Objectives:
This study evaluates commonly available monocytic cell lines as possible systems to study the regulation of IL-1a production by periodontal pathogens. Methods:
Human monocytes isolated by MACS negative selection of cells from venous blood were stimulated for 6 hours as suspension or adherent cultures either in serum free conditions or with 10% human or bovine serum. Concentrations of secreted and cell-associated IL-1a protein were quantified by ELISA. Based on these pilot experiments the monocyte cell lines THP-1, U937 and Monomac-6 were then stimulated as suspension cultures in the presence of 10% bovine serum for 6 hours. Results:
In pilot experiments using isolated monocytes levels of secreted IL-1a produced in response to 10ng/ml Escherichia coli LPS were approximately seven times higher in suspension cultures (657pg/ml) compared to adherent cultures (94pg/ml). Levels of cell-associated IL-1a were also higher in suspension cultures (439pg/ml) than adherent cells (228pg/ml). The addition of serum increased the production of IL-1a but there was no significant difference between bovine and human serum.
Both THP-1 and Monomac-6 cells were highly responsive to supernatants from cultured Porphyromonas gingivalis (W50) and to E.coli LPS. However 53% of the IL-1a from Monomac-6 cells was secreted but only 16% of that made by THP-1 cells. IL-1a protein was undetectable in the stimulated U937 cells. In further experiments, we exposed Monomac-6 cells to culture supernatants of a range of bacteria. Fusobacterium nucleatum, Campylobacter rectus and Actinobacillus actinomycetemcomitans culture supernatants all induced significant IL-1a production (45±2, 18±10 and 23±13 pg/ml respectively). Prevotella intermedius only stimulated low levels of production (0.59±0.7pg/ml ) and IL-1a was undetectable in cultures treated with Strepococcus sangius and Strepococcus mutants. Conclusion:
Monomac-6 cells appear to be a useful model for the study of monocyte responsiveness to bacterial stimulation and will allow the mechanism of IL-1a stimulation to be further investigated.