Jochen Winter

Phl p 5 resorption in human oral mucosa leads to dose-dependent and time-dependent allergen binding by oral mucosal Langerhans cells, attenuates their maturation, and enhances their migratory and TGF-β1 and IL-10–producing properties

BACKGROUND Sublingual immunotherapy (SLIT) is safe and effective as treatment of allergic rhinitis and mild asthma. Oral mucosal Langerhans cells (oLCs) play a central role. However, little is known about allergen binding by oLCs during mucosal allergen resorption and its impact on oLC functions. OBJECTIVE Binding of Phl p 5 to oLCs was studied in a standardized ex vivo model to investigate mechanisms important for SLIT. METHODS Human oral mucosal biopsies were incubated with the grass pollen allergen Phl p 5. Migration, binding of Phl p 5, phenotype and cytokine production, and T-cell priming of Phl p 5-binding oLCs were analyzed. RESULTS Significant uptake required more than 5 minutes, and dose-dependent binding of Phl p 5 to oLCs was saturated at 100 microg/mL Phl p 5. Furthermore, Phl p 5 significantly increased the migratory capacity of oLCs but attenuated their maturation and strongly promoted the release of TGF-beta1 and IL-10 by oLCs themselves as well as by cocultured T cells. CONCLUSION Oral mucosal Langerhans cells bind Phlp5 in a dose-dependent and time-dependent manner, leading to an increased production of tolerogenic cytokines and an enhanced migratory capacity but decelerated maturation of oLCs.

IL-23-producing CD68+ macrophage-like cells predominate within an IL-17-polarized infiltrate in chronic periodontitis lesions

AIM To analyse antigen-presenting cells (APCs), such as dendritic cells (DCs), macrophages (Mo) or B cells depending on the regional site of chronic periodontitis (CP), and to investigate their relation to Th17 cells. MATERIAL AND METHODS Biopsies from oral mucosa as well as the coronal and bottom regions of CP were analysed by immunhistochemistry, immunofluorescence, flow cytometry and real-time PCR. RESULTS A predominance of CD68(+) Mo-like cells and CD20(+) B cells and strong Th17 infiltration was observed in the bottom region of CP lesions, while CD1a(+) DCs were only detected in the coronal regions, where Th17 infiltration was low. Furthermore, CD68(+) Mo-like cells displayed CD163 expression as a typical Mo-marker, but expressed in parallel typical DCs markers, such as CD11c or CD209 and TLR4. Interestingly, Th17-inducing cytokine IL-23p19 was produced by CD68(+) Mo-like cells, but not CD20(+) B cells. Moreover, the stimulation of in vitro generated CD68(+) Mo-like cells by Porphyromonas gingivalis-derived (Pg) lipopolysaccharide resulted in the upregulation of their IL-23p19 mRNA expression, which was inhibited by the blockage of TLR4. CONCLUSIONS In view of these data, a picture emerges that IL-17-producing cells in CP could be in part directed by CD68(+) Mo-like cells, which produce IL-23p19 upon TLR4 activation by Pg.

Interactions of Adiponectin and Lipopolysaccharide from Porphyromonas gingivalis on Human Oral Epithelial Cells

Background Periodontitis is an inflammatory disease caused by pathogenic microorganisms, such as Porphyromonas gingivalis, and characterized by the destruction of the periodontium. Obese individuals have an increased risk for periodontitis and show decreased serum levels of adiponectin. This in-vitro study was established to examine whether adiponectin modulates critical effects of lipopolysaccharide (LPS) from P. gingivalis on oral epithelial cells (OECs). Methodology/Principal Findings The presence of adiponectin and its receptors in human gingival tissue samples and OECs was analyzed by immunohistochemistry and PCR. Furthermore, OECs were treated with LPS and/or adiponectin for up to 72 h, and the gene expression and protein synthesis of pro- and anti-inflammatory mediators, matrix metalloproteinases (MMPs) and growth factors were analyzed by real-time PCR and ELISA. Additionally, cell proliferation, differentiation and in-vitro wound healing were studied. The nuclear translocation of NFκB was investigated by immunofluorescence. Gingival tissue sections showed a strong synthesis of adiponectin and its receptors in the epithelial layer. In cell cultures, LPS induced a significant up-regulation of interleukin (IL) 1β, IL6, IL8, MMP1 and MMP3. Adiponectin abrogated significantly the stimulatory effects of LPS on these molecules. Similarly, adiponectin inhibited significantly the LPS-induced decrease in cell viability and increase in cell proliferation and differentiation. Adiponectin led to a time-dependent induction of the anti-inflammatory mediators IL10 and heme oxygenase 1, and blocked the LPS-stimulated NFκB nuclear translocation. Conclusions/Significance Adiponectin may counteract critical actions of P. gingivalis on oral epithelial cells. Low levels of adiponectin, as observed in obese individuals, may increase the risk for periodontal inflammation and destruction.

Human β-defensins differently affect proliferation, differentiation, and mineralization of osteoblast-like MG63 cells.

Purpose of this study was to investigate whether human β‐defensins (hBDs) affect maturation and proliferation of osteoblast‐like MG63 cells in vitro. Osteoblast‐like MG63 cells were stimulated with hBD‐1, ‐2, and ‐3 under control conditions and with hBD‐2 during experimental inflammation (induced by interleukin‐1β, tumor necrosis factor‐α, toll‐like receptor‐2 and ‐4 agonists). Expression of different osteogenic markers and hBDs were analyzed by real‐time PCR, immunohistochemistry, and enzyme‐linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were monitored. All tested hBDs were expressed on mRNA and protein level in MG63 cells. Only stimulation with hBD‐2 elevated the proliferation rate. hBD‐2 and hBD‐3 positively affected the differentiation of osteoblast‐like cells provided by increased transcript levels of osteogenic markers, up‐regulated ALP enzyme activity and enhanced mineralized nodule formation. All pro‐inflammatory stimuli enhanced interleukin‐6 and hBD‐2 expression and down‐regulated markers of osteoblastic differentiation. In accordance, inflammation increased transcript level of Notch‐1 (an inhibitor of osteoblastic differentiation). hBD‐2 was not able to revert effects of inflammation on differentiation. In bone cells human β‐defensins exhibit further functions than antimicrobial peptide activity. These include stimulation of proliferation and differentiation. Differentiation arrest due to inflammation could not be overcome by hBD‐2 alone. J. Cell. Physiol. 227: 994–1003, 2012.

Human Beta-Defensin-1, -2, and -3 Exhibit Opposite Effects on Oral Squamous Cell Carcinoma Cell Proliferation

The objective of this study was to investigate the impact of human beta-defensins (hBDs) on oral squamous cell carcinoma (OSCC) proliferation and hBD expression in vitro. BHY-OSCC cell lines were stimulated with hBD-1, -2, and -3. Proliferation of BHY cells was ascertained and hBD-mRNA expression was evaluated by real-time PCR. Proliferation of BHY cells decreased by 25% in response to hBD-1 stimulation but increased after stimulation with hBD-2 and -3. HBD-1 stimulation enhanced hBD-3 expression, whereas HBD-2 stimulation decreased early hBD-3 expression. HBD-3 stimulation enhanced hBD-1 expression. HBDs profoundly impact on OSCC proliferation and hBD expression in vitro. Therefore, hBD-1 might function as a tumor suppressor gene in OSCCs, while hBD-2 and -3 might be protooncogenes.

Decreased gene expression of human β-defensin-1 in the development of squamous cell carcinoma of the oral cavity

The aim of this study was to investigate the gene expression of human beta-defensin-1, -2, -3 (hBD-1, -2, -3), interleukin-1beta, tumour necrosis factor-alpha and cyclooxygenase-2 in oral squamous cell carcinoma (OSCC) compared to benign and premalignant lesions as well as healthy controls. Biopsies of healthy gingiva (n=5), irritation fibroma (n=5), leukoplakia (n=5) and OSCC (n=5) were obtained during routine surgical procedures. RNA was extracted according to standard protocols and transcripts of hBD-1, -2, -3, interleukin-1beta, tumour necrosis factor-alpha and cyclooxygenase-2 were analysed by real-time polymerase chain reaction. The expression of hBD-1 was reduced in all lesions (5-fold in irritation fibroma and 2.5-fold in leukoplakia), but most significantly (50-fold) in OSCC. hBD-1 appears to play a role in the development of OSCC. The loss of its function might contribute to the malignant progression of these tumours.

Effects of enamel matrix derivative on periodontal wound healing in an inflammatory environment in vitro

AIM This in vitro study was established to investigate whether the regenerative capacity of periodontal ligament (PDL) cells in the presence of enamel matrix derivative (EMD) is modulated by inflammation. MATERIALS AND METHODS PDL cells were grown in the presence or absence of EMD under normal and inflammatory conditions for up to 14 days. In order to mimic an inflammatory environment, cells were incubated with interleukin (IL)-1β. Cells were also exposed to transforming growth factor (TGF)-β1 and insulin-like growth factor (IGF)-1 under both conditions. For analysis of wound healing, an in vitro wound fill assay was used. The synthesis of growth factors, markers of proliferation, and osteogenic differentiation, as well as collagen was studied by real-time polymerase chain reaction, enzyme-linked immunoassay, and immunoblotting. Mineralization was assessed by alizarine red S and von Kossa staining. RESULTS EMD stimulated significantly the in vitro wound fill rate, cell proliferation and adhesion, synthesis of growth factors, and collagen, as well as mineralization. In the presence of IL-1β, these EMD effects were significantly reduced. IL-1β also inhibited significantly the wound fill rate induced by TGF-β1 and IGF-1. CONCLUSIONS Critical PDL cell functions that are associated with periodontal regeneration are reduced in an inflammatory environment.

Nuclear hBD-1 accumulation in malignant salivary gland tumours

BackgroundWhereas the antimicrobial peptides hBD-2 and -3 are related to inflammation, the constitutively expressed hBD-1 might function as 8p tumour suppressor gene and thus play a key role in control of transcription and induction of apoptosis in malignant epithelial tumours. Therefore this study was conducted to characterise proteins involved in cell cycle control and host defence in different benign and malignant salivary gland tumours in comparison with healthy salivary gland tissue.Methods21 paraffin-embedded tissue samples of benign (n = 7), and malignant (n = 7) salivary gland tumours as well as healthy (n = 7) salivary glands were examined immunohistochemically for the expression of p53, bcl-2, and hBD-1, -2, -3.ResultsHBD-1 was distributed in the cytoplasm of healthy salivary glands and benign salivary gland tumours but seems to migrate into the nucleus of malignant salivary gland tumours. Pleomorphic adenomas showed cytoplasmic as well as weak nuclear hBD-1 staining.ConclusionHBD-1, 2 and 3 are traceable in healthy salivary gland tissue as well as in benign and malignant salivary gland tumours. As hBD-1 is shifted from the cytoplasm to the nucleus in malignant salivary gland tumours, we hypothesize that it might play a role in the oncogenesis of these tumours. In pleomorphic adenomas hBD-1 might be connected to their biologic behaviour of recurrence and malignant transformation.

Human periodontal ligament cells facilitate leukocyte recruitment and are influenced in their immunomodulatory function by Th17 cytokine release.

The objective of this in vitro study was to examine the immunomodulatory impact of human periodontal ligament (PDL) cells on the nature and magnitude of the leukocyte infiltrate in periodontal inflammation, particularly with regard to Th17 cells. PDL cells were challenged with pro-inflammatory cytokines (IL-1ß, IL-17A, and IFN-γ) and analyzed for the expression of cytokines involved in periodontal immunoinflammatory processes (IL-6, MIP-3 alpha, IL-23A, TGFß1, IDO, and CD274). In order to further investigate a direct involvement of PDL cells in leukocyte function, co-culture experiments were conducted. The expression of the immunomodulatory cytokines studied was significantly increased under pro-inflammatory conditions in PDL cells. Although PDL cells did not stimulate leukocyte proliferation or Th17 differentiation, these cells induced the recruitment of leukocytes. The results of our study suggest that PDL cells might be involved in chronic inflammatory mechanisms in periodontal tissues and thus in the transition to an adaptive immune response in periodontitis.

Gene expression of oncogenes, antimicrobial peptides, and cytokines in the development of oral leukoplakia.

OBJECTIVE The aim of this study was to investigate the expression pattern of oncogenes, antimicrobial peptides, and genes involved in inflammation in leukoplakia of the oral cavity compared with healthy gingiva. STUDY DESIGN Biopsies of healthy gingiva (n=20) and leukoplakia (n=20), were obtained during routine surgical procedures. RNA was extracted according to standard protocols. Transcript levels of alpha-defensin (DEFA) 1/3, DEFA-4, S100-A7, deleted-in-oral-cancer (Doc) 1, interleukin (IL) 1beta, IL-6, IL-8, IL-10, tumor necrosis factor (TNF) alpha, cyclooxygenase (Cox) 2, epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor (TGF) beta1, TGF-alpha, collagen-IA1 (Col-1), and tenascin-c were analyzed by real-time reverse-transcription polymerase chain reaction. The proteins encoded by the different genes were visualized by immunostaining. RESULTS Compared with healthy gingiva (set as 1), there was an increased gene expression of DEFA-4 (179.2-fold), S100-A7 (25.4-fold), EGF (24.8-fold), TGF-beta1 (25.2-fold), and tenascin-c (34.3-fold) in oral leukoplakia. The expression of IL-1beta and Doc-1 was decreased (0.01-fold and 0.2-fold, respectively). CONCLUSIONS The combination of an increased expression of the antimicrobial peptide DEFA-4, the oncogene S100-A7, EGF, and tenascin-c, and a decreased Doc-1 expression in oral leukoplakia might characterize its potency of malignant transformation. Chronic inflammation seems not to be involved in the development of this lesion.