RESUMO
The mechanisms driving idiopathic pulmonary fibrosis (IPF) remain undefined, however it is postulated that coagulation imbalances may play a role. The impact of blood-derived clotting factors, including factor XII (FXII) has not been investigated in the context of IPF. Plasma levels of FXII were measured by ELISA in patients with IPF and in age-matched healthy donors. Expression of FXII in human lung tissue was quantified using multiplex immunohistochemistry and Western blotting. Mechanistic investigation of FXII activity was assessed in vitro on primary lung fibroblasts using qPCR and specific receptor/FXII inhibition. The functional outcome of FXII on fibroblast migration was examined by high-content image analysis. Compared with 35 healthy donors, plasma levels of FXII were not higher in patients with IPF (n = 27, P > 0.05). Tissue FXII was elevated in IPF (n = 11) and increased numbers of FXII+ cells were found in IPF (n = 8) lung tissue compared with nondiseased controls (n = 6, P < 0.0001). Activated FXII induced IL6 mRNA and IL-6 protein in fibroblasts that was blocked by anti-FXII antibody, CSL312. FXII induced IL-6 production via PAR-1 and NF-κB. FXII induced migration of fibroblasts in a concentration-dependent manner. FXII is normally confined to the circulation but it leaks from damaged vessels into the lung interstitium in IPF where it 1) induces IL-6 production and 2) enhances migration of resident fibroblasts, critical events that drive chronic inflammation and therefore, contribute to fibrotic disease progression. Targeting FXII-induced fibroblastic processes in IPF may ameliorate pulmonary fibrosis.
Assuntos
Fibrose Pulmonar Idiopática , Fator XII/metabolismo , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Interleucina-6/metabolismo , Pulmão/metabolismoRESUMO
During haematopoiesis, haematopoietic stem cells differentiate into restricted potential progenitors before maturing into the many lineages required for oxygen transport, wound healing and immune response. We have updated Haemopedia, a database of gene-expression profiles from a broad spectrum of haematopoietic cells, to include RNA-seq gene-expression data from both mice and humans. The Haemopedia RNA-seq data set covers a wide range of lineages and progenitors, with 57 mouse blood cell types (flow sorted populations from healthy mice) and 12 human blood cell types. This data set has been made accessible for exploration and analysis, to researchers and clinicians with limited bioinformatics experience, on our online portal Haemosphere: https://www.haemosphere.org. Haemosphere also includes nine other publicly available high-quality data sets relevant to haematopoiesis. We have added the ability to compare gene expression across data sets and species by curating data sets with shared lineage designations or to view expression gene vs gene, with all plots available for download by the user.
Assuntos
Bases de Dados Genéticas , Expressão Gênica/genética , Hematopoese/genética , Transcriptoma/genética , Animais , Biologia Computacional , Células-Tronco Hematopoéticas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/tendências , Humanos , Camundongos , RNA-Seq , SoftwareRESUMO
MDX-1097 is an antibody specific for a unique B cell antigen called kappa myeloma antigen (KMA) that consists of cell membrane-associated free kappa light chain (κFLC). KMA was detected on kappa human multiple myeloma cell lines (κHMCLs), on plasma cells (PCs) from kappa multiple myeloma (κMM) patients and on κPC dyscrasia tissue cryosections. In primary κMM samples, KMA was present on CD38+ cells that were CD138 and CD45 positive and/or negative. MDX-1097 exhibited a higher affinity for KMA compared to κFLC and the latter did not abrogate binding to KMA. MDX-1097-mediated antibody-dependent cellular cytotoxicity (ADCC) and in vitro exposure of target cells to the immunomodulatory drug lenalidomide resulted in increased KMA expression and ADCC. Also, in vitro exposure of peripheral blood mononuclear cells (PBMCs) to lenalidomide enhanced MDX-1097-mediated ADCC. PBMCs obtained from myeloma patients after lenalidomide therapy elicited significantly higher levels of MDX-1097-mediated ADCC than cells obtained prior to lenalidomide treatment. These data establish KMA as a relevant cell surface antigen on MM cells that can be targeted by MDX-1097. The ADCC-inducing capacity of MDX-1097 and its potentiation by lenalidomide provide a powerful rationale for clinical evaluation of MDX-1097 alone and in combination with lenalidomide.
Assuntos
Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos/farmacologia , Cadeias kappa de Imunoglobulina/metabolismo , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Talidomida/análogos & derivados , Anticorpos Monoclonais/metabolismo , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Antineoplásicos/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Imunofenotipagem , Lenalidomida , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Mieloma Múltiplo/diagnóstico , Fenótipo , Plasmócitos/metabolismo , Ligação Proteica , Talidomida/farmacologiaRESUMO
Introduction: Idiopathic pulmonary fibrosis (IPF) is a progressively fibrotic lung condition with poor prognosis. Matrix metalloproteinase-7 (MMP7) is a protein secreted by epithelial cells in IPF lungs. It is not known if MMP7 expression correlates with fibrotic changes in lung tissue. Methods: Tissue samples from lung apices and bases were obtained from 20 IPF patients and 14 non-diseased control (NDC) donors. In formalin-fixed paraffin-embedded sections, histological assessment of fibrosis was performed; overall MMP7 positivity was assessed by immunohistochemistry and MMP7+ cells were quantified using multiplex immunohistochemistry. Protein expression of MMP7 in whole lung lysates was quantified by Western blotting. Bulk tissue transcriptomic profiles of 101 samples were analysed using RNA sequencing technologies. Results: Lung tissue from IPF bases was more fibrotic than in apices. MMP7 protein is elevated in IPF lung base tissue. In IPF whole lung lysates, MMP7 protein levels are increased compared to NDC donors and was increased in IPF lung bases compared to apices. MMP7 protein levels correlated with MMP7 gene expression levels in lung tissue. MMP7 transcript levels were increased in IPF base compared to NDC base lung tissue and increased in IPF base tissue compared to IPF apex tissue. Conclusions: Our cross-sectional study suggests that lung epithelial MMP7 expression increases as the tissue becomes more fibrotic and identifies a potentially nonepithelial or immune-cell source. Mechanisms of disease progression in IPF are still unclear, and our study suggests aberrant MMP7 production may be a histological starting point of lung tissue fibrosis.
RESUMO
Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31) is an immunoglobulin (Ig)-immunoreceptor tyrosine based inhibitory motif (Ig-ITIM) superfamily member that recruits and activates protein-tyrosine phosphatases, predominantly SHP-2 and to a lesser extent, SHP-1. Previously, we have shown that deletion of PECAM-1 results in a hyper-proliferative B-cell phenotype. We wanted to test whether the Ig-ITIM superfamily member, PECAM-1 maintains peripheral tolerance by regulating signalling thresholds of B-cells that control autoantibody production or relaxed negative selection of autoreactive B-cells in bone marrow. In order to address this issue, we utilised the classical model of lysozyme/immunoglobulin transgenic mouse model that defines thresholds for eliminating or inactivating self-reactive B-cells. In this study, we show that breeding of double transgenes: soluble hen egg lysozyme (HEL) and its corresponding high-affinity receptor (HEL-Ig) onto PECAM-1 null background resulted in a spontaneous loss of B-cell tolerance in vivo. The resultant PECAM-1(-/-) Dbl Tg mice displayed elevated levels of anti-HEL immunoglobulin M (IgM) antibodies in the serum compared to PECAM-1+/+ anergic counterparts. Dbl Tg B-cells lacking PECAM-1 showed enhanced B-cell proliferation and calcium flux responses to LPS, IL-4 alone, IgM cross-linking and IL-4 indicating augmentation of antigen-receptor signalling. Thus, PECAM-1 is important in maintaining peripheral tolerance in Dbl Tg B-cells.
Assuntos
Linfócitos B/imunologia , Tolerância Imunológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Animais , Animais Geneticamente Modificados , Autoanticorpos/imunologia , Linfócitos B/efeitos dos fármacos , Medula Óssea/imunologia , Cálcio/imunologia , Proliferação de Células/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Imunoglobulina M/farmacologia , Interleucina-4/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Muramidase/imunologia , Muramidase/farmacologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Transdução de SinaisRESUMO
Homophilic engagement of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) induces 'outside-in' signal transduction that results in phosphorylation events and recruitment and activation of signalling molecules. The formation of signalling scaffolds with PECAM-1 are important signalling events that modulate platelet secretion, aggregation and platelet thrombus formation. In this study, we describe a novel interaction between PECAM-1 and cytosolic calmodulin (CaM) in platelets. Reciprocal co-immunoprecipitation studies revealed that cytosolic CaM is constitutively associated with PECAM-1 in resting, thrombin activated and aggregated human platelets. Our studies demonstrate that CaM directly interacts with a PECAM-1 peptide (594-604) C595A containing the sequences (594)KAFYLRKAKAK(604). This CaM:PECAM-1 interaction has a threefold higher affinity than CaM:GPVI interaction. It is potentiated by the addition of calcium ions, and dissociated by the CaM inhibitor, trifluoperazine. Treatment of platelets with CaM inhibitors triggers cleavage of PECAM-1 in a time- and dose-dependent manner. Furthermore, this membrane proximal portion of PECAM-1 is conserved across mammalian species and the helical representation of basic/hydrophobic residues reveals a charge distribution analogous to other CaM-binding motifs in other proteins. Taken together, these results suggest that this highly charged cluster of amino acids in the PECAM-1 cytoplasmic domain directly interacts with CaM and this novel interaction appears to regulate cleavage of PECAM-1.
Assuntos
Plaquetas/metabolismo , Calmodulina/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Calmodulina/antagonistas & inibidores , Ensaio de Imunoadsorção Enzimática , Humanos , Hidrólise , Dados de Sequência Molecular , Molécula-1 de Adesão Celular Endotelial a Plaquetas/química , Homologia de Sequência de AminoácidosRESUMO
Regulation of B-cell development and activation is imperative to the myriad of activities that perpetuate humoral immunity. This T-cell dependent immune mechanism often relies upon the maintenance of T-cell tolerance, such that the maturity of the antigen-presentating cell, its function and molecular mimicry are contributing factors. Recent findings have implicated the involvement of the B-cell and their corresponding surface co-receptors in regulating autoimmune disease. One candidate receptor, PECAM-1, has demonstrated the ability to downregulate both B and T-cell signalling pathways. The deletion of PECAM-1 in mice has led to a hyper-responsive B-cell phenotype with abnormal B-cell development. Additionally, in vivo functional studies have found that absence of PECAM-1 results in an increased susceptibility to autoimmune disorders of encephalomyelitis and Type I hypersensitivity reactions. Taken together, these findings indicate that PECAM-1 may have an important role in maintaining B-cell tolerance and regulatory function in preventing the onset of autoimmune disease. Elucidating the mechanisms of PECAM-1 function in autoimmune disorders could facilitate development of novel therapeutics.
Assuntos
Doenças Autoimunes/imunologia , Linfócitos B/imunologia , Ativação Linfocitária , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Animais , HumanosRESUMO
Studies of PECAM-1(-/-) mice have identified that PECAM-1 functions as an inhibitory co-receptor to modulate immunological responsiveness. In this study, we describe the in vivo consequences of PECAM-1 deficiency in mouse models of collagen-induced arthritis (CIA) and K/BxN passive transfer model that resembles many of the features of human rheumatoid arthritis. Immunization of PECAM-1(-/-) C57BL/6 (H-2b) mice with chicken collagen type II induced CIA with an incidence of 82% by day 49, while 33%; of wild-type and 100% of DBA/1 mice developed arthritis in a similar time frame. The mean onset of disease for PECAM-1(-/-) C57BL/6 mice was day 32 compared to day 51 for wild-type C57BL/6 mice and day 18 for DBA/1 mice (H-2q susceptible). In terms of disease severity, the mean maximal arthritic index for PECAM-1(-/-) C57BL/6 mice was comparable to DBA/1 mice (8.91 +/- 0.91 vs 11.67 +/- 0.82). This mean maximal index in PECAM-1(-/-) C57BL/6 mice was significantly higher than wild-type C57BL/6 mice (5.00 +/- 0.73). IgG1 and IgG2b antibody responses against CII were elevated in arthritic PECAM-1(-/-) C57BL/6 mice compared to wild-type C57BL/6 mice. Histological examination of arthritic paws of PECAM-1(-/-) C57BL/6 mice revealed inflammatory infiltrates of lymphocytic/monocytic cells and cartilage/bone destruction similar to CIA-induced DBA/1 arthritic paws. In the K/BxN model, the arthritis was not augmented in PECAM-1(-/-) mice compared to wild-type mice. In contrast, in active CIA, PECAM-1(-/-) mice developed severe disease comparable to susceptible DBA/1 mice and profoundly more severe than C57BL/6 mice, where only one third developed a mild/moderate disease. Together these observations suggest that PECAM-1 plays a crucial role in the suppression of development of autoimmune arthritis.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Transferência Adotiva , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/patologia , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Cartilagem/patologia , Colágeno Tipo II/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genéticaRESUMO
Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a newly assigned member of the Ig-immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. In this study, we hypothesized that PECAM-1 plays an essential in vivo role as a counterregulator of immediate hypersensitivity reactions. We found that PECAM-1 was highly expressed on the surface of immature bone marrow mast cells and at a lower density on mature peritoneal mast cells. Examination of skin biopsies from PECAM-1(+/+) and PECAM-1(-/-) mice revealed that absence of PECAM-1 did not affect mast cell development or the capacity of mast cells to populate tissues. To examine whether the absence of PECAM-1 would influence immediate hypersensitivity reactions, PECAM-1(+/+) and PECAM-1(-/-) mice were presensitized with anti-DNP mouse IgE and then challenged 20 h later with DNP-BSA or PBS. PECAM-1(-/-) mice exhibited elevated serum histamine concentrations after Ag stimulation compared with PECAM-1(+/+) mice, indicating an increased severity of systemic IgE-mediated anaphylaxis. PECAM-1(-/-) mice have increased sensitivity to local cutaneous IgE-dependent anaphylaxis compared with PECAM-1(+/+) mice, as assessed by greater tissue swelling of their ears and mast cell degranulation in situ. PECAM-1(-/-) bone marrow mast cells showed enhanced dense granule serotonin release after Fc epsilon RI cross-linking in vitro. These results suggest that PECAM-1 acts as a counterregulator in allergic disease susceptibility and severity and negatively modulates mast cell activation.
Assuntos
Anafilaxia/genética , Anafilaxia/imunologia , Imunoglobulina E/fisiologia , Mastócitos/imunologia , Mastócitos/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Anafilaxia/patologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Degranulação Celular/genética , Degranulação Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Suscetibilidade a Doenças , Edema/genética , Edema/imunologia , Edema/patologia , Imunoglobulina E/metabolismo , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Anafilaxia Cutânea Passiva/genética , Anafilaxia Cutânea Passiva/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Receptores de IgE/imunologia , Receptores de IgE/metabolismo , Índice de Gravidade de Doença , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is an immunoglobulin-immunoreceptor tyrosine-based inhibitory motif (Ig-ITIM) superfamily member that recruits and activates protein-tyrosine phosphatases, SHP-1 and SHP-2, through its intrinsic ITIMs. PECAM-1-deficient (PECAM-1(-/-) ) mice exhibit a hyperresponsive B-cell phenotype, increased numbers of B-1 cells, reduced B-2 cells, and develop autoantibodies. In the periphery, there are reduced mature recirculating B-2 cells and increased B-1a cells within the peritoneal cavity. In addition, PECAM-1(-/-) B cells display hyperproliferative responses to lipopolysaccharide and anti-IgM stimulation and showed enhanced kinetics in their intracellular Ca(++) response following IgM cross-linking. PECAM-1(-/-) mice showed increased serum levels of IgM with elevated IgG isotypes and IgA antidinitrophenol antibody in response to the T-independent antigen, dinitrophenol-Ficoll. Finally, PECAM-1(-/-) mice developed antinuclear antibodies and lupuslike autoimmune disease with age.