Figures & data
Table 1. Clinical parameters
Table 2. Primers
Fig. 1. Analyses of IL-32 and IL-8 expressed in healthy and inflamed human gingival tissue. (A) In order to determine the production of IL-32 by ELISA, both healthy gingival tissue (gingival pocket depth ≤ 3mm; n = 5, two males and three females, aged 29–43 years) and inflamed gingival tissue (gingival pocket depth > 4mm; n = 5, four males and two females, aged 20–55 years) were collected from volunteer patients. The gingival tissue was homogenized for the sample. IL-32 present in the gingival tissue homogenates was measured using ELISA. (B) IL-8 production was measured by ELISA Development kit in accordance with the manufacturer's instructions. The values represent the means and standard deviations of triplicate experiments. *Significantly higher than healthy gingival tissue, by Student's t-test (P<0.01). (C–H) Immunohistochemical analysis of human IL-32 and IL-8 production in the gingival tissue: IL-32 production in human healthy (C, E) and inflamed (D, F) gingival tissues was determined using immunohistochemical staining, following the previously published protocol. For IL-32-positive staining, mouse monoclonal anti-IL-32 was utilized to detect the production of IL-32 (C, D). As a negative control, non-immune mouse IgG was used (E, F). In order to show the inflammation of the tissue, IL-8 was stained following the published procedure. In the positively stained samples, arrows indicate IL-32- and IL-8-positive cells. Original magnification is 200 times. The white bar corresponds to 50 µm.
Fig. 2. In vitro evaluation of IL-32 and IL-8 expression in human gingival fibroblasts. (A) Effects of Porphyromonas gingivalis stimulation on the production of IL-32 mRNA in human gingival fibroblasts: Primary culture of human gingival fibroblasts was stimulated with formalin fixed P. gingivalis (108 cells/ml) for 12 hours, and total RNA was extracted to perform the quantitative RT-PCR for the four different isoforms of IL-32. The mRNA expression of IL-32 was standardized by the ratio against Glyceraldehyde 3 – phosphate dehydrogenase (GAPDH). The values represent the means and standard deviations of triplicate experiments. *Significantly higher than medium control alone without bacteria by Student's t-test (P <0.01). (B) Effect of Porphyromonas gingivalis on IL-32 protein production from HGF: The production of IL-32 in contact with P. gingivalis for 24 hours was monitored using an IL-32 ELISA. The values represent the means and standard deviations of triplicate experiments. *Significantly higher than control without bacteria (the far left bar with #) by Student's t-test (P<0.01). (C) The effects of IL-32 on expression of IL-8 mRNA in HGF: In order to determine the effect of IL-32 in HGF, recombinant human IL-32γ (10 ng/ml) was applied to the culture medium with and without P. gingivalis stimulation. Anti-IL-32 polyclonal antibody (2 µg/ml) or normal goat IgG (2 µg/ml) was also added into the medium for neutralization of IL-32. Total RNA was extracted for quantitative RT-PCR to analyze the IL-8 mRNA expression after 12 hours of stimulation by P. gingivalis. The values represent the means and standard deviations of triplicate experiments. *Significantly higher than control without bacteria (the far left bar with #) by Student's t-test (P<0.01).
