We have developed a direct immunocytochemical technique to identify cytokine and chemokine production in epidermal Langerhans cells (LC) and in vitro derived CD14−, CD1a+, CD83+, CD40+ dendritic cells (DC) at the single cell level. Formaldehyde fixation combined with saponin permeabilization preserved cellular morphology and generated a characteristic juxtanuclear staining signal due to the accumulation of cytokine to the Golgi organelle. This approach was used for the assessment of TNF- α , IL-6, IL-8, IL-10, IL-12, GM-CSF, MIP-1 α , MIP-1 β and RANTES producing cells. In contrast, a diffuse cytoplasmic staining was evident for IL-1ra, IL-1 α and IL-1 β production. IL-1ra and IL-1 α were expressed in 10–25% of unstimulated cultured cells, while all the other cytokines were undetectable. IL-1ra, IL-1 α and IL-1 β were also the dominating cytokines, expressed in up to 85% of the DC, after 3 h of LPS stimulation. A significantly lower number of cells (0–5%) synthesized TNF- α , IL-6, IL-10, IL-12 and GM-CSF. The incidence of chemokine producing cells (IL-8, RANTES, MIP-1 α , MIP-1 β ) peaked 10 h after LPS stimulation in up to 60% of the DC. Both immature CD83− and mature CD83+ DC as well as LC had a similar cytokine production pattern. Thus, in comparison to monocytes, LPS stimulation of DC generated a lower incidence of TNF- α , IL-6, IL-10 and IL-12 producing cells while IL-1 was expressed in a comparable number of cells.
Journal of Immunological Methods – Elsevier
Published: May 1, 1998
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