Tadashi Osaki

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.

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.

Systemic Administration of IL-18 Promotes Diabetes Development in Young Nonobese Diabetic Mice

IL-18 is now identified as a pleiotropic cytokine that acts as a cofactor for both Th1 and Th2 cell development. Type 1 diabetes is considered a Th1-type autoimmune disease, and to date, the suppressive effect of exogenous IL-18 on the development of diabetes has been reported in 10-wk-old nonobese diabetic (NOD) mice. In the present study we administered exogenous IL-18 systemically in 4-wk-old NOD mice using i.m. injection of the IL-18 expression plasmid DNA (pCAGGS-IL-18) with electroporation. Contrary to previous reports, the incidence of diabetes development was significantly increased in NOD mice injected with pCAGGS-IL-18 compared with that in control mice. Systemic and pancreatic cytokine profiles deviated to a Th1-dominant state, and the the frequency of glutamic acid decarboxylase-reactive IFN-γ-producing CD4+ cells was also high in the IL-18 group. Moreover, it was suggested that the promoting effect of IL-18 might be associated with increased peripheral IL-12, CD86, and pancreatic IFN-inducible protein-10 mRNA expression levels. In conclusion, we demonstrate here that IL-18 plays a promoting role as an enhancer of Th1-type immune responses in diabetes development early in the spontaneous disease process, which may contribute to elucidating the pathogenesis of type 1 diabetes.

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.

Megakaryocyte potentiating factor as a tumor marker of malignant pleural mesothelioma: Evaluation in comparison with mesothelin

PURPOSE An early and reliable blood test is one deficiency in diagnosis of malignant pleural mesothelioma (MPM). Megakaryocyte potentiating factor (MPF) and mesothelin variants (MSLN), members of the mesothelin gene family, have been studied as candidate serum markers for MPM. We developed a novel enzyme-linked immunosorbent assay (ELISA) system to compare the diagnostic efficacy of MPF and MSLN in MPM and control groups. EXPERIMENTAL DESIGN MPF and MSLN were assayed with ELISA in 27 consecutive MPM patients and 129 controls including patients with lung cancer and asymptomatic asbestos-exposed subjects. RESULTS Statistically significant elevation of serum MPF and MSLN levels was noted in MPM patients in comparison with every control group. The area under the receiver operating characteristic curve (AUC) was calculated for differentiation of MPM and lung cancer, healthy asbestos-exposed subjects, and healthy adults. While the AUC for serum MPF was 0.879, cut-off=19.1ng/ml (sensitivity=74.1%, specificity=90.4%), the AUC for serum MSLN was 0.713, cut-off=93.5ng/ml (sensitivity=59.3%, specificity=86.2%). Comparison between AUC for MPF and MSLN values shows that MPF is significantly superior to MSLN (p=0.025). Finally, there was a significant correlation between MPF and MSLN values for MPM (Pearsons correlation coefficient=0.77; p<0.001). CONCLUSIONS These findings suggest that diagnostic value of MPF for MPM was better than that of MSLN although both markers showed almost equal specificity for MPM.

Overexpression of SOCS3 exhibits preclinical antitumor activity against malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis for which an effective therapy remains to be established. Our study investigated the therapeutic potential of the suppressor of cytokine signaling 3 (SOCS3), an endogenous inhibitor of intracellular signaling pathways, for treatment of MPM. We infected MPM cells (H226, EHMES‐1, MESO‐1 and MESO‐4) with an adenovirus‐expressing SOCS3 (AdSOCS3) to examine the effect of SOCS3 overexpression on MPM cells. SOCS3 overexpression reduced MPM proliferation and induced apoptosis and partial G0/G1 arrest. SOCS3 also inhibited the proliferation of MPM cells via multiple signaling pathways including Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), extracellular signal‐regulated kinase (ERK), focal adhesion kinase (FAK) and p53 pathways. Notably, AdSOCS3 treatment inhibited tumor growth in an MPM pleural xenograft model. These findings demonstrate that overexpression of SOCS3 has a potent antitumor effect against MPM both in vitro and in vivo and indicate the potential for clinical use of SOCS3 for MPM treatment.

Detailed deletion mapping of the short arm of chromosome 3 in small cell and non-small cell carcinoma of the lung

We constructed a detailed deletion map of the short arm of chromosome 3 (3p) for 55 lung cancer cases by using 17 restriction fragment length polymorphism (RFLP) probes. Initially, we examined 40 small cell lung cancer (SCLC) cases and found three regions of deletion at 3p25-26, 3p21.3 and 3p14-cen, suggesting the possibility of at least three different tumor-suppressor genes on 3p. In order to obtain more detailed deletion area, and to compare the pattern of 3p deletion, we also examined 15 non-small cell lung cancer (NSCLC) cases. Compared to NSCLC cases, most of SCLC cases have widespread deletion on 3p, suggesting multiple tumor-suppressor genes on 3p may be inactivated in this type of cancer. In 3p21.3 area, minimum overlapping area of deletion lays between two probes which are close to each other. These data will be useful to isolate the putative tumor-suppressor genes located on the chromosome 3p.

High frequencies of less differentiated and more proliferative WT1‐specific CD8+ T cells in bone marrow in tumor‐bearing patients: An important role of bone marrow as a secondary lymphoid organ

In tumor‐bearing patients, tumor‐associated antigen (TAA)‐specific CTLs are spontaneously induced as a result of immune response to TAAs and play an important role in anti‐tumor immunity. Wilms’ tumor gene 1 (WT1) is overexpressed in various types of tumor and WT1 protein is a promising pan‐TAA because of its high immunogenicity. In this study, to clarify the immune response to the WT1 antigen, WT1‐specific CD8+ T cells that were spontaneously induced in patients with solid tumor were comparatively analyzed in both bone marrow (BM) and peripheral blood (PB). WT1‐specific CD8+ T cells more frequently existed in BM than in PB, whereas frequencies of naïve (CCR7+ CD45RA+), central memory (CCR7+ CD45RA−), effector‐memory (CCR7− CD45RA−), and effector (CCR7− CD45RA+) subsets were not significantly different between BM and PB. However, analysis of these subsets for the expression of CD57 and CD28, which were associated with differentiation, revealed that effector‐memory and effector subsets of the WT1‐specific CD8+ T cells in BM had less differentiated phenotypes and more proliferative potential than those in PB. Furthermore, CD107a/b functional assay for WT1 peptide‐specific cytotoxic potential and carboxyfluorescein diacetate succinimidyl ester dilution assay for WT1 peptide‐specific proliferation also showed that WT1‐specific CD8+ T cells in BM were less cytotoxic and more proliferative in response to WT1 peptide than those in PB. These results implied that BM played an important role as a secondary lymphoid organ in tumor‐bearing patients. Preferential residence of WT1‐specific CD8+ T cells in BM could be, at least in part, explained by higher expression of chemokine receptor CCR5, whose ligand was expressed on BM fibroblasts on the WT1‐specific CD8+ T cells in BM, compared to those in PB. These results should provide us with an insight into WT1‐specific immune response in tumor‐bearing patients and give us an idea of enhancement of clinical response in WT1 protein‐targeted immunotherapy.