RESUMEN
BACKGROUND AND AIMS: Immortalized cell lines have been long used as substitute for ex vivo murine and human material, but exhibit features that are not found in healthy tissue. True human dendritic cells (DC) cannot be cultured or passaged as opposed to immortalized cell lines. Research in the fields of immunogenic responses and immunotolerance in DCs has increased over the last decade. Autophagy has gained interest in these fields as well, and has been researched extensively in many other cell types as well. Here we have studied the applicability of cell line-derived dendritic cell-like cells of six myeloid cell lines aimed at research focussed on autophagy. METHODS: Six myeloid leukaemia cell lines were differentiated towards cell line-derived dendritic cell-like cells (cd-DC) using GM-CSF, IL-4, Ionomycine and PMA: HL60, KG1, MM6, MV-4-11, THP1 and U937. Autophagy was modulated using Rapamycin, Bafilomycin A1 and 3MA. Cell lines were genotyped for autophagy-related SNPs using RFLP. Marker expression was determined with FACS analysis and cytokine profiles were determined using Human Cytometric Bead Assay. Antigen uptake was assessed using Fluoresbrite microspheres. RESULTS AND DISCUSSION: All researched cell lines harboured SNPs in the autophagy pathways. MM6 and THP1 derived cd-DCs resembled monocyte-derived DCs (moDC) most closely in marker expression, cytokine profiles and autophagy response. The HL60 and U937 cell lines proved least suitable for autophagy-related dendritic cell research. CONCLUSION: The genetic background of cell lines should be taken into account upon studying (the effects of) autophagy in any cell line. Although none of the studied cell lines recapitulate the full spectrum of DC characteristics, MM6 and THP1 derived cd-DCs are most suitable for autophagy-related research in dendritic cells.
Asunto(s)
Adenina/análogos & derivados , Autofagia/efectos de los fármacos , Diferenciación Celular , Células Dendríticas/efectos de los fármacos , Macrólidos/farmacología , Monocitos/inmunología , Sirolimus/farmacología , Adenina/farmacología , Autofagia/genética , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Citometría de Flujo , Genotipo , Células HL-60 , Humanos , Microscopía Fluorescente , Monocitos/metabolismo , Monocitos/patología , Fenotipo , Polimorfismo de Nucleótido Simple , Células THP-1 , Células U937RESUMEN
New peptides for lipopolysaccharide (LPS) and lipoteichoic acid (LTA) neutralization in upper respiratory tract infections were developed and evaluated in terms of efficacy and safety for application in humans. Based on the sequence of the human antimicrobial peptide LL-37 we developed and investigated length variants, substitution analogues and modifications to stabilize the peptides to prevent enzymatic degradation and to improve efficacy. The most promising peptide appears P60.4, a 24 amino acid peptide with similar efficacy as LL-37 in terms of LPS and LTA neutralization and lower pro-inflammatory activity. In addition, the acetylated and amidated version of this peptide shows no toxicity and displays higher or equal antimicrobial activity compared to LL-37.
Asunto(s)
Antiinfecciosos/química , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Lipopolisacáridos/antagonistas & inhibidores , Péptidos/química , Ácidos Teicoicos/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Antiinfecciosos/síntesis química , Antiinfecciosos/uso terapéutico , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Catelicidinas , Modelos Animales de Enfermedad , Cobayas , Lipopolisacáridos/metabolismo , Masculino , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Linfocitos T/efectos de los fármacos , Ácidos Teicoicos/metabolismoRESUMEN
Most antimicrobial peptides have been discovered based on activity-guided purification procedures, which used assays to determine their antimicrobial activity. Nevertheless, recent studies have shown that antimicrobial peptides also exert a range of other functions. Based on these observations, antimicrobial peptides are now not only implicated in host defense against infection but also in other immune reactions, inflammation, and wound-repair processes. The activities of neutrophil defensins and the cathelicidin hCAP-18/LL-37, antimicrobial peptides that are abundantly expressed in the human neutrophil, are the subject of an increasing number of studies. Exposure to neutrophil defensins and hCAP-18/LL-37 results in increases in mediator expression and release, chemotaxis, and proliferation of inflammatory and epithelial cells and fibroblasts, and the mechanisms underlying these effects have been partly elucidated. This review is focused on the effects of neutrophil defensins and hCAP-18/LL-37 on airway epithelial cells.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/fisiología , Enfermedades Pulmonares/fisiopatología , Neutrófilos/fisiología , Mucosa Respiratoria/fisiología , Anticarcinógenos , Humanos , Inflamación , CatelicidinasRESUMEN
Mucosal secretions contain a range of defense effector molecules including antimicrobial peptides and proteinase inhibitors. These molecules play a central role in host defense against infection, and in a variety of immune and inflammatory reactions. The aim of this study was to analyze the levels of neutrophil defensins, the cathelicidin hCAP-18/LL-37, and the proteinase inhibitors secretory leukocyte proteinase inhibitor, SKALP/elafin and cystatin M/E in various mucosal secretions and urine. We show here that especially seminal plasma is characterized by high concentrations of hCAP-18/LL-37, SLPI, SKALP/elafin and cystatin M/E. The results of this study demonstrate that each mucosal secretion is characterized by a unique profile of effector molecules, which may supply individual mucosal secretions with specific properties related to the control of local infection and inflammation.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/inmunología , Péptidos Catiónicos Antimicrobianos/metabolismo , Inmunidad Innata , Moco/inmunología , Inhibidores de Proteasas/inmunología , Catelicidinas , Cistatina M , Cistatinas/inmunología , Cistatinas/metabolismo , Defensinas/inmunología , Defensinas/metabolismo , Femenino , Humanos , Masculino , Membrana Mucosa/inmunología , Membrana Mucosa/metabolismo , Moco/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras , Proteínas/inmunología , Proteínas/metabolismo , Inhibidor Secretorio de Peptidasas LeucocitariasRESUMEN
The respiratory epithelium plays a major role in the primary defense of the airways against infection. It has been demonstrated that bacterial products are involved in the induction of inflammatory reactions of the upper airways. Little is known about the effects of bacterial products on expression of the antimicrobial peptide hCAP-18/LL-37, the only human cathelicidin identified so far. The aim of this study was to investigate the effects of bacterial products from both gram-positive and gram-negative bacteria on the expression of hCAP-18/LL-37 by sinus epithelial cells using an air-exposed tissue culture model. Lipopolysaccharide and lipoteichoic acid both increased hCAP-18/LL-37 expression in cultured sinus epithelium as assessed by immunohistochemistry, where maximal stimulation occurred at 100 ng ml(-1) lipopolysaccharide or 10 microg ml(-1) lipoteichoic acid. The stimulatory effect of lipopolysaccharide and lipoteichoic acid was not restricted to expression of hCAP-18/LL-37, since also mucin expression and IL-8 release from cultured sinus epithelium cells were increased by lipopolysaccharide and lipoteichoic acid. This suggests that bacterial products may stimulate innate immunity in the upper airways.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Células Epiteliales/inmunología , Lipopolisacáridos/inmunología , Senos Paranasales/inmunología , Ácidos Teicoicos/inmunología , Células Cultivadas , Células Epiteliales/metabolismo , Bacterias Gramnegativas/inmunología , Bacterias Grampositivas/inmunología , Humanos , Inmunidad Innata , Interleucina-8/biosíntesis , Mucinas/metabolismo , Senos Paranasales/citología , CatelicidinasRESUMEN
A papillary-structured collagen fibril membrane is created, mimicking the 3D-architecture of the human papillary dermis. Primary human keratinocytes cultured to confluency on papillar-structured films are compared to keratinocytes cultured on flat membranes. Microscopical evaluation reveals the presence of morphologically distinct cells at the base of the papillar structures that are not observed on flat membranes. Gene expression microarrays and RT-qPCR indicate that these cells are in a more proliferative/migrational state, whereas cells on flat membranes have a more differentiated expression profile. Immunohistochemical stainings confirm these results. In conclusion, specific collagen architecture can direct keratinocyte behavior, and this may be used to further improve skin regeneration.
Asunto(s)
Materiales Biomiméticos/química , Colágeno/química , Expresión Génica/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Materiales Biomiméticos/farmacología , Diferenciación Celular/efectos de los fármacos , Colágeno/farmacología , Colágeno/ultraestructura , Dermis/citología , Dermis/efectos de los fármacos , Dermis/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Queratinocitos/citología , Queratinocitos/metabolismo , Membranas Artificiales , Microscopía Electrónica de Rastreo , Análisis de Secuencia por Matrices de Oligonucleótidos , Cultivo Primario de Células , Andamios del Tejido , Cicatrización de Heridas/fisiologíaRESUMEN
BACKGROUND: Inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) are characterized by the presence of eosinophils and neutrophils. However, the mechanisms that mediate the influx of these cells are incompletely understood. Neutrophil products, including neutrophil elastase and antimicrobial peptides such as neutrophil defensins and LL-37, have been demonstrated to display chemotactic activity towards cells from both innate and adaptive immunity. However, chemotactic activity of LL-37 towards eosinophils has not been reported. Therefore, the aim of the present study was to investigate the chemotactic activity of LL-37 for eosinophils and to explore the mechanisms involved in LL-37-mediated attraction of neutrophils and eosinophils. METHODS: Neutrophils and eosinophils were obtained from venous blood of healthy donors. Chemotaxis was studied using a modified Boyden chamber technique. Involvement of formyl-peptide receptors (FPRs) was studied using the antagonistic peptide tBoc-MLP. Activation of the mitogen-activated protein kinase (MAPK) ERK1/2 was studied by Western blotting using antibodies directed against phosphorylated ERK1/2. RESULTS: Our results show that LL-37 chemoattracts both eosinophils and neutrophils. The FPR antagonistic peptide tBoc-MLP inhibited LL-37-induced chemotaxis. Whereas the FPR agonist fMLP activated ERK1/2 in neutrophils, LL-37 did not, indicating that fMLP and LL-37 deliver different signals through FPRs. CONCLUSIONS: LL-37 displays chemotactic activity for eosinophils and neutrophils, and this activity is mediated via an FPR. These results suggest that LL-37 may play a role in inflammatory lung diseases such as asthma and COPD.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Eosinófilos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Receptores de Formil Péptido/inmunología , Agonistas Adrenérgicos beta/farmacología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Péptidos Catiónicos Antimicrobianos/antagonistas & inhibidores , Quimiotaxis de Leucocito/inmunología , Activación Enzimática , Eosinófilos/citología , Eosinófilos/inmunología , Etanolaminas/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fumarato de Formoterol , Humanos , Datos de Secuencia Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/citología , Neutrófilos/enzimología , Neutrófilos/inmunología , Toxina del Pertussis/farmacología , CatelicidinasRESUMEN
Antimicrobial peptides play an important role in innate immunity of the lung by acting as effector molecules in host defence against inhaled pathogens. Various families of antimicrobial peptides have been identified, including the cathelicidins. Cathelicidins are characterized by a conserved N-terminal cathelin domain and a variable C-terminal antimicrobial domain that can be released from the precursor protein after cleavage by proteinases. LL-37 is the C-terminal part of the only human cathelicidin identified to date called human cationic antimicrobial protein (hCAP-18), which is mainly expressed by neutrophils and epithelial cells. In addition to killing a broad spectrum of microorganisms, LL-37 was demonstrated to display various cellular activities related to inflammation including cytotoxicity to host cells, chemotaxis, epithelial cell activation, angiogenesis and epithelial wound repair. Focussing on this recent information, this review discusses the role of LL-37 in infection and inflammation in the lung. In addition, the importance of the fact that antimicrobial peptides such as LL-37 display a range of activities for the design and development of antimicrobial peptides for therapeutic use is discussed.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/inmunología , Enfermedades Pulmonares/inmunología , Catelicidinas , Humanos , Inmunidad InnataRESUMEN
Antimicrobial peptides produced by epithelial cells and neutrophils represent essential elements of innate immunity, and include the defensin and cathelicidin family of antimicrobial polypeptides. The human cathelicidin cationic antimicrobial protein-18 is an antimicrobial peptide precursor predominantly expressed in neutrophils, and its active peptide LL-37 is released from the precursor through the action of neutrophil serine proteinases. LL-37 has been shown to display antimicrobial activity against a broad spectrum of microorganisms, to neutralize LPS bioactivity, and to chemoattract neutrophils, monocytes, mast cells, and T cells. In this study we show that LL-37 activates airway epithelial cells as demonstrated by activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and increased release of IL-8. Epithelial cell activation was inhibited by the MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126, by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, by blocking anti-EGFR and anti-EGFR-ligand Abs, and by the metalloproteinase inhibitor GM6001. These data suggest that LL-37 transactivates the EGFR via metalloproteinase-mediated cleavage of membrane-anchored EGFR-ligands. LL-37 may thus constitute one of the mediators by which neutrophils regulate epithelial cell activity in the lung.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/inmunología , Activación Transcripcional/efectos de los fármacos , Secuencia de Aminoácidos , Catelicidinas , Línea Celular Tumoral , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Receptores ErbB/fisiología , Humanos , Inmunidad Innata/efectos de los fármacos , Interleucina-8/metabolismo , Pulmón/enzimología , MAP Quinasa Quinasa 4 , Metaloproteasas/metabolismo , Metaloproteasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Datos de Secuencia Molecular , Mucosa Respiratoria/enzimología , Activación Transcripcional/inmunología , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The human cathelicidin, hCAP-18, is expressed both in neutrophils and in epithelial cells. hCAP-18 is processed to the antimicrobial peptide LL-37 by proteinase 3 in neutrophils. hCAP-18 is highly expressed in the epididymis with a subsequent high concentration in seminal plasma where the protein is present in its unprocessed and antimicrobially inactive form. We report here that hCAP-18 in seminal plasma is processed to generate a 38-amino acid antimicrobial peptide ALL-38 by the prostate-derived protease gastricsin when incubated at a pH corresponding to the vaginal pH. In accordance with this, seminal plasma derived hCAP-18 was found in its processed form in the vagina following sexual intercourse. The antimicrobial activity of ALL-38 against a variety of microorganisms tested is equal to that of LL-37. This enzymatic activation of a proantimicrobial substance in seminal plasma following exposure to the vaginal milieu represents a novel mechanism to prevent infection following sexual intercourse.