Human gingiva-derived mesenchymal stromal cells attenuate contact hypersensitivity via prostaglandin E2-dependent mechanisms.

The immunomodulatory and anti-inflammatory functions of mesenchymal stromal cells (MSCs) have been demonstrated in several autoimmune/inflammatory disease models, but their contribution to the mitigation of contact hypersensitivity (CHS) remains unclear. Here, we report a new immunological approach using human gingiva-derived MSCs (GMSCs) to desensitize and suppress CHS and the underlying mechanisms. Our results showed that systemic infusion of GMSCs before the sensitization and challenge phase dramatically suppress CHS, manifested as a decreased infiltration of dendritic cells (DCs), CD8(+) T cells, T(H)-17 and mast cells (MCs), a suppression of a variety of inflammatory cytokines, and a reciprocal increased infiltration of regulatory T cells and expression of IL-10 at the regional lymph nodes and the allergic contact areas. The GMSC-mediated immunosuppressive effects and mitigation of CHS were significantly abrogated on pretreatment with indomethacin, an inhibitor of cyclooxygenases. Under coculture condition of direct cell-cell contact or via transwell system, GMSCs were capable of direct suppression of differentiation of DCs and phorbol 12-myristate 13-acetate-stimulated activation of MCs, whereas the inhibitory effects were attenuated by indomethacin. Mechanistically, GMSC-induced blockage of de novo synthesis of proinflammatory cytokines by MCs is mediated partly by the tumor necrosis factor-alpha/prostaglandin E(2) (PGE(2)) feedback axis. These results demonstrate that GMSCs are capable of desensitizing allergic contact dermatitis via PGE(2)-dependent mechanisms.

Human gingiva-derived mesenchymal stem cells elicit polarization of m2 macrophages and enhance cutaneous wound healing.

Increasing evidence has supported the important role of mesenchymal stem cells (MSCs) in wound healing, however, the underlying mechanism remains unclear. Recently, we have isolated a unique population of MSCs from human gingiva (GMSCs) with similar stem cell-like properties, immunosuppressive, and anti-inflammatory functions as human bone marrow-derived MSCs (BMSCs). We describe here the interplay between GMSCs and macrophages and the potential relevance in skin wound healing. When cocultured with GMSCs, macrophages acquired an anti-inflammatory M2 phenotype characterized by an increased expression of mannose receptor (MR; CD206) and secretory cytokines interleukin (IL)-10 and IL-6, a suppressed production of tumor necrosis factor (TNF)-α, and decreased ability to induce Th-17 cell expansion. In vivo, we demonstrated that systemically infused GMSCs could home to the wound site in a tight spatial interaction with host macrophages, promoted them toward M2 polarization, and significantly enhanced wound repair. Mechanistically, GMSC treatment mitigated local inflammation mediated by a suppressed infiltration of inflammatory cells and production of IL-6 and TNF-α, and an increased expression of IL-10. The GMSC-induced suppression of TNF-α secretion by macrophages appears to correlate with impaired activation of NFκB p50. These findings provide first evidence that GMSCs are capable to elicit M2 polarization of macrophages, which might contribute to a marked acceleration of wound healing.

Blocking gastrin and CCK-B autocrine loop affects cell proliferation and apoptosis in vitro.

Gastrin and cholecystokinin-B receptor (CCK-B) were co-expressed in human gastric carcinoma tissues, suggesting that a functional autocrine loop, the gastrin and CCK-B receptor loop, may be presented in gastric cancer cells and play an important role in the pathogenesis and progression of gastric carcinomas. The present study was aimed at studying the effects of blocking the gastrin and CCK-B receptor loop on cell proliferation and apoptosis in gastric cancer cell line SGC-7901 cells (SGC-7901 cells). First, the expression of gastrin and CCK-B receptor mRNAs and gastrin protein in SGC-7901 cells were measured by RT-PCR and immunocytochemistry, respectively. Radioimmunoassay (RIA) was used to detect the concentrations of gastrin in culture medium. The gastrin-CCK-B receptor axis was blocked by using a specific neutralizing antibody against human gastrin and siRNA specifically targeting human CCK-B receptors, respectively. Flow cytometry was used to measure the cell cycle and apoptotic cells, and western blotting was used to measure the expression of CCK-B receptor, caspase-3, and matrix metalloproteinase-2 (MMP-2) in cells. The results showed that SGC-7901 cells not only coexpressed gastrin and CCK-B receptor mRNAs, but also endogenously secreted gastrin protein into the culture medium, thus forming gastrin-CCK-B receptor autocrine loop. Biologically, disrupting gastrin-CCK-B receptor autocrine loop by neutralizing the endogenous gastrin or by knocking down CCK-B receptor expression significantly inhibited the cell proliferation and decreased the percentage of cells residing in the S-phase of the cell cycle, and meanwhile promoted cell apoptosis and increased caspase-3 expression as well as decreased MMP-2 expression. An autocrine loop between endogenously secreted gastrin and CCK-B receptors may play a key role in the regulation of cell proliferation and apoptosis in SGC-7901 cells.

[Effects of green tea extract on expression of human papillomavirus type 16 oncoproteins-induced hypoxia-inducible factor-1alpha and vascular endothelial growth factor in human cervical carcinoma cells].

OBJECTIVE: To investigate the effects of green tea extract (GTE) and its major component (-)-epigallocatechin-3-gallate (EGCG) on the human papillomavirus (HPV) type 16 oncoproteins (E6 and E7)-induced expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in human cervical carcinoma cells. METHODS: Human cervical carcinoma cells of the line C-33A were divided to 4 groups to be transiently transfected with the plasmid HA-pSG5-HPV-16E6 containing the HPV type 16 oncoprotein E6, HA-pSG5-HPV-16 E7, or empty plasmid HA-pSG5, or just exposed to the transfection reagent Lipofectamine 2000 as mock transfection control group. Western blotting and ELISA were used to detect the protein expression of HIF-1alpha and the protein expression of VEGF 18, 24, and 48 h after the transfection. Twenty-four hours after the transfection, the transfected-cells were treated with GTE (40 microg/ml) or EGCG (50 micromol/L) for 8, 16, and 24 h respectively, or treated with GTE of the concentrations of 20, 40, and 80 microg/ml or EGCG of the concentrations of 25, 50, and 100 micromol/L respectively for 16 h. The HIF-1alpha and VEGF protein levels were analyzed by Western blotting and ELISA respectively and the HIF-1alpha and VEGF mRNA levels were determined with real-time PCR. RESULTS: Twenty-four hours after transfection, the HIF-1alpha protein expression of the 16E6 and 16E7 groups were significantly decreased, and the VEGF protein levels of the 16E6 and 16E7 groups were (870+/-66) and (1487+/-51) pg/ml respectively, both significantly higher than that of the empty plasmid group [(366+/-65) pg/ml, both P<0.01]. Treated by 40 microg/ml of GTE or 50 micromol/L of EGCG for 16 h, (1) the HIF-1alpha protein levels of the 16E6 and 16E7 groups were significantly decreased; (2) the VEGF protein levels of the 16E6 group were (476+/-34) and (477+/-65) pg/ml respectively, and the VEGF mRNA levels of the 16E6 group were 1.208+/-0.196 and 1.174+/-0.208 respectively, all significantly lower than those of the untreated group [(870+/-66) pg/ml and 1.805+/-0.081 respectively, all P<0.01]; and (3) the VEGF protein levels of the 16E7 group were (649+/-55) and (514+/-37) pg/ml respectively, and the VEGF mRNA levels of the 16E7 group were 1.442+/-0.136 and 1.399+/-0.064 respectively, all significantly lower than those of untreated group [(1487+/-51) pg/ml and 2.123+/-0.120 respectively, all P<0.01]. The 80 microg/ml of GTE or 100 micromol/L of EGCG showed much stronger effects on the inhibition of VEGF protein expression than 40 microg/ml of GTE or 50 micromol/L of EGCG (both P<0.01). CONCLUSION: GTE and EGCG can remarkably inhibit the HPV-16 oncoproteins-induced expression of HIF-1alpha protein, and VEGF protein and mRNA in human cervical carcinoma cells. Moreover, GTE and EGCG decrease the VEGF protein expression dose-dependently.

Coexpression of cholecystokinin-B/gastrin receptor and gastrin gene in human gastric tissues and gastric cancer cell line.

AIM: To compare the expression patterns of cholecystokinin-B (CCK-B)/gastrin receptor genes in matched human gastric carcinoma and adjacent non-neoplastic mucosa of patients with gastric cancer, inflammatory gastric mucosa from patients with gastritis, normal stomachs from 2 autopsied patients and a gastric carcinoma cell line (SGC-7901), and to explore their relationship with progression to malignancy of human gastric carcinomas. METHODS: RT-PCR and sequencing were employed to detect the mRNA expression levels of CCK-B receptor and gastrin gene in specimens from 30 patients with gastric carcinoma and healthy bordering non-cancerous mucosa, 10 gastritis patients and normal stomachs from 2 autopsied patients as well as SGC-7901. The results were semi-quantified by normalizing it to the mRNA level of beta-actin gene using Lab Image software. The sequences were analyzed by BLAST program. RESULTS: CCK-B receptor transcripts were detected in all of human gastric tissues in this study, including normal, inflammatory and malignant tissues and SGC-7901. However, the expression levels of CCK-B receptor in normal gastric tissues were higher than those in other groups (P<0.05), and its expressions did not correlate with the differentiation and metastasis of gastric cancer (P>0.05). On the other hand, gastrin mRNA was detected in SGC-7901 and in specimens obtained from gastric cancer patients (22/30) but not in other gastric tissues, and its expression was highly correlated with the metastases of gastric cancer (P<0.05). CONCLUSION: Human gastric carcinomas and gastric cancer cell line SGC-7901 cells coexpress CCK-B receptor and gastrin mRNA. Gastrin/CCK-B receptor autocrine or paracrine pathway may possibly play an important role in the progression of gastric cancer.