29644082

RESUMO

OBJECTIVE: We investigated the mechanistic and pharmacological properties of anifrolumab, a fully human, effector-null, anti-type I interferon (IFN) alpha receptor 1 (IFNAR1) monoclonal antibody in development for SLE. METHODS: IFNAR1 surface expression and internalisation on human monocytes before and after exposure to anifrolumab were assessed using confocal microscopy and flow cytometry. The effects of anifrolumab on type I IFN pathway activation were assessed using signal transducer and activator of transcription 1 (STAT1) phosphorylation, IFN-stimulated response element-luciferase reporter cell assays and type I IFN gene signature induction. The ability of anifrolumab to inhibit plasmacytoid dendritic cell (pDC) function and plasma cell differentiation was assessed by flow cytometry and ELISA. Effector-null properties of anifrolumab were assessed in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays with B cells. RESULTS: Anifrolumab reduced cell surface IFNAR1 by eliciting IFNAR1 internalisation. Anifrolumab blocked type I IFN-dependent STAT1 phosphorylation and IFN-dependent signalling induced by recombinant and pDC-derived type I IFNs and serum of patients with SLE. Anifrolumab suppressed type I IFN production by blocking the type I IFN autoamplification loop and inhibited proinflammatory cytokine induction and the upregulation of costimulatory molecules on stimulated pDCs. Blockade of IFNAR1 suppressed plasma cell differentiation in pDC/B cell co-cultures. Anifrolumab did not exhibit CDC or ADCC activity. CONCLUSIONS: Anifrolumab potently inhibits type I IFN-dependent signalling, including the type I IFN autoamplification loop, and is a promising therapeutic for patients with SLE and other diseases that exhibit chronic dysfunctional type I IFN signalling.

19.
Front Immunol ; 8: 1200, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28993780

RESUMO

Autoantibodies directed against citrullinated epitopes of proteins are highly diagnostic of rheumatoid arthritis (RA), and elevated levels of protein citrullination can be found in the joints of patients with RA. Calcium-dependent peptidyl-arginine deiminases (PAD) are the enzymes responsible for citrullination. PAD2 and PAD4 are enriched in neutrophils and likely drive citrullination under inflammatory conditions. PADs may be released during NETosis or cell death, but the mechanisms responsible for PAD activity under physiological conditions have not been fully elucidated. To understand how PADs citrullinate extracellular proteins, we investigated the cellular localization and activity of PAD2 and PAD4, and we report that viable neutrophils from healthy donors have active PAD4 exposed on their surface and spontaneously secrete PAD2. Neutrophil activation by some stimulatory agents increased the levels of immunoreactive PAD4 on the cell surface, and some stimuli reduced PAD2 secretion. Our data indicate that live neutrophils have the inherent capacity to express active extracellular PADs. These novel pathways are distinguished from intracellular PAD activation during NETosis and calcium influx-mediated hypercitrullination. Our study implies that extracellular PADs may have a physiological role under non-pathogenic conditions as well as a pathological role in RA.

20.
PLoS One ; 10(2): e0115828, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25706559

RESUMO

Release of endogenous damage associated molecular patterns (DAMPs), including members of the S100 family, are associated with infection, cellular stress, tissue damage and cancer. The extracellular functions of this family of calcium binding proteins, particularly S100A8, S100A9 and S100A12, are being delineated. They appear to mediate their functions via receptor for advanced glycation endproducts (RAGE) or TLR4, but there remains considerable uncertainty over the relative physiological roles of these DAMPs and their pattern recognition receptors. In this study, we surveyed the capacity of S100 proteins to induce proinflammatory cytokines and cell migration, and the contribution RAGE and TLR4 to mediate these responses in vitro. Using adenoviral delivery of murine S100A9, we also examined the potential for S100A9 homodimers to trigger lung inflammation in vivo. S100A8, S100A9 and S100A12, but not the S100A8/A9 heterodimer, induced modest levels of TLR4-mediated cytokine production from human PBMC. In contrast, for most S100s including S100A9, RAGE blockade inhibited S100-mediated cell migration of THP1 cells and major leukocyte populations, whereas TLR4-blockade had no effect. Intranasal administration of murine S100A9 adenovirus induced a specific, time-dependent predominately macrophage infiltration that coincided with elevated S100A9 levels and proinflammatory cytokines in the BAL fluid. Inflammatory cytokines were markedly ablated in the TLR4-defective mice, but unexpectedly the loss of TLR4 signaling or RAGE-deficiency did not appreciably impact the S100A9-mediated lung pathology or the inflammatory cell infiltrate in the alveolar space. These data demonstrate that physiological levels of S100A9 homodimers can trigger an inflammatory response in vivo, and despite the capacity of RAGE and TLR4 blockade to inhibit responses in vitro, the response is predominately independent of both these receptors.


Assuntos
Calgranulina B/farmacologia , Movimento Celular/fisiologia , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Knockout , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptor 4 Toll-Like/metabolismo
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