Wataru Matsuyama

Activation of Discoidin Domain Receptor 1 Isoform b with Collagen Up-Regulates Chemokine Production in Human Macrophages: Role of p38 Mitogen-Activated Protein Kinase and NF-κB

Macrophages produce an array of proinflammatory mediators at sites of inflammation and contribute to the development of inflammatory responses. Important roles for cytokines, such as IL-1 or TNF-α, and bacterial products, such as LPS, in this process have been well documented; however, the role for the extracellular matrix proteins, such as collagen, remains unclear. We previously reported that discoidin domain receptor 1 (DDR1), a nonintegrin collagen receptor, is expressed during differentiation of human monocytes into macrophages, and the interaction of the DDR1b isoform with collagen facilitates their differentiation via the p38 mitogen-activated protein kinase (MAPK) pathway. In this study, we report that the interaction of DDR1b with collagen up-regulates the production of IL-8, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1 in human macrophages in a p38 MAPK- and NF-κB-dependent manner. p38 MAPK was critical for DDR1b-mediated, increased NF-κB trans-activity, but not for IκB degradation or NF-κB nuclear translocation, suggesting a role for p38 MAPK in the modification of NF-κB. DDR1b-mediated IκB degradation was mediated through the recruitment of the adaptor protein Shc to the LXNPXY motif of the receptor and the downstream TNFR-associated factor 6/NF-κB activator 1 signaling cascade. Taken together, our study has identified NF-κB as a novel target of DDR1b signaling and provided a novel mechanism by which tissue-infiltrating macrophages produce large amounts of chemokines during the development of inflammatory diseases. Intervention of DDR1b signaling may be useful to control inflammatory diseases in which these proteins play an important role.

Regulation of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and TRAIL receptor expression in human neutrophils

Tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) is a member of the TNF superfamily, which is capable of inducing apoptosis in many cell types, including tumour and virus‐infected cells, but rarely in normal cells. Expression of TRAIL mRNA and TRAIL receptors has previously been detected in neutrophils; however, the expression of TRAIL protein and the regulation of TRAIL and TRAIL receptor expression in these cells remain unknown. Here we report, for the first time, that neutrophils constitutively express TRAIL protein on their cell surface and that the TRAIL protein is shed during culture. TNF‐α is a down‐regulator of TRAIL expression, whereas IFN‐γ up‐regulates the expression of TRAIL. Neutrophils did not express a detectable level of TRAIL‐R1 or ‐R4, but constitutively expressed a low, but substantial, level of TRAIL‐R2 and a high level of TRAIL‐R3. Although the level of TRAIL‐R2 was not significantly altered during culture under different experimental conditions, ≈ 30% of TNF‐α‐treated cells rapidly lost their high‐level TRAIL‐R3 expression, whereas the majority of IFN‐γ‐treated cells retained a high level of TRAIL‐R3 expression. Anti‐TRAIL neutralizing antibody significantly inhibited neutrophil apoptosis during cultures in medium alone, or in the presence of TNF‐α or IFN‐γ. Thus, our study identified human neutrophils as a cellular source of TRAIL and suggests that neutrophil‐derived TRAIL may play a role in immune surveillance. Our results also suggest a role for the TRAIL/TRAIL receptor system in neutrophil apoptosis.

Activation of Discoidin Domain Receptor 1 Facilitates the Maturation of Human Monocyte-Derived Dendritic Cells Through the TNF Receptor Associated Factor 6/TGF-β-Activated Protein Kinase 1 Binding Protein 1β/p38α Mitogen-Activated Protein Kinase Signaling Cascade

Maturation of dendritic cells (DCs) is critical for their ability to stimulate resting naive T cells in primary immune responses. Previous studies demonstrated that collagen, such as type I collagen, could facilitate DC maturation; however, the basis of collagen-mediated DC maturation remains unclear. Discoidin domain receptor 1 (DDR1) is a nonintegrin collagen receptor constitutively expressed in a variety of epithelial cells, including tumor cells, and is inducible in leukocytes. In this study, we evaluated the role of DDR1 in DC maturation using human monocyte-derived DCs. Two DDR1 isoforms, DDR1a and DDR1b, were expressed in both immature and mature DCs. Activation of DDR1 on immature DCs resulted in their partial maturation; however, DDR1 activation markedly amplified TNF-α- and LPS-induced phenotypic and functional maturation of DCs through activation of p38 mitogen-activated protein kinase (MAPK), suggesting the involvement of DDR1b in this process. Activation of DDR1b on differentiated DDR1b-overexpressing THP-1 cells or DDR1 on mature DCs induced the formation of TNFR associated factor 6 (TRAF6)/TGF-β-activated kinase 1 binding protein 1β/p38α MAPK complex and p38α autophosphorylation. Transfection of differentiated DDR1b-overexpressing THP-1 cells with dominant negative TRAF6 completely abrogated DDR1b-mediated p38 MAPK phosphorylation, indicating a critical role of TRAF6 in DDR1b-mediated p38 MAPK activation. Taken together, our data suggest that DDR1b-collagen interaction augments the maturation of DCs in a tissue microenvironment through a unique TRAF6/TGF-β-activated kinase 1 binding protein 1β/p38α MAPK signaling cascade and contributes to the development of adaptive immune responses.

Interaction of discoidin domain receptor 1 isoform b (DDR1b) with collagen activates p38 mitogen-activated protein kinase and promotes differentiation of macrophages

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase activated by collagen. DDR1 is constitutively expressed in a variety of normal and transformed epithelial cells and plays a role in cell migration and differentiation through as yet unidentified signaling pathways. We previously reported inducible expression of DDR1 in human leukocytes and suggested a role for the DDR1a isoform in leukocyte migration through extracellular matrix. Here, we evaluated the contribution of DDR1 in the differentiation of the human monocytic THP‐1 cells overexpressing these isoforms and of primary macrophages. Interestingly, collagen activation of DDR1b, but not DDR1a, further promoted phorbol ester‐induced differentiation of THP‐1 cells as determined by reduced cell proliferation and up‐regulated expression of HLA‐DR, CD11c, CD14, and CD40. Collagen activation of DDR1b also induced the recruitment and phosphorylation of Shc and subsequent phosphorylation of p38 mitogen‐activated protein (MAP) kinase and its substrate ATF2. A p38 MAP kinase inhibitor, SB203580, completely inhibited DDR1b‐mediated HLA‐DR expression. Activation of DDR1 endogenously expressed on macrophages also up‐regulated their HLA‐DR expression in a p38 MAP kinase‐dependent manner. Thus, DDR1b in response to collagen transduces signals that promote maturation/differentiation of HLA‐DR‐positive antigen‐presenting cells and contributes to the development of adaptive immunity in a tissue microenvironment.

Suppression of Discoidin Domain Receptor 1 by RNA Interference Attenuates Lung Inflammation

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase whose ligand is collagen. Recently, we have reported the association of DDR1 in the cytokine production of human leukocytes in in vitro and in vivo expression in idiopathic pulmonary fibrosis. However, its role in in vivo inflammation has not been fully elucidated. Small interference RNA (siRNA) can induce specific suppression of in vitro and in vivo gene expression. In this study, using a bleomycin-induced pulmonary fibrosis mouse model, we administered siRNA against DDR1 transnasally and evaluated histological changes, cytokine expression, and signaling molecule activation in the lungs. Histologically, siRNA against DDR1 successfully reduced in vivo DDR1 expression and attenuated bleomycin-induced infiltration of inflammatory cells. Furthermore, it significantly reduced inflammatory cell counts and concentrations of cytokines such as MCP-1, MIP-1α, and MIP-2 in bronchoalveolar lavage fluid. Subsequently, bleomycin-induced up-regulation of TGF-β in bronchoalveolar lavage fluid was significantly inhibited, and collagen deposition in the lungs was reduced. Furthermore, siRNA against DDR1 significantly inhibited bleomycin-induced P38 MAPK activation in the lungs. Considered together, we propose that DDR1 contributes to the development of bleomycin-induced pulmonary inflammation and fibrosis.

Gap junction protein beta 1 (GJB1) mutations and central nervous system symptoms in X-linked Charcot–Marie–Tooth disease

Objectives – To clarify the clinical variability, including central nervous system (CNS) involvement, in X‐linked Charcot–Marie–Tooth disease (CMTX) patients. Materials and methods– We clinically, pathologically and genetically studied six CMTX patients with distinct symptoms and four different GJB1 mutations. Results– One patient with Val63Ile had deafness, low intelligence, saccadic eye movement, upper extremity distal dominant muscle weakness and normal sensation. Another patient with Glu186Lys had severe sensorineural deafness at the age of 6 years, but did not develop muscle weakness until the age of 20 years. Two patients with Arg22Gln had typical CMT1A‐like clinical features, no CNS symptoms and obvious onion bulb formations. Two siblings with deletion of the entire GJB1 gene had mild to moderate lower extremity muscle weakness and sensory disturbance without CNS involvement. Conclusion– These findings suggest that some gain of function mutations of GJB1 may be related to CNS symptoms because the patients with GJB1 deletion only had peripheral neuropathy, although other unknown associated factors may contribute to their clinical phenotypes.

Clinical phenotype in X-linked Charcot-Marie-Tooth disease with an entire deletion of the connexin 32 coding sequence.

To clarify the clinical phenotype and molecular mechanism in X-linked Charcot-Marie-Tooth disease (CMTX) patients with a deletion of the whole connexin 32 (Cx32) coding sequence, we studied a family with this deletion by electrophysiology, Southern blotting and quantitative PCR analyses. Two brothers with no copy of Cx32, 27 and 25 years old, showed steppage gait, moderate muscle atrophy and weakness, and mild sensory disturbance in the distal parts of the legs. The clinical phenotypes in these brothers were not different from those in patients with other types of severe Cx32 mutations. Their mother, with one copy of Cx32, showed very mild muscle weakness and sensory disturbance. An electrophysiological study showed a nonuniform demyelinating neuropathy with some aspects of an axonal-loss neuropathy. Sural nerve biopsy showed loss of myelinated fibers, many relatively thin myelin sheaths, clusters of small myelinated fibers, and some onion bulb formations. The present findings suggest that both a demyelinating process and an axonal involvement were present in the patients with total defect of Cx32 probably due to loss of the function mechanism of Cx32 as the underlying molecular mechanism, because a dominant negative effect theory is not applicable in these patients.

Expression of vascular endothelial growth factor in tuberculous meningitis.

The pathogenesis of tuberculous meningitis is still unclear. Recently, vascular endothelial growth factor (VEGF) was found to be associated with inflammatory diseases and we found the increased serum level of VEGF in pulmonary tuberculosis. We hypothesized that VEGF might be associated with the pathogenesis of tuberculous meningitis and measured serum and cerebrospinal fluid (CSF) levels of VEGF in 28 patients with tuberculous meningitis and 31 non-tuberculous infectious meningitis patients (13 bacterial meningitis patients, eight fungal meningitis patients and 10 patients with viral meningitis) before therapy. We examined the CSF VEGF levels 3 months after in 12 tuberculous meningitis patients. The serum and CSF levels of VEGF were significantly higher in tuberculous meningitis than in other meningitis. The decrease in titer of CSF VEGF paralleled the clinical improvement of tuberculous meningitis. Immunohistochemical staining of autopsied brains demonstrated the presence of VEGF in the inflammatory mononuclear cells of the dense fibroconnective tissue both in the subarachnoid space and surrounding the vasculitis lesion. We found the expression of VEGF in tuberculous meningitis and think that VEGF reflects its activity.

Activation of Discoidin Domain Receptor 1 on CD14-Positive Bronchoalveolar Lavage Fluid Cells Induces Chemokine Production in Idiopathic Pulmonary Fibrosis

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase activated by collagen. We previously reported the functional expression of DDR1 on human monocyte-derived macrophages in vitro; however, information regarding its role in diseases is limited. Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease, and the lesions contain an abundance of collagen. In this study, we examined DDR1 expression on bronchoalveolar lavage fluid (BALF) cells and investigated its functionality using samples obtained from 28 IPF patients, 13 chronic obstructive pulmonary disease patients, and 14 healthy volunteers. The DDR1 expression level in CD14-positive BALF cells was higher in IPF patients than in chronic obstructive pulmonary disease patients or healthy volunteers. The predominant isoform was DDR1b in the IPF group, while DDR1a was predominant in the other two groups. Using immunohistochemical analysis, we also detected DDR1 expression on infiltrating inflammatory cells in the IPF lesion. In IPF patients, DDR1 activation induced the production of MCP-1, IL-8, MIP-1 α, and matrix metalloproteinase-9 (MMP-9) from CD14-positive BALF cells in a p38 MAPK-dependent manner. In contrast, DDR1 activation of CD14-positive BALF cells in the other groups did not induce the production of these chemokines or MMP-9. These chemokines and MMP-9 contribute to the development of IPF and, therefore, we suggest that DDR1 might be associated with the pathogenesis of IPF in the tissue microenvironment.

Discoidin domain receptor 1: A new class of receptor regulating leukocyte-collagen interaction

Previous studies demonstrated that type I collagen, a major component of the extracellular matrix, could influence the differentiation and function of leukocytes; however, it is not clear whether those effects of collagen were based on its interaction with the classic collagen receptors. α1β1 and α2β1 integrins. We recently detected significant upregulation of discoidin domain receptor 1 (DDR 1), a new class of collagen receptor, in human leukocytes, including neutrophils, monocytes, and lymphocytes, in vitro, leading to the hypothesis that the leukocyte-activating effects of collagen might be owing to its interaction with DDR1. In this review, we summarize our recent findings demonstrating that DDR1-collagen interaction facilitates the adhesion, migration, differentiation/maturation, and cytokine/chemokine production of leukocytes. We also describe the intracellular signaling pathways activated by DDR1 interaction with collagen.