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1.
Front Immunol ; 14: 1258291, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37920465

RESUMO

Introduction: Immuno-oncology (IO) research relies heavily on murine syngeneic tumor models. However, whilst the average age for a cancer diagnosis is 60 years or older, for practical purposes the majority of preclinical studies are conducted in young mice, despite the fact that ageing has been shown to have a significant impact on the immune response. Methods: Using aged (60-72 weeks old) mice bearing CT26 tumors, we investigated the impact of ageing on tumor growth as well as the immune composition of the tumor and peripheral lymphoid organs. Results: We found many differences in the immune cell composition of both the tumor and tumor-draining lymph node between aged and young mice, such as a reduction in the naïve T cell population and a decreased intratumoral CD8/Treg ratio in aged animals. We hypothesized that these differences may contribute to impaired anti-cancer immune responses in aged mice and therefore assessed the anti-tumor efficacy of different IO therapies in aged mice, including both co-stimulation (using an anti-OX40 antibody) and immune checkpoint blockade (using anti-PD-L1 and anti-CTLA-4 antibodies). Whilst aged mice retained the capacity to generate anti-tumor immune responses, these were significantly attenuated when compared to the responses observed in young mice. Discussion: These differences highlight the importance of age-related immunological changes in assessing and refining the translational insights gained from preclinical mouse models.


Assuntos
Neoplasias , Camundongos , Animais , Imunoterapia
2.
Trends Immunol ; 44(1): 44-59, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36464584

RESUMO

The human microbiome is recognized as a key factor in health and disease. This has been further corroborated by identifying changes in microbiome composition and function as a novel hallmark in cancer. These effects are exerted through microbiome interactions with host cells, impacting a wide variety of developmental and physiological processes. In this review, we discuss some of the latest findings on how the bacterial component of the microbiome can influence outcomes for different cancer immunotherapy modalities, highlighting identified mechanisms of action. We also address the clinical efforts to utilize this knowledge to achieve better responses to immunotherapy. A refined understanding of microbiome variations in patients and microbiome-host interactions with cancer therapies is essential to realize optimal clinical responses.


Assuntos
Microbiota , Neoplasias , Humanos , Neoplasias/terapia , Neoplasias/microbiologia , Imunoterapia , Bactérias
3.
SLAS Discov ; 27(2): 95-106, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35058180

RESUMO

The field of Immuno-Oncology (IO) is evolving to utilise novel antibody backbones that can co-target multiple cell-surface stimulatory and inhibitory co-receptors (SICR). This approach necessitates a better understanding of SICR co-expression at the single-cell level on IO-relevant tumor-infiltrating leukocyte (TIL) cell types such as T and natural killer (NK) cells. Using high-dimensional flow cytometry we established a comprehensive SICR profile for tumor-resident T and NK cells across a range of human solid tumors where there is a clear need for improved immunotherapeutic intervention. Leveraging the power of our large flow panel, we performed deep-phenotyping of the critical CD8+CD39+ Cytotoxic T Lymphocyte (CTL) population that is enriched for tumor-reactive cytotoxic cells, revealing subsets that are differentiated by their SICR profile, including three that are uniquely defined by NKG2A expression. This study establishes a comprehensive SICR phenotype for human TIL T and NK cells, providing insights to guide the design and application of the next generation of IO molecules.


Assuntos
Neoplasias , Linfócitos T Citotóxicos , Citometria de Fluxo , Humanos , Células Matadoras Naturais , Neoplasias/genética
4.
BMC Cancer ; 22(1): 99, 2022 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-35073853

RESUMO

BACKGROUND: The gut microbiome is implicated as a marker of response to immune checkpoint inhibitors (ICI) based on preclinical mouse models and preliminary observations in limited patient series. Furthermore, early studies suggest faecal microbial transfer may have therapeutic potential, converting ICI non-responders into responders. So far, identification of specific responsible bacterial taxa has been inconsistent, which limits future application. The MITRE study will explore and validate a microbiome signature in a larger scale prospective study across several different cancer types. METHODS: Melanoma, renal cancer and non-small cell lung cancer patients who are planned to receive standard immune checkpoint inhibitors are being recruited to the MITRE study. Longitudinal stool samples are collected prior to treatment, then at 6 weeks, 3, 6 and 12 months during treatment, or at disease progression/recurrence (whichever is sooner), as well as after a severe (≥grade 3 CTCAE v5.0) immune-related adverse event. Additionally, whole blood, plasma, buffy coat, RNA and peripheral blood mononuclear cells (PBMCs) is collected at similar time points and will be used for exploratory analyses. Archival tumour tissue, tumour biopsies at progression/relapse, as well as any biopsies from body organs collected after a severe toxicity are collected. The primary outcome measure is the ability of the microbiome signature to predict 1 year progression-free survival (PFS) in patients with advanced disease. Secondary outcomes include microbiome correlations with toxicity and other efficacy end-points. Biosamples will be used to explore immunological and genomic correlates. A sub-study will evaluate both COVID-19 antigen and antibody associations with the microbiome. DISCUSSION: There is an urgent need to identify biomarkers that are predictive of treatment response, resistance and toxicity to immunotherapy. The data generated from this study will both help inform patient selection for these drugs and provide information that may allow therapeutic manipulation of the microbiome to improve future patient outcomes. TRIAL REGISTRATION: NCT04107168 , ClinicalTrials.gov, registered 09/27/2019. Protocol V3.2 (16/04/2021).


Assuntos
Microbioma Gastrointestinal , Inibidores de Checkpoint Imunológico/uso terapêutico , Consórcios Microbianos , Neoplasias/terapia , Anticorpos Antivirais/análise , Antígenos Virais/análise , Carcinoma Pulmonar de Células não Pequenas/terapia , Progressão da Doença , Fezes/microbiologia , Microbioma Gastrointestinal/imunologia , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Neoplasias Renais/terapia , Neoplasias Pulmonares/terapia , Melanoma/terapia , Consórcios Microbianos/imunologia , Intervalo Livre de Progressão , Estudos Prospectivos , SARS-CoV-2/imunologia , Neoplasias Cutâneas/terapia
5.
Clin Sci (Lond) ; 135(22): 2559-2573, 2021 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-34778899

RESUMO

Granulocyte macrophage colony stimulating factor (GM-CSF) is a key participant in, and a clinical target for, the treatment of inflammatory diseases including rheumatoid arthritis (RA). Therapeutic inhibition of GM-CSF signalling using monoclonal antibodies to the α-subunit of the GM-CSF receptor (GMCSFRα) has shown clear benefit in patients with RA, giant cell arteritis (GCAs) and some efficacy in severe SARS-CoV-2 infection. However, GM-CSF autoantibodies are associated with the development of pulmonary alveolar proteinosis (PAP), a rare lung disease characterised by alveolar macrophage (AM) dysfunction and the accumulation of surfactant lipids. We assessed how the anti-GMCSFRα approach might impact surfactant turnover in the airway. Female C57BL/6J mice received a mouse-GMCSFRα blocking antibody (CAM-3003) twice per week for up to 24 weeks. A parallel, comparator cohort of the mouse PAP model, GM-CSF receptor ß subunit (GMCSFRß) knock-out (KO), was maintained up to 16 weeks. We assessed lung tissue histopathology alongside lung phosphatidylcholine (PC) metabolism using stable isotope lipidomics. GMCSFRß KO mice reproduced the histopathological and biochemical features of PAP, accumulating surfactant PC in both broncho-alveolar lavage fluid (BALF) and lavaged lung tissue. The incorporation pattern of methyl-D9-choline showed impaired catabolism and not enhanced synthesis. In contrast, chronic supra-pharmacological CAM-3003 exposure (100 mg/kg) over 24 weeks did not elicit a histopathological PAP phenotype despite some changes in lung PC catabolism. Lack of significant impairment of AM catabolic function supports clinical observations that therapeutic antibodies to this pathway have not been associated with PAP in clinical trials.


Assuntos
Artrite Reumatoide/metabolismo , COVID-19/terapia , Proteinose Alveolar Pulmonar/imunologia , Surfactantes Pulmonares/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Artrite Reumatoide/terapia , Autoanticorpos/química , Líquido da Lavagem Broncoalveolar , COVID-19/imunologia , Colina/análogos & derivados , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/química , Inflamação , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteinose Alveolar Pulmonar/genética , SARS-CoV-2/imunologia , Tensoativos
6.
J Immunother Cancer ; 9(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34145033

RESUMO

BACKGROUND: Immuno-oncology therapies are now part of the standard of care for cancer in many indications. However, durable objective responses remain limited to a subset of patients. As such, there is a critical need to identify biomarkers that can predict or enrich for treatment response. So far, the majority of putative biomarkers consist of features of the tumor microenvironment (TME). However, in preclinical mouse models, the collection of tumor tissue for this type of analysis is a terminal procedure, obviating the ability to directly link potential biomarkers to long-term treatment outcomes. METHODS: To address this, we developed and validated a novel non-terminal tumor sampling method to enable biopsy of the TME in mouse models based on fine needle aspiration. RESULTS: We show that this technique enables repeated in-life sampling of subcutaneous flank tumors and yields sufficient material to support downstream analyses of tumor-infiltrating immune cells using methods such as flow cytometry and single-cell transcriptomics. Moreover, using this technique we demonstrate that we can link TME biomarkers to treatment response outcomes, which is not possible using the current method of terminal tumor sampling. CONCLUSION: Thus, this minimally invasive technique is an important refinement for the pharmacodynamic analysis of the TME facilitating paired evaluation of treatment response biomarkers with outcomes and reducing the number of animals used in preclinical research.


Assuntos
Biomarcadores Tumorais/metabolismo , Biópsia por Agulha Fina/métodos , Imunoterapia/métodos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos
7.
Eur Respir J ; 54(4)2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31320451

RESUMO

Increased reactive oxygen species (ROS) have been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). This study examined the effect of exogenous and endogenous oxidative stress on macrophage phagocytosis in patients with COPD.Monocyte-derived macrophages (MDMs) were generated from non-smoker, smoker and COPD subjects, differentiated in either granulocyte macrophage-colony stimulating factor (G-Mφ) or macrophage-colony stimulating factor (M-Mφ). Alveolar macrophages were isolated from lung tissue or bronchoalveolar lavage fluid. Macrophages were incubated in ±200 µM H2O2 for 24 h, then exposed to fluorescently labelled Haemophilus influenzae or Streptococcus pneumoniae for 4 h, after which phagocytosis, mitochondrial ROS (mROS) and mitochondrial membrane potential (ΔΨm) were measured.Phagocytosis of bacteria was significantly decreased in both G-Mφ and M-Mφ from COPD patients compared with from non-smoker controls. In non-smokers and smokers, bacterial phagocytosis did not alter mROS or ΔΨm; however, in COPD, phagocytosis increased early mROS and decreased ΔΨm in both G-Mφ and M-Mφ. Exogenous oxidative stress reduced phagocytosis in non-smoker and COPD alveolar macrophages and non-smoker MDMs, associated with reduced mROS production.COPD macrophages show defective phagocytosis, which is associated with altered mitochondrial function and an inability to regulate mROS production. Targeting mitochondrial dysfunction may restore the phagocytic defect in COPD.


Assuntos
Macrófagos Alveolares/imunologia , Mitocôndrias/metabolismo , Fagocitose/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Idoso , Bactérias , Sobrevivência Celular , Feminino , Haemophilus influenzae , Humanos , Técnicas In Vitro , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Masculino , Potencial da Membrana Mitocondrial , Microscopia Confocal , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Streptococcus pneumoniae
8.
Arthritis Res Ther ; 18(1): 287, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27908288

RESUMO

BACKGROUND: Blockade of granulocyte macrophage colony-stimulating factor (GM-CSF) and its receptor (GM-CSFRα) is being successfully tested in trials in rheumatoid arthritis (RA) with clinical results equivalent to those found with neutralization of the current therapeutic targets, TNF and IL-6. To explore further the role of GM-CSF as a pro-inflammatory cytokine, we examined the effect of anti-GM-CSFRα neutralization on myeloid cell populations in antigen-driven arthritis and inflammation models and also compared its effect with that of anti-TNF and anti-IL-6. METHODS: Cell population changes upon neutralization by monoclonal antibodies (mAbs) in the antigen-induced arthritis (AIA) and antigen-induced peritonitis (AIP) models were monitored by flow cytometry and microarray. Adoptive transfer of monocytes into the AIP cavity was used to assess the GM-CSF dependence of the development of macrophages and monocyte-derived dendritic cells (Mo-DCs) at a site of inflammation. RESULTS: Therapeutic administration of a neutralizing anti-GM-CSF mAb, but not of an anti-colony-stimulating factor (anti-CSF)-1 or an anti-CSF-1R mAb, ameliorated AIA disease. Using the anti-GM-CSFRα mAb, the relative surface expression of different inflammatory myeloid populations was found to be similar in the inflamed tissues in both the AIA and AIP models; however, the GM-CSFRα mAb, but not neutralizing anti-TNF and anti-IL-6 mAbs, preferentially depleted Mo-DCs from these sites. In addition, we were able to show that locally acting GM-CSF upregulated macrophage/Mo-DC numbers via GM-CSFR signalling in donor monocytes. CONCLUSIONS: Our findings suggest that GM-CSF blockade modulates inflammatory responses differently to TNF and IL-6 blockade and may provide additional insight into how targeting the GM-CSF/GM-CSFRα system is providing efficacy in RA.


Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Inflamação/imunologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/imunologia , Transferência Adotiva , Animais , Separação Celular , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos
9.
Immunity ; 45(4): 788-801, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27742545

RESUMO

C-type lectin receptors sense a diversity of endogenous and exogenous ligands that may trigger differential responses. Here, we have found that human and mouse Mincle bind to a ligand released by Leishmania, a eukaryote parasite that evades an effective immune response. Mincle-deficient mice had milder dermal pathology and a tenth of the parasite burden compared to wild-type mice after Leishmania major intradermal ear infection. Mincle deficiency enhanced adaptive immunity against the parasite, correlating with increased activation, migration, and priming by Mincle-deficient dendritic cells (DCs). Leishmania triggered a Mincle-dependent inhibitory axis characterized by SHP1 coupling to the FcRγ chain. Selective loss of SHP1 in CD11c+ cells phenocopies enhanced adaptive immunity to Leishmania. In conclusion, Leishmania shifts Mincle to an inhibitory ITAM (ITAMi) configuration that impairs DC activation. Thus, ITAMi can be exploited for immune evasion by a pathogen and may represent a paradigm for ITAM-coupled receptors sensing self and non-self.


Assuntos
Imunidade Adaptativa/imunologia , Células Dendríticas/imunologia , Motivo de Ativação do Imunorreceptor Baseado em Tirosina/imunologia , Lectinas Tipo C/imunologia , Leishmania major/imunologia , Proteínas de Membrana/imunologia , Transdução de Sinais/imunologia , Animais , Antígeno CD11c/imunologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Receptores Fc/imunologia
10.
Eur J Immunol ; 41(10): 3040-53, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21728173

RESUMO

Myeloid cells express a plethora of C-type lectin receptors (CLRs) that can regulate immune responses. CLEC-2 belongs to the Dectin-1 sub-family of CLRs that possess an extracellular C-type lectin-like domain and a single intracellular hemITAM motif. CLEC-2 is highly expressed on mouse and human platelets where it signals via Syk to promote aggregation. We generated a monoclonal antibody (mAb) against mouse CLEC-2 and found that CLEC-2 is additionally widely expressed on leukocytes and that its expression is upregulated during inflammation. MAb-mediated crosslinking of CLEC-2 leads to hemITAM-dependent signaling via Syk, Ca(2+) and NFAT and, in myeloid cells, modulates the effect of toll-like receptor (TLR) agonists to selectively potentiate production of IL-10. A macrophage/dendritic cell-dependent increase in IL-10 is also observed in mice given anti-CLEC-2 mAb together with LPS. Collectively, these data indicate that CLEC-2 is expressed in myeloid cells and acts as a Syk-coupled CLR able to modulate TLR signaling and inflammatory responses.


Assuntos
Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/metabolismo , Células Mieloides/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Anticorpos Monoclonais , Plaquetas , Cálcio/metabolismo , Linhagem Celular , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Dinoprostona/metabolismo , Inflamação , Mediadores da Inflamação/imunologia , Interleucina-10/biossíntese , Lectinas Tipo C/biossíntese , Lectinas Tipo C/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/imunologia , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais , Quinase Syk , Receptores Toll-Like/agonistas
11.
Eur J Immunol ; 41(2): 356-65, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21268006

RESUMO

We have examined the potential to generate bona fide macrophages (MØ) from conditionally immortalised murine bone marrow precursors. MØ can be derived from Hoxb8 conditionally immortalised macrophage precursor cell lines (MØP) using either M-CSF or GM-CSF. When differentiated in GM-CSF (GM-MØP) the resultant cells resemble GM-CSF bone marrow-derived dendritic cells (BMDC) in morphological phenotype, antigen phenotype and functional responses to microbial stimuli. In spite of this high similarity between the two cell types and the ability of GM-MØP to effectively present antigen to a T-cell hybridoma, these cells are comparatively poor at priming the expansion of IFN-γ responses from naïve CD4(+) T cells. The generation of MØP from transgenic or genetically aberrant mice provides an excellent opportunity to study the inflammatory role of GM-MØP, and reduces the need for mouse colonies in many studies. Hence differentiation of conditionally immortalised MØPs in GM-CSF represents a unique in vitro model of inflammatory monocyte-like cells, with important differences from bone marrow-derived dendritic cells, which will facilitate functional studies relating to the many 'sub-phenotypes' of inflammatory monocytes.


Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/citologia , Proteínas de Homeodomínio/genética , Macrófagos/citologia , Células Precursoras de Monócitos e Macrófagos/citologia , Animais , Apresentação de Antígeno/imunologia , Antígenos de Superfície/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Lectinas Tipo C , Lipopeptídeos/farmacologia , Lipopolissacarídeos/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Células Precursoras de Monócitos e Macrófagos/efeitos dos fármacos , Células Precursoras de Monócitos e Macrófagos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico/metabolismo , Ovalbumina/imunologia , Transdução Genética , Zimosan/farmacologia , beta-Glucanas/farmacologia
12.
Nat Immunol ; 8(6): 630-8, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17450144

RESUMO

The C-type lectin dectin-1 binds to yeast and signals through the kinase Syk and the adaptor CARD9 to induce production of interleukin 10 (IL-10) and IL-2 in dendritic cells (DCs). However, whether this pathway promotes full DC activation remains unclear. Here we show that dectin-1-Syk-CARD9 signaling induced DC maturation and the secretion of proinflammatory cytokines, including IL-6, tumor necrosis factor and IL-23, but little IL-12. Dectin-1-activated DCs 'instructed' the differentiation of CD4+ IL-17-producing effector T cells (T(H)-17 cells) in vitro, and a dectin-1 agonist acted as an adjuvant promoting the differentiation of T(H)-17 and T helper type 1 cells in vivo. Infection with Candida albicans induced CARD9-dependent T(H)-17 responses to the organism. Our data indicate that signaling through Syk and CARD9 can couple innate to adaptive immunity independently of Toll-like receptor signals and that CARD9 is required for the development of T(H)-17 responses to some pathogens.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Imunidade Inata/imunologia , Interleucina-17/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Adjuvantes Imunológicos , Animais , Formação de Anticorpos/imunologia , Proteínas Adaptadoras de Sinalização CARD , Candida albicans/imunologia , Diferenciação Celular , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Lectinas Tipo C , Proteínas de Membrana/agonistas , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Quinase Syk
13.
Eur J Immunol ; 37(6): 1600-12, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17458858

RESUMO

Zymosan is a particulate yeast preparation that elicits high levels of IL-2 and IL-10 from dendritic cells (DC) and engages multiple innate receptors, including the Syk-coupled receptor dectin-1 and the MyD88-coupled receptor TLR2. Here, we show that induction of IL-2 and IL-10 by zymosan requires activation of ERK MAP kinase in murine DC. Surprisingly, ERK activation in response to zymosan is completely blocked in Syk-deficient DC and unaffected by MyD88 deficiency. Conversely, ERK activation in response to the TLR2 agonist Pam3Cys is completely MyD88 dependent and unaffected by Syk deficiency. The inability of TLR2 ligands in zymosan to couple to ERK may explain the Syk dependence of the IL-2 and IL-10 response in DC and emphasises the importance of Syk-coupled pattern recognition receptors such as dectin-1 in the detection of yeasts. Furthermore, the lack of receptor compensation observed here suggests that responses induced by complex innate stimuli cannot always be predicted by the signalling pathways downstream of individual receptors.


Assuntos
Células Dendríticas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Tirosina Quinases/fisiologia , Zimosan/farmacologia , Animais , Butadienos/farmacologia , Linhagem Celular , Cisteína/análogos & derivados , Cisteína/farmacologia , Células Dendríticas/efeitos dos fármacos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides/farmacologia , Expressão Gênica/efeitos dos fármacos , Glucanos/farmacologia , Interleucina-10/genética , Subunidade p40 da Interleucina-12/genética , Interleucina-2/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lectinas Tipo C , Lipopeptídeos , Lipoproteínas/farmacologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Proteínas do Tecido Nervoso/genética , Nitrilas/farmacologia , Oligopeptídeos/farmacologia , Peptidoglicano/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Quinase Syk
14.
Mol Cell Biol ; 23(7): 2564-76, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12640137

RESUMO

Myeloid-related protein 14 (MRP-14) and its heterodimeric partner, MRP-8, are cytosolic calcium-binding proteins, highly expressed in neutrophils and monocytes. To understand the function of MRP-14, we performed targeted disruption of the MRP-14 gene in mice. MRP-14(-/-) mice showed no obvious phenotype and were fertile. MRP-8 mRNA but not protein is present in the myeloid cells of these mice, suggesting that the stability of MRP-8 protein is dependent on MRP-14 expression. A compensatory increase in other proteins was not detected in cells lacking MRP-8 and MRP-14. Although the morphology of MRP-14(-/-) myeloid cells was not altered, they were significantly less dense. When Ca(2+) responses were investigated, there was no change in the maximal response to the chemokine MIP-2. At lower concentrations, however, there was reduced responsiveness in MRP-14(-/-) compared with MRP-14(+/+) neutrophils. This alteration in the ability to flux Ca(2+) did not impair the ability of the MRP-14(-/-) neutrophils to respond chemotactically to MIP-2. In addition, the myeloid cell functions of phagocytosis, superoxide burst, and apoptosis were unaffected in MRP-14(-/-) cells. In an in vivo model of peritonitis, MRP-14(-/-) mice showed no difference from wild-type mice in induced inflammatory response. The data indicate that MRP-14 and MRP-8 are dispensable for many myeloid cell functions.


Assuntos
Calgranulina B/fisiologia , Células Mieloides/fisiologia , Animais , Cálcio/metabolismo , Calgranulina A/biossíntese , Calgranulina A/genética , Calgranulina B/genética , Contagem de Células , Células Cultivadas , Quimiocina CXCL2 , Quimiocinas/farmacologia , Relação Dose-Resposta a Droga , Corantes Fluorescentes , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Camundongos , Camundongos Knockout , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Peritonite/induzido quimicamente , Peritonite/genética , Peritonite/imunologia , Fenótipo , RNA Mensageiro/biossíntese
15.
J Biol Chem ; 277(5): 3658-65, 2002 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-11723110

RESUMO

The S100 family proteins MRP-8 (S100A8) and MRP-14 (S100A9) form a heterodimer that is abundantly expressed in neutrophils, monocytes, and some secretory epithelia. In inflamed tissues, the MRP-8/14 complex is deposited onto the endothelium of venules associated with extravasating leukocytes. To explore the receptor interactions of MRP-8/14, we use a model system in which the purified MRP-8/14 complex binds to the cell surface of an endothelial cell line, HMEC-1. This interaction is mediated by the MRP-14 subunit and is mirrored by recombinant MRP-14 alone. The cell surface binding of MRP-14 was blocked by heparin, heparan sulfate, and chondroitin sulfate B, and the binding sites were sensitive to heparinase I and trypsin treatment but not to chondroitinase ABC. Furthermore MRP-8/14 and MRP-14 did not bind to a glycosaminoglycan-minus cell line. MRP-14 has a high affinity for heparin (K(d) = 6.1 +/- 3.4 nm), and this interaction mimicked that with the endothelial cells. We therefore conclude that the MRP-8/14 complex binds to endothelial cells via the MRP-14 subunit interacting chiefly with heparan sulfate proteoglycans. CD36 and RAGE, two other putative receptors for MRP-8/14, were not expressed by HMEC-1 cells. This binding activity may explain the immobilization of the MRP-8/14 complex on endothelium that is observed in vivo.


Assuntos
Antígenos de Diferenciação/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Endotélio Vascular/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Proteínas S100/metabolismo , Animais , Antígenos de Diferenciação/química , Sítios de Ligação , Células CHO , Proteínas de Ligação ao Cálcio/química , Calgranulina A , Calgranulina B , Cátions Bivalentes/farmacologia , Linhagem Celular , Membrana Celular/metabolismo , Cricetinae , Sulfato de Dextrana/farmacologia , Dextranos/farmacologia , Dimerização , Humanos , Imuno-Histoquímica , Cinética , Neutrófilos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas S100/química , Cloreto de Sódio/farmacologia , Transfecção
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