In previous studies carried out on NR8383 cell line cultures (alveolar rat macrophages) enriched with 15 % fetal bovine serum (FBS) we demonstrated that the trophic components spermine and spermidine, added to cell cultures during 18 h, produced a dose-dependent decrease of lipopolysaccharide (LPS)-induced tumour necrosis factor (TNFα) secretion.

We have determined here the effect of these polyamines on the cytokine secretion of NR8383 macrophages under different experimental conditions. We have quantified the secretion of TNFα and monocyte chemoattractant protein-1 (MCP1) after LPS stimulation in macrophages treated with either spermine or spermidine at different times and under different growing conditions.

Cells were plated (10⁶ cells per well) and incubated with several dilutions of spermine (0-1000 µM) and spermidine (0-2000 µM) from 18 h pre- to 1.5 h post-stimulation with 10 ng ml^-1 LPS in 0, 1 and 15 % FBS supplemented cultures. Cells and supernatants were collected after 3 h of LPS stimulation. Cells were used to determine cell viability by flow cytometry and to examine relative levels of TNFα gene expression by RT-PCR after RNA extraction. TNFα and MCP1 secretion in supernatants were measured by a sandwich enzyme immunosorbent assay (ELISA) technique.

Inhibitory concentrations 50 (IC₅₀) for TNFα secretion were about 20-250 µM and 100-1000 µM for spermine and spermidine respectively depending on the different conditions assayed. Maximal inhibition was found when polyamines were added at the same time as LPS and without FBS enrichment. However, both polyamines failed to decrease mRNA levels for TNFα showing a post-transcriptional effect on protein production. When MCP1 secretion was studied, similar results were observed and spermine also exercised the maximal inhibition in cultures without FBS.

In conclusion, spermine and spermidine inhibited the expression of the pro-inflamatory cytokines TNFα and MCP1 under different in vitro assay conditions. The immunomodulatory effect of these polyamines was shown to be inversely related to the incubation time of polyamines and FBS concentration in the medium.

This work was supported by the Ministerio de Educación y Cultura (AGL-2000-0913).