Yumiko Sugawara

Identification of IL-18 and Th17 Cells in Salivary Glands of Patients with Sjögren's Syndrome, and Amplification of IL-17-Mediated Secretion of Inflammatory Cytokines from Salivary Gland Cells by IL-18

IL-18 is a proinflammatory cytokine and plays an important pathogenic role in inflammatory and autoimmune disorders. IL-17 is also a proinflammatory cytokine and IL-17-secreting Th17 cells are involved in autoimmunity. However, the pathological roles of IL-18 and Th17 cells in Sjögren’s syndrome (SS) remain to be elucidated. This study showed that the expression of IL-18 was detected in acinar cells, intraducts, and CD68+ macrophages in salivary glands of SS patients, but not in those of healthy subjects or patients with chronic graft-vs-host disease, by immunohistochemistry, and immunoblot analysis revealed that 24-kDa precursor form of IL-18 (proIL-18) and 18-kDa mature IL-18 were detected in SS salivary glands. The majority of the infiltrating cells in the salivary glands of SS patients were CD4+ T cells, and CD8+ T cells were infiltrated to a lesser extent. The predominant expression of IL-17 was found in infiltrating CD4+ T cells, whereas a small number of infiltrating CD8+ T cells expressed IL-17. Human salivary gland HSY and acinar AZA3 cells constitutively expressed proIL-18 and caspase-1, and a calcium ionophore A23187 induced the secretion of IL-18 from the cells. HSY and AZA3 cells expressed IL-18R and IL-17R on the cell surface, and IL-18 amplified the secretion of IL-6 and IL-8 that were induced by low amounts of IL-17. Primary salivary gland cells from normal subjects partially confirmed these findings. These results suggest that IL-18 and Th17 cells detected in the salivary glands in SS patients are associated with the pathogenesis of SS in the salivary glands.

Toll-like Receptors, NOD1, and NOD2 in Oral Epithelial Cells

Oral epithelium might be the first barrier against oral bacteria in periodontal tissue. We hypothesized that oral epithelium is endowed with innate immune receptors for bacterial components, which play roles in host defense against bacterial infection without being accompanied by excessive inflammatory responses. We found clear expression of Toll-like receptor (TLR)4 as well as TLR2, and strong expression of NOD1 and NOD2 in normal oral epithelial tissues by immunohistochemical analysis. We also showed that primary oral epithelial cells in culture expressed these molecules using PCR, flow cytometry, and immunostaining. In inflamed oral epithelium, cell-surface localizations of TLR2 and TLR4 were more clearly observed than in healthy tissue. Upon stimulation with synthetic ligands for these receptors, the expression of β-defensin 2 was markedly up-regulated. These findings indicate that these molecules in oral epithelial cells are functional receptors that induce antibacterial responses.

Infiltrating CD8+ T Cells in Oral Lichen Planus Predominantly Express CCR5 and CXCR3 and Carry Respective Chemokine Ligands RANTES/CCL5 and IP-10/CXCL10 in Their Cytolytic Granules: A Potential Self-Recruiting Mechanism

Lichen planus is a chronic inflammatory disease of the skin and oral mucosa in which the cell-mediated cytotoxicity is regarded as a major mechanism of pathogenesis. To understand its pathophysiology further, the present study examined the in situ expression of chemokines and chemokine receptors in oral lichen planus. Immunohistochemical analysis of 15 cases has consistently revealed that infiltrating CD4(+) and CD8(+) T cells in the submucosa predominantly expressed CCR5 and CXCR3. Furthermore, infiltrating T cells, particularly CD8(+) T cells, were positive for RANTES/CCL5 and IP-10/CXCL10, the ligands of CCR5 and CXCR3, respectively. By immunoelectron microscopy, these chemokines were localized in the cytolytic granules of CD8(+) T cells. Lesional keratinocytes also overexpressed the ligands of CXCR3, namely, MIG/CXCL9, CXCL10, and I-TAC/CXCL11. Our data suggest that the chemokines signaling via CCR5 and CXCR3, which are known to be selectively expressed by type 1 T cells, are predominantly involved in T-cell infiltration of oral lichen planus. Furthermore, the presence of CCL5 and CXCL10 in the cytolytic granules of tissue-infiltrating CD8(+) T cells expressing CCR5 and CXCR3 reveals a potential self-recruiting mechanism involving activated effector cytotoxic T cells.

Chemically synthesized pathogen‐associated molecular patterns increase the expression of peptidoglycan recognition proteins via toll‐like receptors, NOD1 and NOD2 in human oral epithelial cells

Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition molecules (PRMs) in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PGN) and are suggested to act as anti‐bacterial factors. In humans, four kinds of PGRPs (PGRP‐L, ‐Iα, ‐Iβ and ‐S) have been cloned and all four human PGRPs bind PGN. In this study, we examined the possible regulation of the expression of PGRPs in oral epithelial cells upon stimulation with chemically synthesized  pathogen‐associated  molecular patterns (PAMPs) in bacterial cell surface components: Escherichia coli‐type tryacyl lipopeptide (Pam3CSSNA), E. coli‐type lipid A (LA‐15‐PP), diaminopimelic acid containing desmuramyl peptide (γ‐ d‐glutamyl‐meso‐DAP; iE‐DAP), and muramyldipeptide (MDP). These synthetic PAMPs markedly upregulated the mRNA expression of the four PGRPs and cell surface expression of PGRP‐Iα and ‐Iβ, but did not induce either mRNA expression or secretion of inflammatory cytokines, in oral epithelial cells. Suppression of the expression of Toll‐like receptor (TLR)2, TLR4, nucleotide‐binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the upregulation of PGRP mRNA expression induced by Pam3CSSNA, LA‐15‐PP, iE‐DAP and MDP respectively. These PAMPs definitely activated nuclear factor (NF)‐κB in the epithelial cells, and suppression of NF‐κB activation clearly prevented the induction of PGRP mRNA expression induced by these PAMPs in the cells. These findings suggested that bacterial PAMPs induced the expression of PGRPs, but not proinflammatory cytokines, in oral epithelial cells, and the PGRPs might be involved in host defence against bacterial invasion without accompanying inflammatory responses.

Proinflammatory Cytokines Induce Proteinase 3 as Membrane-Bound and Secretory Forms in Human Oral Epithelial Cells and Antibodies to Proteinase 3 Activate the Cells through Protease-Activated Receptor-2

We wish to retract the article titled “Proinflammatory Cytokines Induce Proteinase 3 as Membrane-Bound and Secretory Forms in Human Oral Epithelial Cells and Antibodies to Proteinase 3 Activate the Cells through Protease-Activated Receptor-2,” by Akiko Uehara, Yumiko Sugawara, Takashi Sasano, Haruhiko Takada, and Shunji Sugawara, The Journal of Immunology, 2004, 173: 4179–4189. This retraction follows an investigation by Tohoku University into scientific misconduct. The investigation pointed out the following: 1. Fig. 1A: HSC-2, KB, and HO-1-u-1 are different cell lines, but the same photo is used for their GAPDH. 2. Fig. 7A: The cells were transfected with PR3or Lamin A/C-specific siRNA, which are different, but the patterns of GAPDH are the same. The first author, who conducted these experiments, could not counter the argument by adducing raw data at the investigation, and the investigation recognized them as scientific misconduct. Therefore, we wish to retract the article. We deeply regret these errors and apologize to the scientific community for the need to retract the article.

Involvement of neutrophil recruitment and protease-activated receptor 2 activation in the induction of IL-18 in mice

Activated neutrophils produce serine proteases, which activate cells through protease‐activated receptor 2 (PAR2). As proteinase 3 (PR3) induces the secretion of interleukin (IL)‐18 from epithelial cells in combination with lipopolysaccharide (LPS) in vitro, we examined whether neutrophils, serine proteases, and PAR2 are involved in the induction of serum IL‐18 and IL‐18‐dependent liver injury in mice treated with heat‐killed Propionibacterium acnes and LPS. LPS‐induced serum IL‐18 levels in P. acnes‐primed mice were reduced significantly by anti‐Gr‐1 injection (depletion of neutrophils and macrophages) but not by a macrophage “suicide” technique, using liposomes encapsulating clodronate. The IL‐18 induction was decreased significantly by coadministration of a serine protease inhibitor [Nafamostat mesilate (FUT‐175)] with LPS. Serum levels of tumor necrosis factor α and liver enzymes induced by P. acnes and LPS were abolished by anti‐Gr‐1 treatment, and concomitantly, liver injury (necrotic change and granuloma formation) and Gr‐1+ cell infiltration into the liver were prevented by the treatment. A deficiency of PAR2 in mice significantly impaired IL‐18 induction by treatment with P. acnes and LPS, and only slight pathological changes in hepatic tissues occurred in the PAR2‐deficient mice treated with P. acnes and LPS. Furthermore, coadministration of exogenous murine PR3 or a synthetic PAR2 agonist (ASKH95) with LPS in the anti‐Gr‐1‐treated mice restored the serum IL‐18 levels to those in control mice treated with P. acnes and LPS. These results indicate that neutrophil recruitment and PAR2 activation by neutrophil serine proteases are critically involved in the induction of IL‐18 and IL‐18‐dependent liver injury in vivo.

Detection and identification of non-Candida albicans species in human oral lichen planus.

Candida species were detected and identified in samples from the buccal mucosa, dorsal surface of the tongue and supragingival plaque of subjects with oral lichen planus (OLP). The Candida in the samples were cultured on selection agars, and identified by sequence analyses of 18S, 5.8S and 25/28S rRNA. The isolation frequency of Candida was higher in subjects with OLP than in those with healthy oral mucosa. Non‐C. albicans were only isolated from people with OLP. These results support the notion that subjects with OLP are more likely to have oral colonization with Candida, and that non‐C. albicans are specifically present in subjects with this condition.

Induction of serum IL-18 with Propionibacterium acnes and lipopolysaccharide in phagocytic macrophage-inactivated mice

IL‐18, an important regulator of immune responses, is expressed in activated macrophages and also in nonimmune cells, such as keratinocytes and epithelial cells. Increased levels of serum IL‐18 are reported in patients with a wide variety of diseases, but it is unclear which type of cell is the major source of serum IL‐18. Here, we showed that the administration of liposomes encapsulating clodronate (Clo‐lip) in mice selectively depleted F4/80+ phagocytic macrophages in the liver and spleen. Serum levels of mature IL‐18 with 18 kDa were increased markedly in mice treated with Propionibacterium acnes and LPS, whereas administration of Clo‐lip and gadolinium chloride, another widely used macrophage inactivator, showed no obvious effect on serum IL‐18 levels, which were marginal in the liver, lung, and spleen and more pronounced in the intestines, especially in the duodenum. Treatment with P. acnes alone induced IL‐18 more than twofold in each organ, and P. acnes and LPS induced a marked increase in IL‐18 levels in the liver and spleen but decreased in the intestines. The administration of Clo‐lip showed only a marginal effect on the IL‐18 levels in these organs. Furthermore, serum levels of liver enzymes and TNF‐α and liver injury (necrotic change and granuloma formation) induced by P. acnes and LPS were reduced moderately by Clo‐lip. These results suggest that phagocytic macrophages do not actively contribute to the induction of serum IL‐18 and liver injury in mice treated with P. acnes and LPS.

Diagnostic Performance of MR Imaging of Three Major Salivary Glands for Sjögren's Syndrome

OBJECTIVE We analyzed the diagnostic performance of the MR imaging findings of the parotid, submandibular, and sublingual glands to discriminate between patients with and without Sjögrens syndrome. METHODS We retrospectively analyzed the correlation between the MR imaging and histopathological findings obtained from 69 patients with clinically suspected Sjögrens syndrome. We evaluated the heterogeneous signal intensity distribution on T1- and T2-weighted images, the multiple high-signal-intensity spots on MR sialograms, and the volume of the parotid, submandibular, and sublingual salivary glands. RESULTS The multiple high-signal-intensity spots in the parotid gland showed the highest sensitivity and diagnostic accuracy (82% and 83%, respectively). In addition, the multiple high-signal-intensity spots and the heterogeneous signal intensity distribution in the submandibular gland showed high specificity (100% and 88%, respectively). The volume of the submandibular gland, but not that of the parotid or sublingual gland, was smaller in patients with Sjögrens syndrome. CONCLUSIONS The presence of multiple high-signal-intensity spots on an MR sialogram in the parotid gland should be considered the best diagnostic indicator for Sjögrens syndrome. The presence of spots, heterogeneity, and the change to smaller volumes in the submandibular gland were also helpful because of their high specificity, particularly in advanced cases.

Sympathetic nerve fibers sprout into rat odontoblast layer, but not into dentinal tubules, in response to cavity preparation

This study was designed to determine if sympathetic nerve fibers exist in dentinal tubules in rat normal dental pulp, and if they sprout into the dentinal tubules in response to artificial cavity preparation in dentin. Sympathetic nerve fibers in rat molar dental pulp were labeled using an anterograde axonal transport technique involving injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the superior cervical ganglion (SCG). They were then observed using light and electron microscopes. In normal dental pulp (control), scattered WGA-HRP reaction products were observed in unmyelinated nerve endings in the odontoblast layer and subodontoblastic region. In injured pulp 3 weeks after cavity preparation, reaction products were about 1.8-times more plentiful in the above areas (versus control pulp). However, no labeled nerve fibers were observed in the dentinal tubules in either control or injured dental pulp. These results indicate that although sympathetic nerve fibers do indeed sprout in rat dental pulp in response to cavity preparation, they do not penetrate into the dentinal tubules in which postganglionic nerve endings derived from the SCG were not originally present.