Influence Of Carbon Monoxide Releasing Molecule On The Expression Of Adhesion Molecules On Human Gingival Fibroblasts Stimulated With Inflammatory Cytokines

Chronic periodontitis (CP) is a kind of destructive oral disease which is the main reason for tooth loss in adults at present. CP is usually characterized by chronic inflammation associated with pathogenic bacteria in sub-gingival plaque, resulting in soft tissue destruction, alveolar bone resorption and tooth loss eventually. Gram-negative bacteria-dominated pathogens in subgingival plaque can not only cause direct damage to periodontal tissue, but also stimulate host immune response via the lipopolysaccharide (LPS) and the subsequent inflammatory cytokines. One of the prominent features of chronic periodontitis is dense inflammatory cell accumulation, e.g. monocyte/macrophage, lymphocyte, as well as their cytokines in extravascular periodontal connective tissues. In particular, proinflammatory cytokines TNF-a and IL-1βhave been demonstrated to be the most important ones involved in periodontal tissue destruction.The migration and infiltration of immuno-active cells is a highly regulated process that is dependent on various cell surface adhesion molecules on vascular endothelial cells, inflammatory cells and fibroblasts. In particular, the critical roles of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and endothelial leukocyte adhesion molecule (E-selectin) in inflammation have been widely studied.Fibroblasts are the most abundant cells in periodontal connective tissue. In addition to its role in maintaining periodontal tissue integrity and homeostasis, its role in regulating local inflammatory response has been well investigated. Under inflammatory condition, immuno-responsive cells, e.g. leukocytes and lymphocytes, adhere to and then infiltrate through vascular wall as a consequence of up-regulation of surface adhesion molecules on endothelial cells. Similarly, ICAM-1, VCAM-1 and E-selectin are also highly regulated in gingival fibroblasts in response to inflammatory chemokines, such as TNF-α, IL-1βand bacterial endotoxin LPS. The up-regulation of these adhesion molecules on gingival fibroblasts seems to play a crucial role in further leukocyte extravasations into the inflammatory periodontal connective tissues. Gingival fibroblast therefore represents an important target for intervention in periodontical disease therapy.Carbon monoxide (CO), an endogenous by-product of heme metabolism by heme oxygenase (HO), was shown to play important roles in multi-cellular events, including inhibition of cell proliferation, cell apoptosis and suppression of inflammation. The effect of CO was mediated by induction of HO-1, regulation of p38 MAPK signalling pathway and activation of guanylate cyclase. Recently, Motterlini et al reported a class of compounds, termed CO releasing molecules (CORMs), which are able to release CO in a controllable manner under physiological conditions. In particular, CORM-3 is fully water-soluble, can rapidly liberate CO once dissolved in physiological solutions. These molecules might therefore be of therapeutic interest to exert important pharmacological activities by carrying and delivering CO in a controllable fashion.In the present study, we investigated the influence of CORM-3 on the expression of adhesion molecules on human gingival fibroblasts stimulated by TNF-αand IL-1βconcurrently, and the possible mechanism by which CORM-3 exerts this effect.Material and methodsPartⅠ: Influence of CORM-3 on the expression of adhesion molecules on human gingival fibroblasts stimulated with TNF-αand IL-1βHuman gingival fibroblasts (HGF) were established from the explants of normal gingival tissues obtained during routine clinical procedures. Explants were washed and then minced with scissors into small pieces (1-3mm2) and cultured in DMEM supplemented with 10% FBS, penicillin 50IU/ml and streptomycin 50IU/ml in humidified air with 5% CO2 at 37℃. Cellular lactate dehydrogenase (LDH) assay was used to test the cytotoxicity of CORM-3 at 500μM to HGF or PBMC. To mimic the inflammatory environment, TNF-a at 50ng.ml-1 and IL-1βat lOng.ml-1 were used to stimulate HGF concurrently in the presence or absence of CORM-3 for different time periods. Expression of ICAM-1, VCAM-1 and E-selectin at mRNA and protein level was studied by means of RT-PCR and Western blot, respectively. To demonstrate that the regulation of CORM-3on the expression of adhesion molecules on HGF is via CO, degassed CORM-3 was also included in the experiment. Functional attachment assay was designed to check whether the down-regulation of CORM-3 on the expression of adhesion molecule can lead to a decreased attachment of peripheral blood mononuclear cells (PBMC) to HGF. PBMC were isolated from freshly drawn healthy human blood by volunteers by gradient density centrifugation. CFSE-DA was applied to label PBMC and the attachment of PBMC to HGF in the presence or absence of CORM-3 was checked by fluorescence microscopy.Part II:The mechanism of the down-regulation of the adhesion molecules on HGF by CORM-3To elucidate whether guanylate cyclase (GC) pathway was involved in the regulatory effect of CORM-3, an inhibitor of GC, ODQ was included in the experiment. HGF were co-transfected with a reporter construct containing NF-κB response elements (6-κB.luc) and renilla luciferase reporter, Dual-Glo Luciferase Assay System was used to assess the NF-κB activity of HGF in the response to inflammatory cytokines in the presence or absence of CORM-3. Nuclear protein was extracted and Western blot was applied to check the nuclear expression of NF-KB-p50 and NF-KB-p65. The possible role of mitogen-activated protein kinase (MAPK) system, including extracellular regulated protein kinases (ERK), p38 and JNK, was also studied in the experiment.ResultsPart I:Based on the previous study and the preliminary results, CORM-3 at 500μM was set as the working concentration in the experiment. Results from LDH assay showed that 500μM CORM-3 was no cytotoxicity to either HGF or PBMC. Western blotting analysis and PT-PCR results showed that the protein and mRNA expression of ICAM-1, VCAM-1 or E-selectin was up-regulated dramatically by 12hr concurrent treatment of TNF-a (50ng/ml) and IL-1β(10ng/ml), respectively. While induction of the adhesion molecules by these cytokines was significantly attenuated by CORM-3 at a concentration of 500μM. The modulatory effect of CORM-3 was mediated via CO since the degassed CORM-3 lost its effect. The down-regulation of adhesion molecules by CORM-3 also led to a decreased attachment of PBMC to HGF, evidenced by the decreased number of green-fluorescence PBMC to CORM-3 pretreated HGF in the functional attachment assay. Pre-treatment of the HGF with TNF-a and IL-1βin the presence of neutralizing antibody against ICAM-1 and VCAM-1 also significantly inhibited the PBMC adhesion.Part II:Down-regulation of ICAM-1 was not mediated via cGMP since ODQ, a specific inhibitor of GC by oxidation of enzyme heme, did not abrogate the effect of CORM-3. Dual-Glo luciferase activity assay showed that within 2 hr of TNF-a and IL-1βco-stimulation, NF-KB-binding activity was not significantly influenced by CORM-3. While NF-κB binding activity was significantly reduced at later time points of TNF-a and IL-1βco-stimulation in CORM-3-treated cells. In the mean time, the nuclear expression of NF-κB p65, rather than p50, was significantly decreased by CORM-3 treatment. TNF-a and IL-1βconcurrent stimulation also induced the expression of phosphorylated p38 dramatically, which was significantly attenuated by CORM-3 treatment. Though the expression of phosphorylated ERK and JNK was also augmented by TNF-αand IL-1βconcurrent stimulation, it was not influenced by CORM-3.ConclusionsIn the present study, we investigated the anti-inflammatory effect of CORM-3 on human gingival fibroblasts. The main findings of this study were the following. First, CORM-3, via its released CO, inhibited the up-regulation of ICAM-1, VCAM-1 and E-selectin after TNF-αand IL-1βconcurrent stimulation on human gingival fibroblasts, which also resulted in a decreased mononuclear cell adhesion to these cells. Second, initial activation of the NF-κB pathway by TNF-αand IL-1β co-stimulation was not influenced by CORM-3. Instead, CORM-3 suppressed sustained activation of this pathway, which was reflected by a decreased nuclear NF-KB-p65 expression. Third, CORM-3 inhibited MAPK p38 phosphorylation in response to proinflammatory cytokine stimulation.Since up-regulation of adhesion molecules on HGF plays a key role in recruitment and retention of immuno-active cells at sites of periodontal inflammatory disease, which has been regarded as one of the most important determinants of disease progression. The results of this study bode well for the application of CORM-3 as an anti-inflammatory agent to inhibit NF-κB activity and to suppress the expression of adhesion molecules on HGF, which suggests a promising potential of CORM-3 in inflammatory periodontal disease treatment.