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1.
Eur J Immunol ; 54(7): e2350824, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38593339

RESUMO

Antibody-based CD47 blockade aims to activate macrophage phagocytosis of tumor cells. However, macrophages possess a high degree of phenotype heterogeneity that likely influences phagocytic capacity. In murine models, proinflammatory (M1) activation increases macrophage phagocytosis of tumor cells, but in human models, results have been conflicting. Here, we investigated the effects of proinflammatory polarization on the phagocytic response of human monocyte-derived macrophages in an in vitro model. Using both flow cytometry-based and fluorescence live-cell imaging-based phagocytosis assays, we observed that mouse monoclonal anti-CD47 antibody (B6H12) induced monocyte-derived macrophage phagocytosis of cancer cells in vitro. Proinflammatory (M1) macrophage polarization with IFN-γ+LPS resulted in a severe reduction in phagocytic response to CD47 blockade. This reduction coincided with increased expression of the antiphagocytic membrane proteins LILRB1 and Siglec-10 but was not rescued by combination blockade of the corresponding ligands. However, matrix metalloproteinase inhibitors (TAPI-0 or GM6001) partly restored response to CD47 blockade in a dose-dependent manner. In summary, these data suggest that proinflammatory (M1) activation reduces phagocytic response to CD47 blockade in human monocyte-derived macrophages.


Assuntos
Antígeno CD47 , Macrófagos , Fagocitose , Humanos , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Antígeno CD47/antagonistas & inibidores , Macrófagos/imunologia , Macrófagos/metabolismo , Fagocitose/imunologia , Ativação de Macrófagos/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Inflamação/imunologia , Anticorpos Monoclonais/farmacologia , Camundongos , Animais , Linhagem Celular Tumoral , Neoplasias/imunologia , Interferon gama/metabolismo , Interferon gama/imunologia
2.
Immunology ; 170(3): 344-358, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37291897

RESUMO

Macrophages are important innate immune cells with the ability to adapt their phenotype to environmental cues. Research on human macrophages often uses monocyte-derived macrophages cultured in vitro, but it is unclear if culture medium affects macrophage phenotype. The objective of this study was to determine the impact of culture medium composition on monocyte-derived macrophage phenotype. Monocyte-derived macrophages were generated in different formulations of culture media (RPMI 1640, DMEM, MEM, McCoy's 5a and IMDM). Viability, yield and cell size were monitored, and RT-qPCR, flow cytometry or ELISA was used to compare levels of phenotype markers (CD163, CD206, CD80, TNFα, IL-10, SIRPα, LILRB1 and Siglec-10). Yield, cell size, gene expression, membrane protein levels and release of soluble proteins were all affected by changes in culture medium composition. The most pronounced effects were observed after culture in DMEM, which lacks the non-essential amino acids asparagine, aspartic acid, glutamic acid and proline. Supplementation of DMEM with non-essential amino acids either fully or partly reversed most effects of DMEM on macrophage phenotype. The results suggest culture medium composition and amino acid availability affect the phenotype of human monocyte-derived macrophages cultured in vitro.


Assuntos
Aminoácidos , Macrófagos , Humanos , Meios de Cultura/metabolismo , Fenótipo , Aminoácidos/metabolismo , Citometria de Fluxo/métodos , Monócitos
3.
FASEB J ; 34(11): 14960-14976, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32924185

RESUMO

Atherosclerosis is an inflammatory disease characterized by the accumulation of macrophages in the vessel wall. Macrophages depend on their polarization to exert either pro-inflammatory or anti-inflammatory effects. Macrophages of the anti-inflammatory phenotype express high levels of CD163, a scavenger receptor for the hemoglobin-haptoglobin complex. CD163 can also bind to the pro-inflammatory cytokine TWEAK. Using ApoE-deficient or ApoE/CD163 double-deficient mice we aim to investigate the involvement of CD163 in atherosclerosis development and its capacity to neutralize the TWEAK actions. ApoE/CD163 double-deficient mice displayed a more unstable plaque phenotype characterized by an increased lipid and macrophage content, plaque size, and pro-inflammatory cytokine expression. In vitro experiments demonstrated that the absence of CD163 in M2-type macrophages-induced foam cell formation through upregulation of CD36 expression. Moreover, exogenous TWEAK administration increased atherosclerotic lesion size, lipids, and macrophages content in ApoE-/- /CD163-/- compared with ApoE-/- /CD163+/+ mice. Treatment with recombinant CD163 was able to neutralize the proatherogenic effects of TWEAK in ApoE/CD163 double-deficient mice. Recombinant CD163 abolished the pro-inflammatory actions of TWEAK on vascular smooth muscle cells, decreasing NF-kB activation, cytokines and metalloproteinases expression, and macrophages migration. In conclusion, CD163-expressing macrophages serve as a protective mechanism to prevent the deleterious effects of TWEAK on atherosclerotic plaque development and progression.


Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação Mielomonocítica/fisiologia , Aterosclerose/patologia , Citocina TWEAK/metabolismo , Células Espumosas/patologia , Macrófagos/patologia , Placa Aterosclerótica/patologia , Receptores de Superfície Celular/fisiologia , Animais , Aterosclerose/etiologia , Aterosclerose/metabolismo , Citocinas/metabolismo , Feminino , Células Espumosas/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Knockout para ApoE , Placa Aterosclerótica/etiologia , Placa Aterosclerótica/metabolismo
4.
Int J Mol Sci ; 21(15)2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32752088

RESUMO

The macrophage is a key cell in the pro- and anti-inflammatory response including that of the inflammatory microenvironment of malignant tumors. Much current drug development in chronic inflammatory diseases and cancer therefore focuses on the macrophage as a target for immunotherapy. However, this strategy is complicated by the pleiotropic phenotype of the macrophage that is highly responsive to its microenvironment. The plasticity leads to numerous types of macrophages with rather different and, to some extent, opposing functionalities, as evident by the existence of macrophages with either stimulating or down-regulating effect on inflammation and tumor growth. The phenotypes are characterized by different surface markers and the present review describes recent progress in drug-targeting of the surface marker CD163 expressed in a subpopulation of macrophages. CD163 is an abundant endocytic receptor for multiple ligands, quantitatively important being the haptoglobin-hemoglobin complex. The microenvironment of inflammation and tumorigenesis is particular rich in CD163+ macrophages. The use of antibodies for directing anti-inflammatory (e.g., glucocorticoids) or tumoricidal (e.g., doxorubicin) drugs to CD163+ macrophages in animal models of inflammation and cancer has demonstrated a high efficacy of the conjugate drugs. This macrophage-targeting approach has a low toxicity profile that may highly improve the therapeutic window of many current drugs and drug candidates.


Assuntos
Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Inflamação/genética , Terapia de Alvo Molecular , Neoplasias/genética , Receptores de Superfície Celular/genética , Humanos , Inflamação/patologia , Inflamação/terapia , Macrófagos/metabolismo , Macrófagos/patologia , Neoplasias/patologia , Neoplasias/terapia , Microambiente Tumoral/genética
5.
Cancer Immunol Immunother ; 68(3): 489-502, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30637473

RESUMO

Tumor-associated macrophages (TAMs) are of major importance in cancer-related immune suppression, and tumor infiltration by CD163pos TAMs is associated with poor outcome in most human cancers. Therefore, therapeutic strategies for reprogramming TAMs from a tumor-supporting (M2-like) phenotype towards a tumoricidal (M1-like) phenotype are of great interest. Activation of the transcription factor STAT3 within the tumor microenvironment is associated with worse prognosis, and STAT3 activation promotes the immunosuppressive phenotype of TAMs. Therefore, we aimed to develop a drug for inhibition of STAT3 specifically within human TAMs by targeting the endocytic CD163 scavenger receptor, which is highly expressed on TAMs. Here, we report the first data on a CD163-targeted STAT3-inhibitory drug consisting of corosolic acid (CA) packaged within long-circulating liposomes (LCLs), which are CD163-targeted by modification with monoclonal anti-CD163 antibodies (αCD163)-CA-LCL-αCD163. We show, that activation of STAT3 (by phosphorylation) was inhibited by CA-LCL-αCD163 specifically within CD163pos cells, with minor effect on CD163neg cells. Furthermore, CA-LCL-αCD163 inhibited STAT3-regulated gene expression of IL-10, and increased expression of TNFα, thus indicating a pro-inflammatory effect of the drug on human macrophages. This M1-like reprogramming at the mRNA level was confirmed by significantly elevated levels of pro-inflammatory cytokines (IFNγ, IL-12, TNFα, IL-2) in the culture medium. Since liposomes are attractive vehicles for novel anti-cancer drugs, and since direct TAM-targeting may decrease adverse effects of systemic inhibition of STAT3, the present results encourage future investigation of CA-LCL-αCD163 in the in vivo setting.


Assuntos
Macrófagos/fisiologia , Monócitos/fisiologia , Receptores de Superfície Celular/antagonistas & inibidores , Fator de Transcrição STAT3/antagonistas & inibidores , Triterpenos/administração & dosagem , Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Células Cultivadas , Citocinas/biossíntese , Composição de Medicamentos , Humanos , Interleucina-10/farmacologia , Lipossomos , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Triterpenos/toxicidade
6.
J Immunol ; 198(10): 3775-3789, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28483986

RESUMO

Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a diverse variety of ligands including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the United States National Institute of Allergy and Infectious Diseases, National Institutes of Health, to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of nonself or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. This classification was discussed at three national meetings and input from participants at these meetings was requested. The following manuscript is a consensus statement that combines the recommendations of the initial workshop and incorporates the input received from the participants at the three national meetings.


Assuntos
Receptores Depuradores/classificação , Receptores Depuradores/fisiologia , Animais , Endocitose , Humanos , Ligantes , Camundongos , National Institute of Allergy and Infectious Diseases (U.S.)/normas , Fagocitose , Receptores Imunológicos/fisiologia , Receptores Depuradores Classe A/fisiologia , Transdução de Sinais , Terminologia como Assunto , Estados Unidos
7.
Nature ; 501(7467): 430-4, 2013 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-23965626

RESUMO

The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic ß-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.


Assuntos
Apolipoproteínas/sangue , Apolipoproteínas/metabolismo , Lipoproteínas HDL/sangue , Lipoproteínas HDL/metabolismo , Trypanosoma brucei gambiense/fisiologia , África , Animais , Animais Geneticamente Modificados , Apolipoproteína L1 , Apolipoproteínas/antagonistas & inibidores , Apolipoproteínas/toxicidade , Membrana Celular/química , Membrana Celular/metabolismo , Cisteína Proteases/metabolismo , Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Hemólise , Humanos , Interações Hidrofóbicas e Hidrofílicas , Metabolismo dos Lipídeos , Lipoproteínas HDL/antagonistas & inibidores , Lipoproteínas HDL/química , Lipoproteínas HDL/toxicidade , Parasitos/patogenicidade , Parasitos/fisiologia , Estrutura Secundária de Proteína , Soro/química , Soro/parasitologia , Trypanosoma brucei gambiense/efeitos dos fármacos , Trypanosoma brucei gambiense/patogenicidade , Tripanossomíase Africana/parasitologia , Glicoproteínas Variantes de Superfície de Trypanosoma/química , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo
8.
Trends Biochem Sci ; 39(2): 82-90, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24393667

RESUMO

Nutrients, biological waste-products, toxins, pathogens, and other ligands for endocytosis are typically captured by multidomain receptors with multiligand specificity. Upon internalization, the receptor-ligand complex segregates, followed by lysosomal degradation of the ligand and recycling of the receptor. Endosomal acidification and calcium efflux lead to the essential ligand-receptor affinity switch and separation. Recent data, including crystal structures of receptor-ligand complexes, now reveal how calcium, in different types of domain scaffolds, functions in a common way as a removable 'lynchpin' that stabilizes favorable positioning of ligand-attractive receptor residues. In addition to explaining how calcium depletion can cause ligand-receptor dissociation, the new data add further insight into how acidification contributes to dissociation through structural changes that affect the receptor calcium sites.


Assuntos
Cálcio/metabolismo , Endocitose/genética , Endossomos/metabolismo , Células Eucarióticas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Transporte Biológico , Sinalização do Cálcio , Células Eucarióticas/citologia , Regulação da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética
9.
J Neurosci ; 36(36): 9375-90, 2016 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-27605613

RESUMO

UNLABELLED: Increasing evidence supports a decisive role for inflammation in the neurodegenerative process of Parkinson's disease (PD). The immune response in PD seems to involve, not only microglia, but also other immune cells infiltrated into the brain. Indeed, we observed here the infiltration of macrophages, specifically CD163+ macrophages, into the area of neurodegeneration in the 6-hydroxydopamine (6-OHDA) PD model. Therefore, we investigated the therapeutic potential of the infiltrated CD163+ macrophages to modulate local microglia in the brain to achieve neuroprotection. To do so, we designed liposomes targeted for the CD163 receptor to deliver dexamethasone (Dexa) into the CD163+ macrophages in the 6-OHDA PD model. Our data show that a fraction of the CD163-targeted liposomes were carried into the brain after peripheral intravenous injection. The 6-OHDA-lesioned rats that received repeated intravenous CD163-targeted liposomes with Dexa for 3 weeks exhibited better motor performance than the control groups and had minimal glucocorticoid-driven side effects. Furthermore, these animals showed better survival of dopaminergic neurons in substantia nigra and an increased number of microglia expressing major histocompatibility complex II. Therefore, rats receiving CD163-targeted liposomes with Dexa were partially protected against 6-OHDA-induced dopaminergic neurodegeneration, which correlated with a distinctive microglia response. Altogether, our data support the use of macrophages for the modulation of brain neurodegeneration and specifically highlight the potential of CD163-targeted liposomes as a therapeutic tool in PD. SIGNIFICANCE STATEMENT: The immune response now evident in the progression of Parkinson's disease comprises both local microglia and other immune cells. We provide evidence that CD163+ macrophages can be a target to modulate brain immune response to achieve neuroprotection in the 6-hydroxydopamine model. To do so, we targeted the CD163+ population, which to a low but significant extent infiltrated in the neurodegenerating area of the brain. Specially designed liposomes targeted for the CD163 receptor were loaded with glucocorticoids and injected peripherally to modify the infiltrated CD163 cells toward an anti-inflammatory profile. This modification of the CD163 population resulted in a distinctive microglial response that correlated with decreased dopaminergic cell death and better motor performance.


Assuntos
Anti-Inflamatórios/farmacologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Glucocorticoides/metabolismo , Microglia/efeitos dos fármacos , Doença de Parkinson/patologia , Receptores de Superfície Celular/metabolismo , Adrenérgicos/toxicidade , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Modelos Animais de Doenças , Feminino , Hidrocortisona/sangue , Lipossomos/farmacologia , Locomoção/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Oxidopamina/toxicidade , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/etiologia , Ratos , Ratos Sprague-Dawley , Tirosina 3-Mono-Oxigenase/metabolismo
10.
J Biol Chem ; 291(3): 1103-14, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26601954

RESUMO

MFAP4 (microfibrillar-associated protein 4) is an extracellular glycoprotein found in elastic fibers without a clearly defined role in elastic fiber assembly. In the present study, we characterized molecular interactions between MFAP4 and elastic fiber components. We established that MFAP4 primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin and fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site-directed mutagenesis disclosed residues Phe(241) and Ser(203) in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved in proper elastic fiber organization.


Assuntos
Proteínas de Transporte/metabolismo , Desmosina/metabolismo , Tecido Elástico/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Microfibrilas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Tropoelastina/metabolismo , Substituição de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Fibrilina-1 , Fibrilinas , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Ligantes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tropoelastina/química , Tropoelastina/genética
11.
J Infect Dis ; 214(8): 1198-204, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27354366

RESUMO

BACKGROUND: CD163, a monocyte- and macrophage-specific scavenger receptor, is shed as soluble CD163 (sCD163) during the proinflammatory response. Here, we assessed the association between plasma sCD163 levels and progression to AIDS and all-cause mortality among individuals infected with human immunodeficiency virus type 1 (HIV). METHODS: Plasma sCD163 levels were measured in 933 HIV-infected individuals. Hazard ratios (HRs) with 95% confidence intervals (CIs) associated with mortality were computed by Cox proportional hazards regression. RESULTS: At baseline, 86% were receiving antiretroviral treatment, 73% had plasma a HIV RNA level of <50 copies/mL, and the median CD4(+) T-cell count was 503 cells/µL. During 10.5 years of follow-up, 167 (17.9%) died. Plasma sCD163 levels were higher in nonsurvivors than in survivors (4.92 mg/L [interquartile range {IQR}, 3.29-8.65 mg/L] vs 3.16 mg/L [IQR, 2.16-4.64 mg/L]; P = .0001). The cumulative incidence of death increased with increasing plasma sCD163 levels, corresponding to a 6% or 35% increased risk of death for each milligram per liter or quartile increase, respectively, in baseline plasma sCD163 level (adjusted HR, 1.06 [95% CI, 1.03-1.09] and 1.35 [95% CI, 1.13-1.63], respectively). CONCLUSIONS: Plasma sCD163 was an independent marker of all-cause mortality in a cohort of HIV-infected individuals, suggesting that monocyte/macrophage activation may play a role in HIV pathogenesis and be a target of intervention.


Assuntos
Antígenos CD/sangue , Antígenos de Diferenciação Mielomonocítica/sangue , Infecções por HIV/sangue , Infecções por HIV/mortalidade , Plasma/metabolismo , Receptores de Superfície Celular/sangue , Adulto , Antirretrovirais/uso terapêutico , Biomarcadores/sangue , Progressão da Doença , Feminino , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Humanos , Ativação de Macrófagos/fisiologia , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Modelos de Riscos Proporcionais , Receptores de Superfície Celular/metabolismo
12.
J Proteome Res ; 15(12): 4591-4600, 2016 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-27758107

RESUMO

Loss-of-function mutations in the transmembrane ABCC6 transport protein cause pseudoxanthoma elasticum (PXE), an ectopic, metabolic mineralization disorder that affects the skin, eye, and vessels. ABCC6 is assumed to mediate efflux of one or several small molecule compounds from the liver cytosol to the circulation. Untargeted metabolomics using liquid chromatography-mass spectrometry was employed to inspect liver cytosolic extracts from mice with targeted disruption of the Abcc6 gene. Absence of the ABCC6 protein induced an altered profile of metabolites in the liver causing accumulation of compounds as more features were upregulated than downregulated in ABCC6-deficient mice. However, no differences of the identified metabolites in liver could be detected in plasma, whereas urine reflected some of the changes. Of note, N-acetylated amino acids and pantothenic acid (vitamin B5), which is involved in acetylation reactions, were accumulated in the liver. None of the identified metabolites seems to explain mineralization in extrahepatic tissues, but the present study now shows that abrogated ABCC6 function does cause alterations in the metabolic profile of the liver in accordance with PXE being a metabolic disease originating from liver disturbance. Further studies of these changes and the further identification of yet unknown metabolites may help to clarify the liver-related pathomechanism of PXE.


Assuntos
Transportadores de Cassetes de Ligação de ATP/deficiência , Fígado/metabolismo , Metabolômica/métodos , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Citosol/química , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Mutação , Pseudoxantoma Elástico/genética
13.
J Hepatol ; 64(4): 813-22, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26639396

RESUMO

BACKGROUND & AIMS: Activation of liver macrophages plays a key role in liver and systemic inflammation and may be involved in development and prognosis of acute-on-chronic liver failure (ACLF). We therefore measured the circulating macrophage activation markers soluble sCD163 and mannose receptor (sMR) and related them to the short-(1-3 months) and long-term (6 months) mortality in the cirrhosis patients of the CANONIC study. METHODS: Eighty-six cirrhosis patients had no ascites and no ACLF, 580 had ascites but no ACLF; 100, 66, and 19 had ACLF-grade-I (ACLF-I), ACLF-II, and ACLF-III, respectively. The patients' clinical course was registered and their MELD, CLIF-C Acute Decompensation (AD), and CLIF-C ACLF-scores computed at inclusion. RESULTS: We found a stepwise increase (p<0.001) in median sCD163 (5.68 (IQR: 3.86-9.60); 8.26 (5.02-12.34); 9.50 (5.37-17.91); 15.68 (10.12-19.42); 20.18 (15.26-32.20) mg/L) and sMR (0.60 (0.40-0.84); 0.81 (0.57-1.12); 0.81 (0.61-1.26); 1.17 (0.89-1.62); 1.41 (1.14-1.79)mg/L) with increasing grades of ACLF. Both sCD163 and sMR were independently associated with short and long-term mortality and showed equal or higher predictive accuracy than MELD, CLIF-C ACLF and CLIF-C AD scores. Addition of the macrophage markers to the clinical scores improved the prognostic efficacy: In ACLF patients sCD163 improved prediction of short-term mortality (C-index: 0.74 (0.67-0.80)) and in patients without ACLF sMR improved prediction of long-term mortality (C-index: 0.80 (0.76-0.85)). CONCLUSIONS: The severity related increase in sCD163 and sMR and close association with mortality suggest a primary importance of inflammatory activation of liver macrophages in the emergence and course of ACLF. Accordingly, supplementation of the macrophage biomarkers to the platform of the clinical scores improved the prognostic performance beyond that of the original scores.


Assuntos
Insuficiência Hepática Crônica Agudizada/mortalidade , Cirrose Hepática/mortalidade , Ativação de Macrófagos , Insuficiência Hepática Crônica Agudizada/imunologia , Adulto , Idoso , Antígenos CD/sangue , Antígenos de Diferenciação Mielomonocítica/sangue , Biomarcadores , Feminino , Humanos , Lectinas Tipo C/análise , Cirrose Hepática/imunologia , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/análise , Pessoa de Meia-Idade , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/sangue
14.
Rheumatology (Oxford) ; 55(10): 1871-9, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27330157

RESUMO

OBJECTIVE: Co-stimulatory T cell cytokines are important in the progression of RA. This study investigates the interplay between 4-1BB, a disintegrin and metalloprotease-17 (ADAM17) and galectin-9 (Gal-9) in RA. METHODS: Stimulated mononuclear cells from patients with chronic RA (n = 12) were co-incubated with tissue inhibitor of metalloproteinase, 4-1BB ligand and Gal-9. Plasma samples were examined for soluble 4-1BB (s4-1BB) in newly diagnosed, treatment-naïve patients with RA (n = 97). The 28-joint DAS with CRP (28DAS-CRP), total Sharp score, erosion score and joint space narrowing were used to evaluate treatment outcome serially over a 2-year period. RESULTS: RA CD4(+) and CD8(+) synovial T cells express high levels of 4-1BB. The addition of TNF-α to cultured synovial mononuclear cells increased shedding of 4-1BB. 4-1BB ligand only increased TNF-α shedding in combination with Gal-9. RNA interference-mediated knockdown of ADAM17 or the addition of an ADAM17 inhibitor reduced the 4-1BB shedding. Shedding of 4-1BB was not influenced by Gal-9. Plasma levels of s4-1BB were increased in early RA and correlated with the number of swollen joints at baseline. After 3 months of treatment, the plasma levels of s4-1BB were equal to those of the controls. Baseline plasma levels of s4-1BB were inversely correlated with DAS28-CRP after 2 years of treatment, but not with total Sharp score, erosion score or joint space narrowing. CONCLUSION: ADAM17 induces 4-1BB shedding in RA. Gal-9 is pivotal for the function of 4-1BB and induction of TNF-α. Furthermore, high plasma levels of s4-1BB were associated with the number of swollen joints, but also with a low DAS28-CRP after 2 years treatment in early RA.


Assuntos
Ligante 4-1BB/fisiologia , Proteína ADAM17/fisiologia , Artrite Reumatoide/etiologia , Galectinas/fisiologia , Metaloproteinase 17 da Matriz/fisiologia , Ligante 4-1BB/metabolismo , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Progressão da Doença , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Leucócitos Mononucleares , Estudos Longitudinais , Metotrexato/uso terapêutico , Líquido Sinovial/química , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/biossíntese
15.
J Immunol ; 192(5): 1997-2006, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24563502

RESUMO

Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a variety of ligands, including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the U.S. National Institute of Allergy and Infectious Diseases, National Institutes of Health to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of non-self or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. The discussion and nomenclature recommendations described in this report only refer to mammalian scavenger receptors. The purpose of this article is to describe the proposed mammalian nomenclature and classification developed at the workshop and to solicit additional feedback from the broader research community.


Assuntos
Receptores Depuradores/classificação , Animais , Humanos , Receptores Depuradores/imunologia , Terminologia como Assunto
16.
Nature ; 464(7287): 445-8, 2010 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-20237569

RESUMO

Cobalamin (Cbl, vitamin B(12)) is a bacterial organic compound and an essential coenzyme in mammals, which take it up from the diet. This occurs by the combined action of the gastric intrinsic factor (IF) and the ileal endocytic cubam receptor formed by the 460-kilodalton (kDa) protein cubilin and the 45-kDa transmembrane protein amnionless. Loss of function of any of these proteins ultimately leads to Cbl deficiency in man. Here we present the crystal structure of the complex between IF-Cbl and the cubilin IF-Cbl-binding-region (CUB(5-8)) determined at 3.3 A resolution. The structure provides insight into how several CUB (for 'complement C1r/C1s, Uegf, Bmp1') domains collectively function as modular ligand-binding regions, and how two distant CUB domains embrace the Cbl molecule by binding the two IF domains in a Ca(2+)-dependent manner. This dual-point model provides a probable explanation of how Cbl indirectly induces ligand-receptor coupling. Finally, the comparison of Ca(2+)-binding CUB domains and the low-density lipoprotein (LDL) receptor-type A modules suggests that the electrostatic pairing of a basic ligand arginine/lysine residue with Ca(2+)-coordinating acidic aspartates/glutamates is a common theme of Ca(2+)-dependent ligand-receptor interactions.


Assuntos
Fator Intrínseco/química , Fator Intrínseco/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Vitamina B 12/química , Vitamina B 12/metabolismo , Ácido Aspártico/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cristalografia por Raios X , Ácido Glutâmico/metabolismo , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Eletricidade Estática
17.
Biochem J ; 465(2): 259-70, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25360794

RESUMO

Gelatinase B/matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) cleaves many substrates and is produced by most cell types as a zymogen, proMMP-9, in complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1). Natural proMMP-9 occurs as monomers, homomultimers and heterocomplexes, but our knowledge about the overall structure of proMMP-9 monomers and multimers is limited. We investigated biochemical, biophysical and functional characteristics of zymogen and activated forms of MMP-9 monomers and multimers. In contrast with a conventional notion of a dimeric nature of MMP-9 homomultimers, we demonstrate that these are reduction-sensitive trimers. Based on the information from electrophoresis, AFM and TEM, we generated a 3D structure model of the proMMP-9 trimer. Remarkably, the proMMP-9 trimers possessed a 50-fold higher affinity for TIMP-1 than the monomers. In vivo, this finding was reflected in a higher extent of TIMP-1 inhibition of angiogenesis induced by trimers compared with monomers. Our results show that proMMP-9 trimers constitute a novel structural and functional entity that is differentially regulated by TIMP-1.


Assuntos
Precursores Enzimáticos/química , Metaloproteinase 9 da Matriz/química , Modelos Moleculares , Complexos Multiproteicos/química , Inibidor Tecidual de Metaloproteinase-1/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo
18.
J Immunol ; 188(5): 2399-409, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22279103

RESUMO

CD163-L1 belongs to the group B scavenger receptor cysteine-rich family of proteins, where the CD163-L1 gene arose by duplication of the gene encoding the hemoglobin scavenger receptor CD163 in late evolution. The current data demonstrate that CD163-L1 is highly expressed and colocalizes with CD163 on large subsets of macrophages, but in contrast to CD163 the expression is low or absent in monocytes and in alveolar macrophages, glia, and Kupffer cells. The expression of CD163-L1 increases when cultured monocytes are M-CSF stimulated to macrophages, and the expression is further increased by the acute-phase mediator IL-6 and the anti-inflammatory mediator IL-10 but is suppressed by the proinflammatory mediators IL-4, IL-13, TNF-α, and LPS/IFN-γ. Furthermore, we show that CD163-L1 is an endocytic receptor, which internalizes independently of cross-linking through a clathrin-mediated pathway. Two cytoplasmic splice variants of CD163-L1 are differentially expressed and have different subcellular distribution patterns. Despite its many similarities to CD163, CD163-L1 does not possess measurable affinity for CD163 ligands such as the haptoglobin-hemoglobin complex or various bacteria. In conclusion, CD163-L1 exhibits similarity to CD163 in terms of structure and regulated expression in cultured monocytes but shows clear differences compared with the known CD163 ligand preferences and expression pattern in the pool of tissue macrophages. We postulate that CD163-L1 functions as a scavenger receptor for one or several ligands that might have a role in resolution of inflammation.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Endocitose/imunologia , Mediadores da Inflamação/fisiologia , Macrófagos/imunologia , Macrófagos/patologia , Receptores de Superfície Celular/metabolismo , Animais , Antígenos CD/biossíntese , Antígenos CD/fisiologia , Antígenos de Diferenciação Mielomonocítica/biossíntese , Antígenos de Diferenciação Mielomonocítica/fisiologia , Diferenciação Celular/imunologia , Células Cultivadas , Células HEK293 , Células HL-60 , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Células Jurkat , Células de Kupffer/imunologia , Células de Kupffer/patologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , Macrófagos/metabolismo , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/patologia , Neuroglia/imunologia , Neuroglia/patologia , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/fisiologia , Células U937
19.
Mol Ther ; 20(8): 1550-8, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22643864

RESUMO

Synthetic glucocorticoids are potent anti-inflammatory drugs but serious side effects such as bone mobilization, muscle mass loss, immunosuppression, and metabolic alterations make glucocorticoid therapy a difficult balance. The therapeutic anti-inflammatory effect of glucocorticoids relies largely on the suppressed release of tumor-necrosis factor-α and other cytokines by macrophages at the sites of inflammation. We have now developed a new biodegradable anti-CD163 antibody-drug conjugate that specifically targets the glucocorticoid, dexamethasone to the hemoglobin scavenger receptor CD163 in macrophages. The conjugate, that in average contains four dexamethasone molecules per antibody, exhibits retained high functional affinity for CD163. In vitro studies in rat macrophages and in vivo studies of Lewis rats showed a strong anti-inflammatory effect of the conjugate measured as reduced lipopolysaccharide-induced secretion of tumor-necrosis factor-α. The in vivo potency of conjugated dexamethasone was about 50-fold that of nonconjugated dexamethasone. In contrast to a strong systemic effect of nonconjugated dexamethasone, the equipotent dose of the conjugate had no such effect, measured as thymus lymphocytes apoptosis, body weight loss, and suppression of endogenous cortisol levels. In conclusion, the study shows antibody-drug conjugates as a future approach in anti-inflammatory macrophage-directed therapy. Furthermore, the data demonstrate CD163 as an excellent macrophage target for anti-inflammatory drug delivery.


Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Dexametasona/química , Dexametasona/uso terapêutico , Inflamação/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Animais , Anti-Inflamatórios/síntese química , Anticorpos/química , Anticorpos/uso terapêutico , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Western Blotting , Células CHO , Células Cultivadas , Cricetinae , Feminino , Citometria de Fluxo , Inflamação/induzido quimicamente , Lipopolissacarídeos/toxicidade , Ratos , Receptores de Superfície Celular/metabolismo
20.
Cancers (Basel) ; 15(6)2023 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-36980716

RESUMO

More than 80% of human cancers originate in epithelial tissues. Loss of epithelial cell characteristics are hallmarks of tumor development. Receptor-mediated endocytosis is a key function of absorptive epithelial cells with importance for cellular and organismal homeostasis. LRP2 (megalin) is the largest known endocytic membrane receptor and is essential for endocytosis of various ligands in specialized epithelia, including the proximal tubules of the kidney, the thyroid gland, and breast glandular epithelium. However, the role and regulation of LRP2 in cancers that arise from these tissues has not been delineated. Here, we examined the expression of LRP2 across 33 cancer types in The Cancer Genome Atlas. As expected, the highest levels of LRP2 were found in cancer types that arise from LRP2-expressing absorptive epithelial cells. However, in a subset of tumors from these cancer types, we observed epigenetic silencing of LRP2. LRP2 expression showed a strong inverse correlation to methylation of a specific CpG site (cg02361027) in the first intron of the LRP2 gene. Interestingly, low expression of LRP2 was associated with poor patient outcome in clear cell renal cell carcinoma, papillary renal cell carcinoma, mesothelioma, papillary thyroid carcinoma, and invasive breast carcinoma. Furthermore, loss of LRP2 expression was associated with dedifferentiated histological and molecular subtypes of these cancers. These observations now motivate further studies on the functional role of LRP2 in tumors of epithelial origin and the potential use of LRP2 as a cancer biomarker.

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