RESUMO
BACKGROUND: Neurotrophins are produced by various cells upon different stimuli and participate in the initiation and regulation of inflammation in various diseases including allergy and asthma, but little is known about the production and control of neurotrophins by dendritic cells (DCs). The aim of this study was to assess whether DCs produce the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and whether inflammatory stimuli or allergens are able to induce the production of neurotrophic factors. METHODS: Monocyte-derived dendritic cells (MoDCs) were generated from different donors. The neurotrophins NGF and BDNF were demonstrated by RT-PCR, Western blotting, flow cytometry analysis and fluorescence microscopy. MoDCs were cultured and stimulated with lipopolysaccharide (LPS) or allergen for 24 h. The supernatants and cells were collected. Measurement for NGF and BDNF was performed by ELISA. RESULTS: DCs express mRNA for the neurotrophins NGF and BDNF. Proteins were detectable by Western blot, FACS analysis and fluorescence microscopy. LPS led to an up-regulation of BDNF, while NGF was unaffected. Cell lysates demonstrated an increased amount of BDNF after stimulation with LPS or allergen, while NGF was not affected significantly. CONCLUSIONS: DCs are a source of neurotrophins. LPS selectively regulates the production of BDNF. Allergen stimulation leads to an LPS-independent regulation. This contributes to a complex involvement of neurotrophins in allergic diseases.
Assuntos
Alérgenos/farmacologia , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Células Dendríticas/metabolismo , Hipersensibilidade/sangue , Lipopolissacarídeos/farmacologia , Fator de Crescimento Neural/biossíntese , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/genética , Diferenciação Celular , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Monócitos/patologia , Fator de Crescimento Neural/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
BACKGROUND: Neurotrophins are involved in inflammatory reactions influencing several cells in health and disease including allergy and asthma. Dendritic cells (DCs) play a major role in the induction of inflammatory processes with an increasing role in allergic diseases as well. OBJECTIVE: The aim of this study was to investigate the influence of neurotrophins on DC function. METHODS: Monocyte-derived dendritic cells were generated from allergic and non-allergic donors. Neurotrophin receptors were demonstrated by western blotting, flow cytometry and fluorescence microscopy. Activation of small GTPases was evaluated by pull-down assays. DCs were incubated with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and supernatants were collected for measurement of IL-4, IL-6, IL-10, IL-12p70, TNF-alpha and TGF-beta. RESULTS: Receptor proteins were detectable by western blot, fluorescence activated cell sorting analysis and fluorescence microscopy. Signalling after neurotrophin stimulation occurred in a ligand-specific pattern. NGF led to decreased RhoA and increased Rac activation, while BDNF affected RhoA and Rac activity in a reciprocal fashion. Cells of allergics released a significantly increased amount of IL-6, while for healthy subjects a significantly higher amount of IL-10 was found. CONCLUSION: These data indicate that DCs are activated by the neurotrophins NGF and BDNF by different pathways in a receptor-dependant manner. These cells then may initiate inflammatory responses based on allergic sensitization releasing preferred cytokines inducing tolerance or a T-helper type 2 response.