Ritva Häyrinen-Immonen

Tumor Necrosis Factor-a and its Receptors, p55 and p75, in Gingiva of Adult Periodontitis

Tumor necrosis factor-a. (TNF-a), a pro-inflammatory cytokine, can stimulate matrix metalloproteinase synthesis and osteoclastic bone resorption. We hypothesized that elevated expression of TNF-a and its p55 and p75 receptors (TNF-R) in gingival tissue might associate with periodontitis. Immunohistochemistry was used for the study of the localization of TNF-a and its p55 and p75 TNF-R in adult periodontitis (AP) gingival tissue, in comparison with that in healthy control specimens. TNF-a and p55 TNF-R were detected in sulcular epithelial basal cells and in monocyte/macrophages, fibroblasts, and endothelial cells in the AP gingival tissue specimens, but mainly in fibroblasts and endothelial cells in control specimens. P75 TNF-R was occasionally found in monocyte/macrophage-like cells in gingival tissue specimens. The percentage of TNF-a-containing cells was not increased in AP compared with controls (13.2% ± 6.1% vs. 12.8% ± 7.6%), but, due to the increased cellularity of AP samples, the number of TNF-a positive cells/mm2 was clearly increased (1621 ± 663 vs. 664 ±191, p > 0.001). Thus, AP gingival tissue has an elevated expression of TNF-a and especially its p55 receptor, suggesting that TNF-a may contribute to tissue degradation in periodontitis.

Long-lasting allergic patch test reaction caused by gold

Allergic contact dermatitis caused by gold is rare, and only isolated cases have been reported. Patch Jesting with gold may cause a long‐lasing reaction. The purpose of this study is to describe a well‐studied case of gold allergy caused by denial gold crowns. A gold‐sensitized patient and a non‐sensitized control subject were examined using patch tests, immunohistochemistry. electron microscopy and blast transformation reactions. Sodium thiosulfate, auranofin and sodium thiomalate gave positive patch test reactions. Immunohistochemistry and electron microscopy were performed from biopsies taken from allergic patch lest reactions caused by gold sodium thiosulfate 1 day and 17 days after applying the patches, from normal skin and from a 17‐day‐old allergic patch test reaction caused by ammonium persulfate. Down‐regulation had taken place by 17 days in the allergic ammonium persulfate reaction, but not in the 17‐day allergic gold lest reaction. The patient reacted to all but one of the gold‐induced blast transformation tests, sodium chloroaurate being non‐inductive. The non‐sensitized control subject did not exhibit any reactions. In conclusion, gold sodium thiosulfate, gold sodium thiomalate and auranofin can be used as patch test substances for gold allergy, though long‐lasting allergic patch test reactions may develop. In vitro gold salt induced blast transformation is an alternative test for gold allergy. The slow down‐regulation of the allergic patch test reactions needs to be studied further.

Increased density of lymphocytes bearing γ/δ T-cell receptors in recurrent aphthous ulceration (RAU)

Lymphocytes bearing the T-cell receptor (TCR) gamma/delta are increased in the peripheral blood of patients with recurrent aphthous ulcers (RAU) and Behcets disease. In this study, we examined whether the density of TCR-gamma/delta bearing lymphocytes was also increased locally in RAU lesions. Ten RAU lesions from ten patients were compared with ulcer-free mucosa from sites contralateral to the lesions, and with 10 samples of clinically healthy oral mucosa taken from 10 healthy volunteers. Samples were labeled with a panel of monoclonal antibodies specific to CD3, alpha/beta TCR and gamma/delta TCR in avidin-biotin-peroxidase complex (ABC) staining. Lymphocytes expressing gamma/delta TCRs were very low in non-lesional mucosa and clinically healthy mucosa. By contrast, gamma/delta T-cells were numerous and observed in all RAU lesions especially within the epithelium, inflammatory infiltrates and at perivascular locations. The count of gamma/delta T-cells was high in connective tissue of RAU (200 +/- 126 cells/mm2) compared with connective tissue of controls (4+/-4 cells/mm2; P<0.0001) or non-lesional mucosa (5+/-7 cells/mm2). Interestingly, the density of gamma/delta T-cells was also high in the epithelium of RAU (70+/-34 cells/mm2) compared with the epithelium of non-lesional mucosa (2.8+/-06 cells/mm2; P<0.0001) or epithelium of healthy controls (1.2+/-1.5 cells/mm2; P<0.0001). Moreover, the mean percentage of gamma/delta+ T-cells among total CD3+ lymphocytes was increased in the connective tissue area from 4% and 5% in controls and non-lesional mucosa, respectively, to 19% in RAU. In epithelial areas, the average percentage was increased from 2% and 6% in controls and non-lesional mucosa, respectively, to 36% in RAU. These data showed that gamma/delta T-cells are more numerous in RAU lesions and such an increase was purely restricted to RAU inflammatory areas.

Collagenase and stromelysin in recurrent aphthous ulcers (RAU)

Six patients with recurrent aphthous ulcers were studied for the presence of matrix metalloproteinases (MMP) 1, 3, and 8 in the lesions and in the clinically unaffected control mucosa obtained from the opposite side. MMP-type specific antisera were applied in the avidin-biotin-peroxidase complex staining method. Neutrophil-type collagenase (MMP-8) was found intracellularly in the connective tissue under the necrotized epithelium, and also laterally to the ulcer in association with the basement membrane. Fibroblast-type collagenase (MMP-1) and stromelysin (MMP-3) were found in the epithelial cells adjacent to the ulcerous lesion. They were found also in the endothelium of capillary blood vessels and postcapillary venules and also in some macrophage- and fibroblast-like mononuclear cells in the lamina propria laterally to the ulcer. A small number of MMP-1 and MMP-3 positive cells were noted in the control biopsies obtained from the clinically uninvolved control mucosa. These findings suggest regional differences in the distribution of the two main collagenases, implying distinct roles in tissue destruction and remodeling.

IL-17C and its receptor IL-17RA/IL-17RE identify human oral epithelial cell as an inflammatory cell in recurrent aphthous ulcer

BACKGROUND Recurrent aphthous ulcer (RAU) is an ulcerative disease of non-keratinized oral mucosa. Colon and bronchial epithelial cells produce interleukin-17C (IL-17C) upon stimulation of Toll-like receptor 2 (TLR2), TLR3 and TLR5, which are highly expressed in epithelial cells in RAU lesions. We therefore investigated the eventual presence and function of IL-17C in cultured human oral keratinocytes (HOK) and control biopsies compared to RAU lesions. METHODS Expression of IL-17A, IL-17C, IL-17RA and IL-17RE was analysed in cultured HOK cells using quantitative real-time polymerase chain reaction (qRT-PCR). HOK cells were stimulated with IL-17C and analysed for IL-8 and tumour necrosis factor-α (TNF-α) using qRT-PCR. Control mucosa (n = 5) was immunostained for IL-17A, IL-17C, IL-8, TNF-α and mast cell tryptase and compared with RAU lesions (n = 5) using the mean grey scale value. RESULTS IL-17C, but no IL-17A, mRNA was found in cultured HOK cells. Components of the heterodimeric IL-17RA/IL-17RE receptor for IL-17C were also highly expressed. Stimulation of HOK with IL-17C increased TNF-α mRNA (P = 0.03; IL-8 increase was not statistically significant). HOK in RAU lesions stained intensively for IL-17C compared to controls (P = 0.006). This was associated with increased epithelial immunostaining of TNF-α (P = 0.04) and IL-8 (P = 0.02). Most of the inflammatory cells which stained for IL-17A in control mucosa and RAU lesions were also mast cell tryptase positive. CONCLUSION IL-17C is highly expressed in epithelial cells in RAU lesions, where it seems to stimulate oral keratinocytes via IL-17RA/IL-17RE to produce pro-inflammatory cytokines. Human oral epithelial cells are probably important inflammatory cells in RAU.

Recurrent aphthous ulcers—a Toll‐like receptor–mediated disease?

BACKGROUND Recurrent aphthous ulcer (RAU) is characterized by acute and painful inflammatory ulcerations, which heal spontaneously but tend to recur. Many pathogens have been proposed as causative agents, but none has been consistently proven. According to our hypothesis, RAU is an autoinflammatory disorder triggered by pathogen-associated molecular patterns (PAMPs) shared by different pathogenic and commensal microbes. METHODS PAMP-reactive Toll-like receptors (TLRs) were mapped in oral epithelium in healthy controls compared to RAU. RESULTS In controls, the superficial epithelium formed a TLR(-), a PAMP non-reactive physical barrier zone, but all TLRs were found deeper in the epithelium, usually restricted to suprabasal and basal cell layers. In RAU, the epithelial TLR polarity was lost: TLRs 1, 2, 5, 7, and 8 were found throughout the epithelium, but also TLRs 4, 6, and 10 extended higher up than normally, whereas TLR-3 was almost lost in RAU. In RAU lesions, connective tissue stroma was heavily infiltrated by TLR(+) inflammatory cells. CONCLUSIONS Normal TLR architecture prevents inflammatory responses against normal microbes but still contains a deep TLR(+) , PAMP-reactive dormant defense zone. In RAU, the TLR(+), PAMP-reactive zone extends to surface or subsurface exposed to microbial PAMPs. TLR reactivity is further enhanced by recruitment of inflammatory leukocytes forming a new deep line of defense. The organization of the TLR system in healthy mucosa and its changes in RAU are compatible with active pathogenic involvement of TLRs, which together with the typical clinical picture and course suggest that RAU is a TLR-mediated disease.

Epithelial Cell Apoptosis in Recurrent Aphthous Ulcers

A recurrent aphthous ulcer (RAU) is a common inflammatory ulcerative lesion affecting oral mucosa. We studied the eventual apoptosis of epithelial cells from the point of view of ulcer and inflammation. RAU lesions and healthy mucosa samples were immunostained for caspase-3 and high-mobility group box 1 (HMGB1). DNA nicks were identified using TUNEL staining. We studied the effects of tumor necrosis factor α (TNFα) and interferon γ (IFNγ) on the toll-like receptor 2 and 4 (TLR2 and TLR4) expression of human oral SCC-25 keratinocytes. We also studied the effects of self-DNA, all-thiol-HMGB1, and disulfide-HMGB1 on epithelial cells, with or without IFNγ. At the edge of RAU lesions, all epithelial cell layers were caspase-3+, TUNEL+, and HMGB-1+ and had widened intercellular spaces. In contrast, healthy epithelial cells were negative for caspase-3 and TUNEL staining. HMGB1 was seen in only the basal cell layers, and the cells retained close cell-to-cell contacts. Self-DNA increased TNF-α mRNA (P = 0.02) in SCC-25 cells. Both TNFα and IFNγ (P = 0.01) increased TLR2. Upon TNFα stimulation, SCC-25 cells lost their nuclear HMGB1 staining. HMGB1 did not increase IL-8, IL-6, or TNF-α mRNA in SCC-25 cells, which was unaffected by the presence of IFNγ. We conclude that in healthy epithelium, the most superficial cells at the end of their life cycle are simply desquamated. In contrast, RAU is characterized by top-to-bottom apoptosis such that dead cells may slough off, leading to an ulcer. Because of a lack of scavenging anti-inflammatory macrophages, apoptotic cells probably undergo secondary necrosis releasing proinflammatory danger signals, which may contribute to the peripheral inflammatory halo. This is supported by self-DNA-induced TNFα synthesis. In contrast to TLR4- and TLR2-binding lipopolysaccharide used as a positive control, disulfide-HMGB1 did not stimulate proinflammatory cytokines.