Effect of Colchicine on Inducible Nitric Oxide Synthase Activity and Nitric Oxide Production of Mice Induced by <i>Aggregatibacter actinomycetemcomitans</i>

Objective: Colchicine induced a non-protective Th2-like immunity in Aggregatibacter actinomycetemcomitans-stimulated murine immune response. The aim of the present study was to determine whether colchicine affects inducible nitric oxide synthase (iNOS) activity and nitric oxide (NO) production in A. actinomycetemcomitans-immunized mice. Materials and Methods: BALB/c mice were sham-immunized (group I) or immunized with heat-killed A. actinomycetemcomitans (group II-VII). Colchicine was injected intraperitoneally before (group III), on the same day of (group IV), or after (group V) the primary immunization and on the same day of (group VI) or after (group VII) the secondary immunization. In vitro, spleen cells from either sham- or heat-killed A. actinomycetemcomitan-immunized animals were cultured and stimulated with heat-killed A. actinomycetemcomitans in the presence or absence of colchicine with or without addition of L-arginine, Db-cAMP, forskolin or interferon-γ (IFN-γ). The levels of splenic iNOS activity and both serum and culture supernatant NO levels were assessed. Results: The results showed that colchicine did inhibit both splenic iNOS activity and serum NO levels only when the drug was injected at the same time as the immunization (group IV and VI). Splenic iNOS activity and NO levels on antigen-stimulated spleen cell cultures were also suppressed by colchicine, even in the presence of L-arginine, Db-AMP or forskolin. IFN-γ only partially restored iNOS activity and NO levels in the antigen and colchicine-treated spleen cell cultures. Conclusion: This study suggests, therefore, that colchicine may suppress the iNOS activity and NO production in A. actinomycetemcomitans-immunized mice in vivo and in vitro.

The Role of Prostaglandin E2 on Osteoblast Proliferation Induced by Hydroxyapatite

Objective: Prostaglandin E2 (PGE2) plays a crucial role in regulating bone cell differentiation and proliferation. The aim of the present study was to determine whether PGE2 may regulate osteoblast proliferation induced by hydroxyapatite. Materials and Methods: Osteoblasts (HOS cell line) pre-treated with cyclooxygenase (COX) inhibitors (indomethacin, aspirin and nimesulide) were then cultured. The cells were also pre-treated with or without nimesulide and then cultured with PGE2. The cell cultures were also treated with SQ22536 (adenylyl cyclase inhibitor) and added with Db-cAMP (cAMP analog), and/or PGE2. KT5720 [protein kinase A (PKA) inhibitor.], Db-cAMP and/or forskolin (adenylyl cyclase activator)-treated cultures were used to assess the role of PKA. The role of EP2 and/or EP4 was determined by using EP2 antagonist (PF-04418948) and EP4 antagonist (L-161,982) with PGE2. All cells were cultured with or without hydroxyapatite. The levels of PGE2 and cAMP were detected from the culture supernantants and the cell proliferation was assessed colorimetrically. Results: Nimesulide and indomethacin but not aspirin suppressed partially the cell proliferation but fully PGE2 production. PGE2 abrogated nimesulide-mediated suppression of cell proliferation. The cell proliferation was enhanced by low but suppressed by high concentration of PGE2. Moreover, the SQ22536-mediated suppression of cell proliferation was abolished by Db-cAMP but not PGE2. Conversely, PGE2, Db-cAMP or forskolin failed to eliminate KT5720-mediated suppression of cell proliferation. The effect of PGE2 on cell proliferation and cAMP levels was mediated predominantly via EP2 and to a lesser extent, EP4. The results of the controls for all experiments were significantly lower than hydroxyapatite-stimulated cell cultures. Conclusion: These results suggest thatPGE2, acting via a COX-2-, cAMP-PKA- and both EP2 and EP4-dependent pathway may partially regulate hydroxyapatite-induced human osteoblasts in an autocrine fashion.