Muneharu Maruyama

EFFECT OF EICOSAPENTAENOIC AND DOCOSAHEXAENOIC ACID ON NATURAL-KILLER-CELL ACTIVITY IN HUMAN PERIPHERAL-BLOOD LYMPHOCYTES

The effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on natural killer (NK) cell activity in human peripheral blood lymphocytes were studied. The direct addition of trieicosapentaenoyl-glycerol (EPA-TG) or tridocosahexaenoylglycerol (DHA-TG) emulsion to a cytotoxicity assay system significantly suppressed NK cell activity. The addition of lipoxygenase inhibitor AA861 also inhibited NK cell activity. The inhibition was proportional to the concentration of EPA-TG emulsion. DHA-TG emulsion, or AA861. The presence of both EPA-TG emulsion or DHA-TG emulsion and AA861 at the same time led to a greater inhibitory effect on NK cell activity than when these emulsions were used separately. The inhibitory effect caused by these lipids or lipoxygenase blockade could not be reversed by adding back exogenous leukotrienes to the assay system. Preincubation of effector cells with EPA-TG or DHA-TG emulsion resulted in a significant inhibition of their NK cell activity. NK cell activity of human lymphocytes was markedly decreased after the infusion of EPA-TG emulsion into healthy volunteers. Thus, in vivo use of EPA-TG or DHA-TG emulsion may influence immune reactivity of the host, although the mechanism has not yet been elucidated.

Activation of peroxisome proliferator-activated receptor γ inhibits TNF-α-mediated osteoclast differentiation in human peripheral monocytes in part via suppression of monocyte chemoattractant protein-1 expression

Tumor necrosis factor-alpha (TNF-alpha) plays critical roles in bone resorption at the site of inflammatory joints. The aim of this study is to evaluate the effect of peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonists, a new class of anti-inflammatory compounds, on TNF-alpha-mediated osteoclastogenesis in human monocytes. Human monocytes were differentiated into osteoclasts in the presence of TNF-alpha and macrophage colony-stimulating factor. Tartrate-resistant acid phosphatase (TRAP) staining and a pit formation assay using dentin were used for the identification of activated osteoclasts. The protein and gene expressions of transcription factors were determined by immunofluorescence and real-time RT-PCR analysis, respectively. TNF-alpha-induced osteoclast generation from human peripheral monocytes in a dose-dependent manner, and the induction was not inhibited by osteoprotegerin, a decoy receptor for receptor activator of NF-kappaB ligand. The addition of PPAR-gamma agonists, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) or ciglitazone, to the culture resulted in a remarkably reduced number of generated osteoclasts. In addition, both agonists inhibited the protein and gene expressions of nuclear factor of activated T-cell isoform c1 (NFATc1), c-Fos, c-Jun and NF-kappaB p65, which are known to be associated with osteoclastogenesis. GW9662, an antagonist of PPAR-gamma, fully rescued ciglitazone-induced inhibition, but did not affect 15d-PGJ2-induced inhibition. Monocyte chemoattractant protein-1 (MCP-1), a CC chemokine related to osteoclastogenesis, was induced during TNF-alpha-mediated osteoclast differentiation, and the neutralizing antibody to MCP-1 reduced osteoclast formation by about 40%. 15d-PGJ2 and ciglitazone blocked the induction of MCP-1 by TNF-alpha. Moreover, the addition of MCP-1 rescued the inhibition of TRAP-positive multinucleated cell (TRAP-MNCs) formation by 15d-PGJ2 and ciglitazone, although generated TRAP-MNCs had no capacity to resorb dentin slices. Our data demonstrate that 15d-PGJ2 and ciglitazone down-regulate TNF-alpha-mediated osteoclast differentiation in human cells, in part via suppression of the action of MCP-1. These PPAR-gamma agonists may be a promising therapeutic application for rheumatoid arthritis and inflammatory bone-resorbing diseases.

Interleukin-1α and tumor necrosis factor α synergistically stimulate prostaglandin E2-dependent production of interleukin-11 in rheumatoid synovial fibroblasts

OBJECTIVE: Interleukin-11 (IL-11), an IL-6-type cytokine, is thought to be involved in bone resorption via osteoclast differentiation. Here, we characterized the combined effect of IL-1alpha and tumor necrosis factor alpha (TNFalpha), major cytokines in the rheumatoid synovium, on the production of IL-11 by cultured rheumatoid synovial fibroblasts (RSFs). METHODS: The amounts of IL-11, IL-6, and prostaglandin E2 (PGE2) were measured by enzyme-linked immunosorbent assay. IL-11 messenger RNA (mRNA) levels were determined by Northern blotting. Protein expression of cytosolic phospholipase A2 (cPLA2), cyclooxygenase 2 (COX-2), and protein kinase C (PKC) isoforms were determined by Western blotting. RESULTS: IL-1alpha and TNFalpha synergistically stimulated RSFs to produce IL-11 at both the mRNA and protein levels. This synergistic effect was completely inhibited by indomethacin. The inhibition was prevented by PGE2, indicating that the synergistic effect of IL-1alpha and TNFalpha was PGE2-mediated. The cooperative effects of these 2 cytokines were also observed in the production of PGE2 and the expression of 2 regulatory enzymes in PGE2 production, cPLA2 and COX-2. The synergistic induction of IL-11 by IL-1alpha and TNFalpha was completely inhibited by a potent inhibitor of all isoforms of PKC, GF109203X. In contrast, phorbol myristate acetate, which induced a down-regulation of PKC, degrading all PKC isoforms except atypical PKC, did not affect the induction of IL-11. CONCLUSION: These findings suggest that IL-1alpha and TNFalpha synergistically stimulate the production of IL-11 via their effects on PGE2 production in the rheumatoid joint, and that atypical PKC may be another target for down-regulation of IL-11, the bone resorption-associated cytokine.

Reactive oxygen intermediates stimulate interleukin-6 production in human bronchial epithelial cells

Reactive oxygen intermediates (ROIs) play an important role in the initiation and progression of lung diseases. In this study, we investigated whether ROIs were involved in the induction of interleukin (IL)-6 in human bronchial epithelial cells. We exposed normal human bronchial epithelial cells as well as a human bronchial epithelial cell line, HS-24, to ROIs. We measured the amount of IL-6 in the culture supernatants using ELISA and the IL-6 mRNA levels using RT-PCR. Superoxide anions (O-2), but not hydrogen peroxide (H2O2), increased IL-6 production. To examine whether it is a cell type-specific mechanism of airway epithelial cells, the experiments were also performed in human lung fibroblasts, WI-38-40. In WI-38-40 cells, neither O-2 nor H2O2 increased IL-6 production. In contrast, tumor necrosis factor (TNF)-alpha (200 U/ml) induced IL-6 at the protein and mRNA levels in both airway epithelial cells and lung fibroblasts. This cytokine-induced IL-6 production was significantly suppressed by several antioxidants, including dimethyl sulfoxide (DMSO), in airway epithelial cells. In WI-38-40 cells, DMSO was not able to suppress IL-6 production induced by TNF-alpha. Pretreatment with DMSO recovered the TNF-alpha-induced depletion of intracellular reduced glutathione in HS-24 cells. These findings indicate that oxidant stress specifically induces IL-6 production in human bronchial epithelial cells and that in these cells ROIs may be involved in IL-6 production after stimulation with cytokines such as TNF-alpha. Presumably, ROIs participate in the local immune response in lung diseases via IL-6 release from bronchial epithelial cells.

Cigarette smoking decreases interleukin-8 secretion by human alveolar macrophages

Cigarette smoking can impair pulmonary immune function, and hence influences the development of lung diseases. Interleukin-8 (IL-8) is a proinflammatory peptide and a potent chemotactic factor for neutrophils, and is produced by both immune and non-immune cells including monocytes and alveolar macrophages (AM). We investigated the effect of cigarette smoking on the secretion of IL-8 by human AM. The IL-8 concentration in bronchoalveolar lavage fluid (BALF) was much higher in smokers than in non-smokers (18.4 +/- 3.9 vs 4.1 +/- 1.0 pg ml-1; P < 0.005). However, spontaneous IL-8 secretion by cultured AM was lower in smokers than in non-smokers (46.8 +/- 12.7 vs 124.1 +/- 24.0 ng ml-1; P < 0.01). When stimulated with lipopolysaccharide (LPS), AM from smokers secreted significantly less IL-8 than those from non-smokers at all tested concentrations of LPS. In contrast, the amount of IL-8 secreted by peripheral blood monocytes with or without LPS stimulation was comparable in smokers and non-smokers. These observations indicate that smoking decreases IL-8 secretion by AM, which may modify or decrease the inflammatory response in the lung.

Clinical Significance of the Number of Positive Tumor Markers in Assisting the Diagnosis of Lung Cancer with Multiple Tumor Marker Assay

The clinical significance of multiple tumor marker assay in assisting the diagnosis of lung cancer was assessed in 67 patients with primary lung cancer, and 115 with nonmalignant pulmonary disease. The tumor markers studied were carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), squamous cell carcinoma-related antigen (SCC), and tissue polypeptide antigen (TPA). The positive rates for all of the tumor markers were significantly higher in the lung cancer group than in the nonmalignant pulmonary disease group. The sensitivity was 31-66%, the specificity was more than 90% for all five markers, and the accuracy was 69-82%. Among the markers, the positive rate of CEA was best correlated with adenocarcinoma (Ad), NSE with small cell carcinoma (Sm), SCC with squamous cell carcinoma (Sq), CA19-9 with Ad, and TPA with Ad. In multiple tumor marker assay, as the number of combined markers was increased, the sensitivity of the assay became higher and the specificity became lower, resulting in a lower accuracy. However, when more than two markers were positive, the relative possibility of lung cancer was increased 90-100%. The number of positive tumor markers in multiple tumor marker assay indicated that it would be of auxiliary value for the diagnosis of lung cancer.

Doxorubicin induces expression of multidrug resistance-associated protein 1 in human small cell lung cancer cell lines by the c-jun N-terminal kinase pathway.

Multidrug resistance (MDR) is a major impediment to successful chemotherapy for lung cancer. Overexpression of multidrug resistance‐associated protein 1 (MRP1) appears to be involved in MDR development in lung cancer cells. A number of chemotherapeutic agents including doxorubicin (DOX) were reported to induce MRP1 expression in human lung cancer cells. In our study, we investigated the mechanism by which DOX induces MRP1 expression in human small cell lung cancer (SCLC) cell lines, GLC4 and NCI‐H82. These cells expressed MRP1 protein at low levels and were sensitive to DOX. Doxorubicin at 50 nM induced a marked increase in MRP1 expression in 24 hr, and stimulated c‐jun N‐terminal kinase (JNK) activity. Treatment with a JNK inhibitor, SP600125, significantly inhibited MRP1 induction. Furthermore, transfection with JNK1 and JNK2 antisense oligonucleotides markedly inhibited DOX‐induced MRP1 expression. Chromatin immunoprecipitation assays revealed an enhanced recruitment of phosphorylated c‐jun to the MRP1 promoter containing the AP‐1 site upon DOX stimulation, which was inhibited by pretreatment with SP600125. Surprisingly, GLC4 cells exposed to DOX for 24 hr maintained increased MRP1 expression and resistance to DOX for at least 3 weeks. Pretreatment with SP600125 before DOX stimulation blocked the appearance of the MDR phenotype as well as MRP1 induction in GLC4 cells. These findings suggest that JNK activation may play an essential role for the induction of MRP1 protein in human SCLC cells by chemotherapeutic agents and that combined treatment of a JNK inhibitor with anticancer drugs may prevent the development of MDR by the abrogation of MRP1 induction.

Resting T cells negatively regulate osteoclast generation from peripheral blood monocytes

There is accumulating evidence that T cells may be involved in osteoclastogenesis in a variety of murine systems. However, the precise role of human T cells in the regulation of osteoclast generation is still unclear. To address this issue, we investigated the effect of resting peripheral T cells on receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast generation from human peripheral monocytes. Although osteoclasts were not generated in the culture of human peripheral blood mononuclear cells (PBMC) in the presence of RANKL and macrophage colony-stimulating factor (M-CSF), the addition of cyclosporine A (CsA), a potent inhibitor of T-cell function, resulted in the formation of an increasing number of lacunae resorption on dentine, suggesting T cells may inhibit osteoclast formation. In a coculture of T cells and monocytes, which were isolated from PBMC, T cells inhibited the osteoclast generation from monocytes, as determined by tartrate-resistant acid phosphatase (TRAP) staining and a pit assay using dentine. This inhibition of osteoclast generation by T cells was also observed in a culture of the parathyroid hormone-stimulated SaOS4/3 osteoblast cell line and monocytes. The culture in Transwell plates revealed that the cell-to-cell interaction was not required for the inhibition, suggesting that T-cell cytokines may be responsible for the inhibition. Among inhibitory T-cell cytokines on osteoclastogenesis, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma) were actively produced by CD4 T cells but not CD8 T cells in the coculture of T cells with monocytes, and the neutralizing antibodies to these cytokines partially rescued the T-cell-induced inhibition of osteoclast formation. Although CsA did not affect RANKL-induced osteoclast generation in the culture of monocytes alone, it completely rescued the T-cell-induced inhibition of osteoclast formation and strongly inhibited the production of GM-CSF and IFN-gamma. Thus, we demonstrate that resting T cells negatively regulate the osteoclast generation via production of GM-CSF and IFN-gamma by CD4 T cells and that CsA stimulates the osteoclast generation through the inhibition of the production of these cytokines. These findings provide new insight into therapeutic strategies for immunosuppression-induced bone loss in transplant and other diseases.

Detection of primary and metastatic lesions by [18F]fluoro-2-deoxy-D-glucose PET in a patient with thymic carcinoid

We present a case of thymic carcinoid, in which primary and metastatic lesions of lymph nodes and bones could be detected by [18F]fluoro‐2‐deoxy‐D‐glucose (FDG)‐PET, but not by 123I‐meta‐iodobenzylguanidine (123I‐MIBG) SPECT, or by 99mTc‐methylene diphosphonate (99mTc‐MDP) bone scintigraphy. FDG‐PET may be a useful tool for managing thymic carcinoids in patients with negative results on 123I‐MIBG SPECT or 99mTc‐MDP imaging.

Hereditary angioedema with a de novo mutation of exon 8 in the C1 inhibitor gene showing recurrent edema of the hands around the peripheral joints: Importance for the differential diagnosis of joint swelling

We describe a patient with hereditary angioedema (HAE), showing recurrent edema around the peripheral joints. Her symptoms began at the age of 18 with hand swelling distal to the wrist joints. Until she was referred to our hospital 3 years after her initial symptoms, she was still undiagnosed, although she was suspected of having rheumatoid arthritis. Laboratory examination showed reduced levels of CH50 and C4 with normal C3 levels. The C1 inhibitor (C1-INH) was decreased to 5 mg/ml, with remarkably reduced activity. Although these findings were compatible with a diagnosis of HAE, there were no episodes of skin edema in her family. To establish the diagnosis, we carried out DNA analysis of the C1-INH gene, which revealed a newly identified de novo mutation of G to A at nucleotide 16869 in exon 8. As described in this patient, localized edema around the peripheral joints may be the only manifestation of HAE. HAE should therefore be taken into consideration for the differential diagnosis of joint swelling.