Article

Monocytes of allergics and non-allergics produce, store and release the neurotrophins NGF, BDNF and NT-3

Humboldt-Universität zu Berlin, Berlín, Berlin, Germany
Regulatory Peptides (Impact Factor: 2.01). 02/2005; 124(1-3):19-25. DOI: 10.1016/j.regpep.2004.06.024
Source: PubMed

ABSTRACT Recent studies have shown that neurotrophins (NTs) are involved in inflammatory processes. Elevated plasma levels of NTs were found allergic diseases with the highest levels in allergic asthma. However, the exact cellular sources involved in the regulation and release of neurotrophins in allergic inflammation are still not well defined.
The aim of this study was to assess whether monocytes of allergic and non-allergic subjects produce, store and release the neurotrophins NGF, BDNF and NT-3.
Monocytes of allergic and non-allergic donors were purified by immunomagnetic selection. APAAP-staining for the presence of NTs and their receptors was performed. RT-PCR and Western blot evaluated the production and storage of NTs. Monocytes were incubated and supernatants were collected for measurement of neurotrophic factors after stimulation with lipopolysaccharide (LPS) as inflammatory stimulus. The neurotrophin content in lysates and cell culture supernatants was determined by ELISA.
Human monocytes express the neurotrophins NGF, BDNF and NT-3 but also their specific receptors TrkA, TrkB and TrkC. RT-PCR amplification of isolated mRNA demonstrated expression of the examined neurotrophins. Proteins were detectable by Western blot. NTs were found in the monocyte lysates and supernatants at different levels in allergic and non-allergic donors. Cell stimulation with LPS leads to release of NGF and NT3.
Monocytes, produce, store and release NGF, BDNF and NT-3. They are a possible source of elevated neurotrophin levels found in allergy and asthma.

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In summary these results show, that primary cell culture of the murine tracheal epithalial cells could be done. These cells are functionally active and produce NGF as well as BDNF. The cytokines IL-1ß, IL-13 and IL-4 do not play a role in regulation of the Neurotrophin production. Das Allergisches Asthma bronchiale (BA) ist eine Modellkrankheit für neuro-immunologische Interaktionen, und ist charakterisiert durch eine chronische Entzündung der Atemwege, Entwicklung von Atemwegshyperaktivität und reversibler Broncho-Obstruktion. Neurotrophine (NGF, BDNF, NT-3 und NT-4) sind wichtige Mediatoren auch im Immunsystem, und es wird vermutet, dass sie Neuro-Immuninteraktionen in der chronischen Entzündung der Atemwege steuern. Untersuchungen an Tieren haben gezeigt, dass bei allergischer Entzündung der Atemwege Neurotrophine hochreguliert werden. Es wurde auch gezeigt, dass menschliche Lungen-Epithelzellen (A549) NGF produzieren können. Ziel dieser Studie war, die Rolle von Atemwegs-Epithelzellen in der Regulation der Neurotrophinproduktion bei allergischen Atemwegserkrankungen zu untersuchen. Um die Produktion von Neurotrophinen von Maus-Trachealepithelzellen zu untersuchen, wurde ein Protokoll für das Züchten der Mäuse-Trachealepithelzellen unter Air-liquid Interface Bedingungen entwickelt. Hierfür wurde ein früher publiziertes Protokoll modifiziert. Epithelzellen der Trachea von Wildtypmäusen wurden mit einem spezifischen Medium dissoziiert, das eine DNAse und Pronase enthält. Diese Zellen wurden über der semipermeablen Membran des Zellkultureinsatzes in Anwesenheit von Wachstumfaktoren gezüchtet. Um die Reinheit jener Zellen zu untersuchen, wurden kultivierte Zellen mit Pancytokeratin (Epithelzellen Marker) Einzelzellen zu unterschiedlichen Zeitpunkten gefärbt. Die basale Produktion von Neurotrophinen wurde zu unterschiedlichen Zeitpunkten gemessen (6, 12, 24, 48, und 72 Stunden). Der Effekt der Zytokine IL-1ß, IL-13, und IL-4 auf die Neurotrophinproduktion wurde dosisabhängig untersucht und quantitativ durch Enzyme-linked Immunosorbent Assay (ELISA) gemessen. Das gleiche Protokoll wurde auf murine LA 4 Epithel-Krebszellen angewendet. Es wurde festgestellt, dass >95% der kultivierten Zellen Pancytokeratin positiv waren, und für mehr als vierzig Tage in der Kultur überlebten. Indem wir die basale Produktion von Neurotrophine durch diese Zellen untersuchten, fanden wir heraus, dass NGF und BDNF Produktion zeitabhängig hochreguliert wurden, während NT-3 und NT-4 nicht produziert wurden. Spezifische mRNA für NGF, BDNF waren in allen Proben durch RT-PCR nachweisbar. In kultivierten Primerzellen konnte keine Effekte von IL-1ß, IL-13, und IL-4, auch bei ansteigenden Konzentrationen, auf die Neurotrophinproduktion nachgewiesen werden. Allerdings hatten diese Zytokine Effekte auf die Neurotrophin-Produktion von LA-4 Epithelzellen. Es gab eine konzentrationsabhängige Hochregulationweise von NGF und BDNF nach Inkubation mit IL-1ß, IL-13, und IL-4, in LA 4 Zellen. Zusammenfassend zeigen diese Resultate, das Primärzellkulturen mit murinen Tracheal-Epithelzellen angelegt werden können. Diese Zellen sind funktionell aktiv und produzieren NGF sowie BDNF. Die Zytokine IL-1ß, IL-13 und IL-4 spielen keine Rolle in der Regulation dieser Neurotrophinproduktion.