Isao Tachibana

Tetraspanins CD9 and CD81 function to prevent the fusion of mononuclear phagocytes

Tetraspanins CD9 and CD81 facilitate the fusion between gametes, myoblasts, or virus-infected cells. Here, we investigated the role of these tetraspanins in the fusion of mononuclear phagocytes. Expression of CD9 and CD81 and their complex formation with integrins were up-regulated when blood monocytes were cultured under normal conditions. Under fusogenic conditions in the presence of Con A, CD9 and CD81 up-regulation was inhibited, and their complex formation with integrins was down-regulated. Anti-CD9 and -CD81 antibodies, which were previously shown to inhibit the fusion of gametes, myoblasts, and virus-infected cells, unexpectedly promoted the fusion of monocytes and alveolar macrophages. However, these effects were not due to altered cell adhesion, aggregation, or cytokine production. When stimulated in vitro or in vivo, alveolar macrophages and bone marrow cells of CD9- and CD81-null mice formed larger numbers of multinucleated cells than those of wild-type mice. Finally, CD9/CD81 double-null mice spontaneously developed multinucleated giant cells in the lung and showed enhanced osteoclastogenesis in the bone. These results suggest that CD9 and CD81 coordinately prevent the fusion of mononuclear phagocytes.

NAG-2, a Novel Transmembrane-4 Superfamily (TM4SF) Protein That Complexes with Integrins and Other TM4SF Proteins

Transmembrane-4 superfamily (TM4SF) proteins form complexes with integrins and other cell-surface proteins. To further characterize the major proteins present in a typical TM4SF protein complex, we raised monoclonal antibodies against proteins co-immunoprecipitated with CD81 from MDA-MB-435 breast cancer cells. Only two types of cell-surface proteins were recognized by our 35 selected antibodies. These included an integrin (α6β1) and three different TM4SF proteins (CD9, CD63, and NAG-2). The protein NAG-2 (novelantigen-2) is a previously unknown 30-kDa cell-surface protein. Using an expression cloning protocol, cDNA encoding NAG-2 was isolated. When aligned with other TM4SF proteins, the deduced amino acid sequence of NAG-2 showed most identity (34%) to CD53. Flow cytometry, Northern blotting, and immunohistochemistry showed that NAG-2 is widely present in multiple tissues and cell types but is absent from brain, lymphoid cells, and platelets. Within various tissues, strongest staining was seen on fibroblasts, endothelial cells, follicular dendritic cells, and mesothelial cells. In nonstringent detergent, NAG-2 protein was co-immunoprecipitated with other TM4SF members (CD9 and CD81) and integrins (α3β1and α6β1). Also, two-color immunofluorescence showed that NAG-2 was co-localized with CD81 on the surface of spread HT1080 cells. These results confirm the presence of NAG-2 in specific TM4SF·TM4SF and TM4SF-integrin complexes.

Proteomics-based identification of α-enolase as a tumor antigen in non-small lung cancer

Autoantibodies against tumor antigens represent one type of biomarker that may be assayed in serum for detection of cancer and monitoring of disease progression. In the present study, we used a proteomics‐based approach to identify novel tumor antigens in non‐small cell lung cancer (NSCLC). By combining two‐dimensional electrophoresis, western blotting, mass spectrometry and enzyme‐linked immunosorbent assay technology, we detected autoantibodies against α‐enolase in a subset of NSCLC patients’ sera. When ‘Mean ODhealthy control sera + 3 SDhealthy control sera’ was used as the cut‐off point, the prevalence of this autoantibody was 27.7% in patients with NSCLC (26 of 94), 1.7% in healthy control subjects (1 of 60), and not detectable in sera from 15 patients with small cell lung cancer, 18 patients with gastrointestinal cancer and nine patients with Mycobacterium avium complex infection of lung. Immunohistochemical staining showed that expression of α‐enolase was increased in cancer tissues of NSCLC patients, and flow cytometric analysis confirmed the expression of α‐enolase at the surface of cancer cells. The combined detection of autoantibodies against α‐enolase, carcinoembryonic antigen and cytokeratin 19 fragment (CYFRA21‐1) enhanced sensitivity for the diagnosis of NSCLC. Therefore, autoantibodies against α‐enolase may constitute a promising biomarker for NSCLC. (Cancer Sci 2007; 98: 1234–1240)

Expression of tetraspanins in human lung cancer cells: frequent downregulation of CD9 and its contribution to cell motility in small cell lung cancer

Small cell lung cancer (SCLC) invades locally and metastasizes distantly extremely early when compared with nonsmall cell lung cancer (NSCLC). The underlying molecular mechanisms, however, have not been elucidated. Accumulating evidence suggests that downregulation of several members of tetraspanins is associated with progression of solid tumors, thus indicating poor prognosis. Here we screened 30 lung cancer cell lines for expression of tetraspanins, CD9, CD63, CD81, CD82, CD151, and NAG-2. Flow cytometry revealed that, among these proteins, CD9 is broadly expressed in NSCLC lines, but is absent or highly reduced in most SCLC lines (P<0.0001). Using the Boyden chamber and videomicroscopic cell motility assays, we showed that stable transfection of CD9 into an SCLC line, OS3-R5, reduced cell motility on fibronectin. Furthermore, by transient transfection of green fluorescent protein (GFP)-tagged CD9 into three other SCLC lines, we observed that SCLC cells expressing GFP-CD9 were uniformly less motile than untransfected cells. CD9 or GFP-CD9 was associated with β1 integrins and distributed at the tumor cell periphery and cell–cell contacts, suggesting that CD9 modifies β1 integrin function to reduce motility. These findings suggest that low expression of CD9 may contribute to the highly invasive and metastatic phenotype of SCLC.

Early [18F]Fluorodeoxyglucose Positron Emission Tomography at Two Days of Gefitinib Treatment Predicts Clinical Outcome in Patients with Adenocarcinoma of the Lung

Purpose: Positron emission tomography (PET) with [18F]fluorodeoxyglucose (FDG) is increasingly used in early assessment of tumor response after chemotherapy. We investigated whether a change in [18F]FDG uptake at 2 days of gefitinib treatment predicts outcome in patients with lung adenocarcinoma. Experimental Design: Twenty patients were enrolled. [18F]FDG-PET/computed tomographic (CT) scan was carried out before and 2 days after gefitinib treatment. Maximum standardized uptake values (SUV) were measured, and post-gefitinib percentage changes in SUV were calculated. Early metabolic response (SUV decline < −25%) was compared with morphologic response evaluated by CT scan and with progression-free survival (PFS). Results: At 2 days of gefitinib treatment, 10 patients (50%) showed metabolic response, 8 had metabolic stable disease, and 2 had progressive metabolic disease. Percentage changes of SUV at 2 days were correlated with those of tumor size in CT at 1 month (R2 = 0.496; P = 0.0008). EGFR gene was assessable in 15 patients, and of 12 patients with EGFR mutations, 8 showed metabolic response at 2 days and 6 showed morphologic response at 1 month. None of 3 patients with wild-type EGFR showed metabolic or morphologic response. Metabolic response at 2 days was not statistically associated with PFS (P = 0.095), but when a cutoff value of −20% in SUV decline was used, metabolic responders had longer PFS (P < 0.0001). Conclusion: Early assessment of [18F]FDG tumor uptake with PET at 2 days of gefitinib treatment could be useful to predict clinical outcome earlier than conventional CT evaluation in patients with lung adenocarcinoma. Clin Cancer Res; 18(1); 220–8. ©2011 AACR.

Suppressor of cytokine signalling-1 gene silencing in acute myeloid leukaemia and human haematopoietic cell lines.

The aim of this study was to investigate whether the suppressor of cytokine signalling (SOCS)‐1 can act as a tumour suppressor when functioning as a negative regulator of the Janus family tyrosine kinases (JAKs), which have been reported to play important roles in leukaemogenesis. For this purpose, we carried out molecular analysis of the SOCS‐1 gene in human acute myeloid leukaemia (AML) and human haematopoietic cell lines. Sequencing alterations in the coding region were found in two of 90 primary AML samples and one of 17 cell lines. Hypermethylation of the SOCS‐1 gene was also observed in 72% of primary cases and 52% of cell lines and aberrant methylation strongly correlated with reduced expression. Transfection of SOCS‐1 into Jurkat cells harbouring the mutation and methylation suppressed cell growth at a low serum concentration. These findings indicate that SOCS‐1 is frequently silenced in haematopoietic malignancies, mainly as a result of hypermethylation, and suggest that SOCS‐1 may be able to function as a tumour suppressor.

Sustained interleukin-6 signalling leads to the development of lymphoid organ-like structures in the lung

A variety of pathological changes are seen in lymphoproliferative disorders of the lung but the histogenesis of these abnormalities is not yet fully understood. We previously showed that adenovirus vector‐mediated transient expression of both the human interleukin‐6 (IL‐6) and IL‐6 receptor (IL‐6R) genes, but not the IL‐6 gene alone, in the rat lung induced lymphocytic alveolitis. In the present study, we explored the lung pathology of human IL‐6 and IL‐6R double transgenic mice to elucidate the effects of prolonged IL‐6 signalling on the lung. The transgenic animals developed mononuclear cell accumulation in peribronchovascular regions, but little infiltration into alveolar spaces. Immunohistochemical analysis revealed that the cellular accumulations contained not only mixtures of inflammatory cells but also lymphoid tissue‐like structures. As the expression of CXCL13/BLC, the indispensable chemokine for lymphoid organogenesis, was recognized in the B cell follicles of the pulmonary lesions, we speculate that this chemokine plays an inductive role in the development of the lymphoid tissue‐like structures. These structures were distinguished from bronchus‐associated lymphoid tissues (BALTs) by their location and by the lack of lymphoepithelium, which is a characteristic of BALT. These findings imply that IL‐6 signalling may play a role in the pathogenesis of lymphoproliferative disorders of the lung. Copyright

Tetraspanin CD9 Negatively Regulates Lipopolysaccharide-Induced Macrophage Activation and Lung Inflammation

Tetraspanins facilitate the formation of multiple molecular complexes at specialized membrane microdomains and regulate cell activation and motility. In the present study, the role of tetraspanin CD9 in LPS-induced macrophage activation and lung inflammation was investigated in vitro and in vivo. When CD9 function was ablated with mAb treatment, small interfering RNA transfection, or gene knockout in RAW264.7 cells or bone marrow-derived macrophages, these macrophages produced larger amounts of TNF-α, matrix metalloproteinase-2, and -9 upon stimulation with LPS in vitro, when compared with control cells. Sucrose gradient analysis revealed that CD9 partly colocalized with the LPS-induced signaling mediator, CD14, at low-density light membrane fractions. In CD9 knockout macrophages, CD14 expression, CD14 and TLR4 localization into the lipid raft, and their complex formation were increased whereas IκBα expression was decreased when compared with wild-type cells, suggesting that CD9 prevents the formation of LPS receptor complex. Finally, deletion of CD9 in mice enhanced macrophage infiltration and TNF-α production in the lung after intranasal administration of LPS in vivo, when compared with wild-type mice. These results suggest that macrophage CD9 negatively regulates LPS response at lipid-enriched membrane microdomains.

Double Deficiency of Tetraspanins CD9 and CD81 Alters Cell Motility and Protease Production of Macrophages and Causes Chronic Obstructive Pulmonary Disease-like Phenotype in Mice

CD9 and CD81 are closely related tetraspanins that regulate cell motility and signaling by facilitating the organization of multimolecular membrane complexes, including integrins. We show that CD9 and CD81 are down-regulated in smoking-related inflammatory response of a macrophage line, RAW264.7. When functions of CD9 and CD81 were ablated with monoclonal antibody treatment, small interfering RNA transfection, or gene knock-out, macrophages were less motile and produced larger amounts of matrix metalloproteinase (MMP)-2 and MMP-9 than control cells in vitro. In line with this, CD9/CD81 double-knock-out mice spontaneously developed pulmonary emphysema, a major pathological component of chronic obstructive pulmonary disease (COPD). The mutant lung contained an increased number of alveolar macrophages with elevated activities of MMP-2 and MMP-9 and progressively displayed enlarged airspace and disruption of elastic fibers in the alveoli. Secretory cell metaplasia, a finding similar to goblet cell metaplasia in cigarette smokers, was also observed in the epithelium of terminal bronchioles. With aging, the double-knockout mice showed extrapulmonary phenotypes, including weight loss, kyphosis, and osteopenia. These results suggest that the tetraspanins CD9 and CD81 regulate cell motility and protease production of macrophages and that their dysfunction may underlie the progression of COPD.

Involvement of Endothelial Apoptosis Underlying Chronic Obstructive Pulmonary Disease―like Phenotype in Adiponectin-null Mice: Implications for Therapy

RATIONALE Chronic obstructive pulmonary disease is frequently complicated with comorbidities, such as cardiovascular disease, osteoporosis, and body weight loss, but the causal link remains unclear. OBJECTIVES To investigate the role of adiponectin in the pathogenesis of chronic obstructive pulmonary disease and its potential use in therapy. METHODS Adiponectin localization and dynamics in the lung were analyzed in an elastase-induced emphysema model. Next, the lung of adiponectin-knockout mice, extrapulmonary effects, and the underlying mechanism were investigated. Finally, we tested whether exogenous adiponectin could ameliorate the emphysematous change in adiponectin-knockout mice. MEASUREMENTS AND MAIN RESULTS Adiponectin expression in lung vasculature and plasma concentration of adiponectin were reduced after elastase-instillation. Notably, adiponectin-knockout mice showed progressive alveolar enlargement and increased lung compliance. They further exhibited not only systemic inflammation, but also extrapulmonary phenotype, such as body weight loss, fat atrophy, and osteoporosis. Moreover, endothelial apoptosis was enhanced in the lungs of adiponectin-knockout mice, as evidenced by caspase-3 activity. Consistent with this, expressions of vascular endothelial growth factor receptor-2 and platelet endothelial cell adhesion molecule-1 on endothelial cells were decreased in the adiponectin-knockout mice. Finally, adenovirus-mediated adiponectin supplementation ameliorated the emphysematous phenotype. CONCLUSIONS Adiponectin-knockout mice develop progressive chronic obstructive pulmonary disease-like phenotype with systemic inflammation and extrapulmonary phenotypes. Hypoadiponectinemia could thus play a critical role in the progression of chronic obstructive pulmonary disease and concomitant comorbidities through endothelial dysfunction. Together, adiponectin could be a novel target for chronic obstructive pulmonary disease therapy.