Differentiation of human dendritic cells from monocytes in vitro using granulocyte-macrophage colony stimulating factor and low concentration of interleukin-4

Abstract

Several laboratories have developed culture systems that allow the generation of large numbers of human dendritic cells (DC) from monocytes using granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-4 (IL-4). In this work we provided evidence that GM-CSF (100 ng/ml) in combination with a low concentration of IL-4 (5 ng/ml) was efficient in the generation of immature, non-adherent, monocyte-derived DC as the same concentration of GM-CSF, and ten times higher concentration of IL-4 (50 ng/ml). This conclusion was based on the similar phenotype profile of DC such as the expression of CD1a, CD80, CD86, and HLA-DR, down-regulation of CD14, and the absence of CD83, as well as on their similar allostimulatory activity for T cells. A higher number of cells remained adherent in cultures with lower concentrations of IL-4 than in cultures with higher concentrations of the cytokine. However, most of these adherent cells down-regulated CD14 and stimulated the proliferation of alloreactive T cells. In contrast adherent cells cultivated with GM-CSF alone were predominantly macrophages as judged by the expression of CD14 and the inefficiency to stimulate alloreactive T cells. DC generated in the presence of lower concentrations of IL-4 had higher proapoptotic potential for the Jurkat cell line than DC differentiated with higher concentrations of IL-4, suggesting their stronger cytotoxic, anti-tumor effect.

U više laboratorija su uspostavljeni sistemi za kultivaciju velikog broja humanih dendritičnih ćelija (DĆ) od monocita korišćenjem faktora stimulacije granulocitno--makrofagnih kolonija (GM-CSF) i interleukina-4 (IL-4). U ovom radu je pokazano da je kombinacija GM-CSF (100 ng/ml) i mala koncentracija IL-4 (5 ng/ml) podjednako efikasna za dobijanje nezrelih, neadherentnih, DĆ monocitnog porekla kao i kombinacija GM-CSF sa deset puta većom koncentracijom IL-4 (50 ng/ml). Ovaj zaključak izveden je na osnovu sličnog fenotipskog profila DĆ (ispoljavanje CD1a, CD80, CD86 i HLA-DR, smanjenje ekspresije CD14 i odsustva CD83), kao i slične alostimulatorne aktivnosti ovih ćelija za limfocite T. U kulturama sa nižim koncentracijama IL-4 prisutan je bio veći broj adherentnih ćelija nego u kulturama sa većim koncentracijama IL-4. Međutim, većina ovih ćelija je smanjivala ekspresiju CD14 i stimulisala proliferaciju aloreaktivnih limfocita T. Nasuprot njima adherentne ćelije, diferentovane samo u prisustvu GM-CSF, koje su ispoljavale CD14 i nisu imale sposobnost stimulacije aloreakativnih limfocita T pokazivale su karakteristike makrofaga. DĆ obrazovane u prisustvu manjih koncentracija IL-4 imale su veći potencijal za indukciju apoptoze Jurkat ćelijske linije, a time i snažniji citotoksični, antitumorski efekat nego DĆ diferentovane u prisustvu većih koncentracija IL-4.