Objectives: Interleukin-32 (IL-32) has recently been identified as a pro-inflammatory mediator capable of inducing cytokine and chemokine expression via p38 MAPK, NF-κB intracellular pathways. Furthermore, IL-32 expression correlates with levels of inflammation in inflammatory disease for example, rheumatoid arthritis. The aim of this study was to investigate IL-32 expression in monocytes in response to lipopolysaccharide (LPS) from Porphyromonas gingivalis and to determine whether IL-32 played a role in the pathogenesis of periodontal disease. Therefore serum and saliva samples from patients with periodontitis were analysed to determine whether IL-32 levels correlated with disease severity.
Methods: THP-1 monocytes were stimulated with P. gingivalis LPS (100ng/ml) for 2, 6 or 24 hours. RNA was extracted from the cells and IL-32 gene expression was quantified by real-time PCR. Intracellular expression of IL-32 and nucleoli were detected using IL-32 and fibrillarin specific antibodies respectively. Antibody binding was visualised using FITC and TRITC conjugated secondary antibodies and fluorescent confocal microscopy. Secreted cytokine levels in tissue culture supernatants were quantified by ELISA for TNF-α and IL-32.
Results: IL-32 gene expression was highly upregulated following stimulation with P. gingivalis LPS with maximal expression levels occurring between 6 and 24 hours. Confocal microscopy revealed that IL-32 protein was present in unstimulated cells as well as in those treated with P. gingivalis LPS. IL-32 was found to localise to the cytosol and co-localises with fibrillarin; a protein found only in nucleoli. No secreted IL-32 was detected in tissue culture supernatants from monocytes or in serum and saliva samples from individuals with periodontitis.
Conclusion: P. gingivalis highly upregulates IL-32 at the mRNA level. However, secreted IL-32 was not detected in tissue culture supernatants or in serum. These results indicate that IL-32 may have an intracellular role in immune responses but a wider role in periodontal disease remains to be determined.