Yuya Terashima

Overproduction of Th2-specific chemokines in NC/Nga mice exhibiting atopic dermatitis–like lesions

We have examined the expression of chemokines and their receptors in the atopic dermatitis-like (AD-like) lesions of NC/Nga mice. Such lesions develop when the mice are kept in conventional conditions, but not when they are kept isolated from specific pathogens. The thymus- and activation-regulated chemokine TARC is unexpectedly highly expressed in the basal epidermis of 14-week-old mice with lesions, whereas it is not expressed in the skin without lesions. Production of TARC by keratinocytes was confirmed by culturing murine keratinocytic cell line cells (PAM212) with TNF-alpha, IFN-gamma, or IL-1beta. Expression of another Th2 chemokine, macrophage-derived chemokine (MDC), was observed in the skin from mice kept in both conventional and pathogen-free conditions, but expression of MDC was increased severalfold in the skin with lesions. The cellular origin of MDC was identified to be dermal dendritic cells. Infiltration of the skin by IL-4-producing T cells and mast cells, and the increase of CCR4 mRNA in the skin, coincided with the development of AD lesions. These observations indicate that TARC and MDC actively participate in the pathogenesis of AD-like lesions in NC/Nga mice and that these Th2 chemokines could be novel targets for intervention therapy of AD in humans.

Peyer's patch is the essential site in initiating murine acute and lethal graft-versus-host reaction.

Acute graft-versus-host disease (a-GVHD) is initiated primarily by immunologically competent cytotoxic T cells (CTLs) that express anti-host specificities. However, the host lymphoid compartment in which these precursor CTLs are initially stimulated remains unclear. Here we show that gut Peyers patches (PPs) are required to activate anti-host CTL responses in a well characterized murine acute graft-versus-host reaction (a-GVHR) model, involving transfer of parent lymphocytes into F1 hybrid recipients. The a-GVHR was prevented when recruitment of donor T cells into PP was interrupted either by disrupting the gene encoding chemokine receptor CCR5 or by blocking integrin α4β7–MAdCAM-1 (mucosal vascular addressin) interactions. Mice deficient for PPs failed to develop a-GVHD in two models of disease induction. Thus, blockade of CTL generation in PPs might offer new strategies for circumventing a-GVHD.

ADAMTS-1 is an active metalloproteinase associated with the extracellular matrix.

Cellular disintegrin and metalloproteinases (ADAMs) are a family of genes with a sequence similar to the snake venom metalloproteinases and disintegrins. ADAMTS-1 is a unique ADAM family protein with respect to the presence of thrombospondin type I motifs and the capacity to bind to the extracellular matrix. Because ADAMTS-1 has a potential zinc-binding motif in the metalloproteinase domain, we examined in this study whether ADAMTS-1 is an active metalloproteinase by means of the proteinase trapping mechanism of α2-macroglobulin. We found that the soluble type of ADAMTS-1 protein is able to form a covalent-binding complex with α2-macroglobulin. Furthermore, the point mutation within the zinc-binding motif of ADAMTS-1 protein eliminates its capacity to bind to α2-macroglobulin. These data demonstrate that the metalloproteinase domain of ADAMTS-1 is catalytically active. In addition, we showed that the removal of the pro-domain from the ADAMTS-1 precursor is impaired in the furin-deficient cell line, LoVo, and that the processing ability of the cells is restored by the co-expression of the furin cDNA. These data provide evidence that the ADAMTS-1 precursor is processed in vivo by furin endopeptidase in the secretory pathway. Consequently, ADAMTS-1 is an active metalloprotease that is associated with the extracellular matrix. This study strongly suggests that ADAMTS-1 may play a role in the inflammatory process through its protease activity.

Structural Basis of the Interaction between Chemokine Stromal Cell-derived Factor-1/CXCL12 and Its G-protein-coupled Receptor CXCR4

The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1 β-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply buried binding pocket in the CXCR4 transmembrane region by the “fly-casting” mechanism. This is the first structural analyses of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand systems, for which the structural studies are still challenging.

Pivotal function for cytoplasmic protein FROUNT in CCR2-mediated monocyte chemotaxis

Ligation of the chemokine receptor CCR2 on monocytes and macrophages with its ligand CCL2 results in activation of the cascade consisting of phosphatidylinositol-3-OH kinase (PI(3)K), the small G protein Rac and lamellipodium protrusion. We show here that a unique clathrin heavy-chain repeat homology protein, FROUNT, directly bound activated CCR2 and formed clusters at the cell front during chemotaxis. Overexpression of FROUNT amplified the chemokine-elicited PI(3)K–Rac–lamellipodium protrusion cascade and subsequent chemotaxis. Blocking FROUNT function by using a truncated mutant or antisense strategy substantially diminished signaling via CCR2. In a mouse peritonitis model, suppression of endogenous FROUNT markedly prevented macrophage infiltration. Thus, FROUNT links activated CCR2 to the PI(3)K–Rac–lamellipodium protrusion cascade and could be a therapeutic target in chronic inflammatory immune diseases associated with macrophage infiltration.

Dramatic decrease of circulating levels of monocyte chemoattractant protein-1 in Kawasaki disease after gamma globulin treatment.

Kawasaki disease (KD) is a systemic vasculitis preferentially affecting coronary arteries. Extensive monocytes/macrophages infiltrate in the vascular lesions, implying the involvement of a chemotactic cytokine in their recruitment. We investigated the role of monocyte chemoattractant protein‐1 (MCP‐1, also termed monocyte chemotactic and activating factor) in KD. In the immunohistochemical studies using the cardiac tissues of patients with fatal KD, MCP‐1 but not interleukin (IL) ‐8 or macrophage inflammatory protein‐1α was localized at the extracellular matrix associated with mononuclear cellular infiltration. The sites of MCP‐1 expression correlated with the distribution of the acute inflammation, including early coronary vasculitis. In prospectively studied patients with KD, circulating levels of MCP‐1, IL‐8, tumor necrosis factor a (TNF‐α), and IL‐1α were elevated in 73, 77, 57, and 0% of samples before gamma globulin (GG) treatment (400 mg/kg × 5 days = total 2 g/kg), respectively, compared with respective control values. GG treatment correlated with a rapid decrease in the circulating levels of MCP‐1 (P = 0.001) but not IL‐8 (P = 0.19) or TNF‐α (P = 0.33). In the sensitive Western blotting, MCP‐1 bound to GG. Furthermore, GG inhibited the MCP‐1‐induced Ca2+ influx in a human monocytic cell line in vitro. These findings suggest a role of MCP‐1 in KD, and indicate that GG treatment may block MCP‐1 activity, thus alleviating KD vasculitis. J. Leukoc. Biol. 65: 566–572; 1999.

Chemerin activates fibroblast-like synoviocytes in patients with rheumatoid arthritis

IntroductionChemerin is a chemotactic agonist identified as a ligand for ChemR23 that is expressed on macrophages and dendritic cells (DCs). In this study, we analyzed the expression of chemerin and ChemR23 in the synovium of rheumatoid arthritis (RA) patients and the stimulatory effects of chemerin on fibroblast-like synoviocytes (FLSs) from RA patients.MethodsChemerin and ChemR23 expression in the RA synovium was ascertained by immunohistochemistry and Western blot analysis. Chemerin expression on cultured FLSs was analyzed by ELISA. ChemR23 expression on FLSs was determined by immunocytochemistry and Western blot analysis. Cytokine production from FLSs was measured by ELISA. FLS cell motility was evaluated by utilizing a scrape motility assay. We also examined the stimulating effect of chemerin on the phosphorylation of mitogen-activated protein kinase (MAPK), p44/42 mitogen-activated protein kinase (ERK1/2), p38MAPK, c-Jun N-terminal kinase (JNK)1/2 and Akt, as well as on the degradation of regulator of NF-κB (IκBα) in FLSs, by Western blot analysis.ResultsChemerin was expressed on endothelial cells and synovial lining and sublining cells. ChemR23 was expressed on macrophages, immature DCs and FLSs and a few mature DCs in the RA synovium. Chemerin and ChemR23 were highly expressed in the RA synovium compared with osteoarthritis. Chemerin and ChemR23 were expressed on unstimulated FLSs. TNF-α and IFN-γ upregulated chemerin production. Chemerin enhanced the production of IL-6, chemokine (C-C motif) ligand 2 and matrix metalloproteinase 3 by FLSs, as well as increasing FLS motility. The stimulatory effects of chemerin on FLSs were mediated by activation of ERK1/2, p38MAPK and Akt, but not by JNK1/2. Degradation of IκB in FLSs was not promoted by chemerin stimulation. Inhibition of the ERK1/2, p38MAPK and Akt signaling pathways significantly suppressed chemerin-induced IL-6 production. Moreover, blockade of the p38MAPK and Akt pathways, but not the ERK1/2 pathway, inhibited chemerin-enhanced cell motility.ConclusionsThe interaction of chemerin and ChemR23 may play an important role in the pathogenesis of RA through the activation of FLSs.

NMR Analyses of the Interaction between CCR5 and Its Ligand Using Functional Reconstitution of CCR5 in Lipid Bilayers

CC-chemokine receptor 5 (CCR5) belongs to the G protein-coupled receptor (GPCR) family and plays important roles in the inflammatory response. In addition, its ligands inhibit the HIV infection. Structural analyses of CCR5 have been hampered by its instability in the detergent-solubilized form. Here, CCR5 was reconstituted into high density lipoprotein (rHDL), which enabled CCR5 to maintain its functions for >24 h and to be suitable for structural analyses. By applying the methyl-directed transferred cross-saturation (TCS) method to the complex between rHDL-reconstituted CCR5 and its ligand MIP-1alpha, we demonstrated that valine 59 and valine 63 of MIP-1alpha are in close proximity to CCR5 in the complex. Furthermore, these results suggest that the protective influence on HIV-1 infection of a SNP of MIP-1alpha is due to its change of affinity for CCR5. This method will be useful for investigating the various and complex signaling mediated by GPCR, and will also provide structural information about the interactions of other GPCRs with lipids, ligands, G-proteins, and effector molecules.

An Anti-Inflammatory Drug, Propagermanium, May Target GPI-Anchored Proteins Associated with an MCP-1 Receptor, CCR2

Monocyte chemoattractant protein-1 (MCP-1) promotes the migration and activation of monocytes and plays a pivotal role in the development of chronic inflammation. Propagermanium (3-oxygermylpropionic acid polymer) has been used as a therapeutic agent against chronic hepatitis B in Japan. We report here that propagermanium specifically inhibits in vitro chemotactic migration of monocytes by MCP-1. Propagermanium did not inhibit binding of MCP-1 to a human monocytic cell line, THP-1 cells, or affect intracellular Ca(2+) mobilization or the cAMP concentration in MCP-1-treated THP-1 cells. The effect of propagermanium seems to require glycosylphosphatidylinositol (GPI)-anchored proteins, as cleavage of GPI anchors by phosphatidylinositol-phospholipase C (PI-PLC) eliminated the inhibitory activity of propagermanium. Anti-GPI-anchored protein antibodies, such as anti-CD55 and anti-CD59, reduced staining of C-C chemokine receptor 2 (CCR2) with an anti-CCR2 antibody against the N-terminus of CCR2 in a flow cytometric analysis, and these antibodies also selectively inhibited MCP-1-induced migration of THP-1 cells. Furthermore, under fluorescence microscopy, GPI-anchored proteins colocalized with CCR2 on THP-1 cells. These results suggest that propagermanium may target GPI-anchored proteins that are closely associated with CCR2 to selectively inhibit the MCP-1-induced chemotaxis, thus providing a mechanistic basis for the anti-inflammatory effects of the drug.

Robust Antitumor Effects of Combined Anti–CD4-Depleting Antibody and Anti–PD-1/PD-L1 Immune Checkpoint Antibody Treatment in Mice

Ueha, Yokochi, Ishiwata, and colleagues show in three mouse tumor models that CD4 depletion led to tumor-specific CTL proliferation in the draining lymph node and increased tumor infiltration of PD-1+CD8+ T cells; it also synergized with PD-1/PD-L1 blockade to suppress tumor growth and prolong survival. Depletion of CD4+ cells in tumor-bearing mice has strong antitumor effects. However, the mechanisms underlying these effects and the therapeutic benefits of CD4+ cell depletion relative to other immunotherapies have not been fully evaluated. Here, we investigated the antitumor effects of an anti–CD4-depleting mAb as a monotherapy or in combination with immune checkpoint mAbs. In B16F10, Colon 26, or Lewis lung carcinoma subcutaneous tumor models, administration of the anti-CD4 mAb alone had strong antitumor effects that were superior to those elicited by CD25+ Treg depletion or other immune checkpoint mAbs, and which were completely reversed by CD8+ cell depletion. CD4+ cell depletion led to the proliferation of tumor-specific CD8+ T cells in the draining lymph node and increased infiltration of PD-1+CD8+ T cells into the tumor, with a shift toward type I immunity within the tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, particularly anti–PD-1 or anti–PD-L1 mAbs, synergistically suppressed tumor growth and greatly prolonged survival. To our knowledge, this work represents the first report of robust synergy between anti-CD4 and anti–PD-1 or anti–PD-L1 mAb therapies. Cancer Immunol Res; 3(6); 631–40. ©2015 AACR.