Fumiko Marttila-Ichihara

Vimentin function in lymphocyte adhesion and transcellular migration

Although the adhesive interactions of leukocytes with endothelial cells are well understood, little is known about the detailed mechanisms underlying the actual migration of leukocytes across the endothelium (diapedesis). Leukocytes have been shown to use both paracellular and transcellular routes for transendothelial migration. Here we show that peripheral blood mononuclear cells (PBMCs; T- and B-lymphocytes) preferentially use the transcellular route. The intermediate filaments of both endothelial cells and lymphocytes formed a highly dynamic anchoring structure at the site of contact between these two cell types. The initiation of this process was markedly reduced in vimentin-deficient (vim−/−) PBMCs and endothelial cells. When compared with wild-type PBMCs, vim−/− PBMCs showed a markedly reduced capacity to home to mesenteric lymph nodes and spleen. Furthermore, endothelial integrity was compromised in vim−/− mice, demonstrating that intermediate filaments also regulate the barrier that governs leukocyte extravasation. Absence of vimentin resulted in highly aberrant expression and distribution of surface molecules critical for homing (ICAM-1 and VCAM-1 on endothelial cells and integrin-β1 on PBMCs). These data show that intermediate filaments are active in lymphocyte adhesion and transmigration.

Altered purinergic signaling in CD73 deficient mice inhibits tumor progression

CD73/ecto‐5′‐nucleotidase dephosphorylates extracellular AMP into adenosine, and it is a key enzyme in the regulation of adenosinergic signaling. The contribution of host CD73 to tumor growth and anti‐tumor immunity has not been studied. Here, we show that under physiological conditions CD73‐deficient mice had significantly elevated ATPase and ADPase activities in LN T cells. In a melanoma model, the growth of primary tumors and formation of metastasis were significantly attenuated in mice lacking CD73. Among tumor‐infiltrating leukocytes there were fewer Tregs and mannose receptor‐positive macrophages, and increased IFN‐γ and NOS2 mRNA production in CD73‐deficient mice. Treatment of tumor‐bearing animals with soluble apyrase, an enzyme hydrolyzing ATP and ADP, significantly inhibited tumor growth and accumulation of intratumoral Tregs and mannose receptor‐positive macrophages in the WT C57BL/6 mice but not in the CD73‐deficient mice. Pharmacological inhibition of CD73 with α,β‐methylene‐adenosine‐5′‐diphosphate in WT mice retarded tumor progression similarly to the genetic deletion of CD73. Together these data show that increased pericellular ATP degradation in the absence of CD73 activity in the host cells is a novel mechanism controlling anti‐tumor immunity and tumor progression, and that the purinergic balance can be manipulated therapeutically to inhibit tumor growth.

Vascular Adhesion Protein-1 Enhances Tumor Growth by Supporting Recruitment of Gr-1(+)CD11b(+) Myeloid Cells into Tumors

Cancer growth is regulated by several nonmalignant cell types, such as leukocytes and endothelial cells, which reside in the stroma of the tumor. Vascular adhesion protein-1 (VAP-1) is an amine oxidase enzyme that is expressed on the surface of endothelial cells. It supports leukocyte traffic into inflamed tissues, but nothing is known about its possible role in cancer biology in vivo. Here, we report that B16 melanoma and EL-4 lymphoma remain smaller in VAP-1-deficient mice than in wild-type controls. We found an unexpected defect in tumor angiogenesis in the absence of VAP-1. VAP-1 also selectively enhanced the recruitment of Gr-1+CD11b+ myeloid cells into the tumors. Generation of mice expressing enzymatically inactive VAP-1 showed that the oxidase activity of VAP-1 was necessary to support neoangiogenesis, myeloid cell recruitment, and tumor growth in vivo. These data describe VAP-1 as the first adhesion molecule known to be involved in the recruitment of Gr-1+CD11b+ myeloid cells into tumors. They also suggest that VAP-1 is a potential new tool for immunotherapy of tumors that could be exploited to reduce tumor burden by controlling the traffic of Gr-1+CD11b+ myeloid cells.

Siglec-9 is a novel leukocyte ligand for vascular adhesion protein-1 and can be used in PET imaging of inflammation and cancer

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1-dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the ⁶⁸Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.

Small-Molecule Inhibitors of Vascular Adhesion Protein-1 Reduce the Accumulation of Myeloid Cells into Tumors and Attenuate Tumor Growth in Mice

Vascular adhesion protein-1 (VAP-1) is an endothelial, cell surface–expressed oxidase involved in leukocyte traffic. The adhesive function of VAP-1 can be blocked by anti–VAP-1 Abs and small-molecule inhibitors. However, the effects of VAP-1 blockade on antitumor immunity and tumor progression are unknown. In this paper, we used anti–VAP-1 mAbs and small-molecule inhibitors of VAP-1 in B16 melanoma and EL-4 lymphoma tumor models in C57BL/6 mice. Leukocyte accumulation into tumors and neoangiogenesis were evaluated by immunohistochemistry, flow cytometry, and intravital videomicroscopy. We found that both anti–VAP-1 Abs and VAP-1 inhibitors reduced the number of leukocytes in the tumors, but they targeted partially different leukocyte subpopulations. Anti–VAP-1 Abs selectively inhibited infiltration of CD8-positive lymphocytes into tumors and had no effect on accumulation of myeloid cells into tumors. In contrast, the VAP-1 inhibitors significantly reduced only the number of proangiogenic Gr-1+CD11b+ myeloid cells in melanomas and lymphomas. Blocking of VAP-1 by either means left tumor homing of regulatory T cells and type 2 immune-suppressing monocytes/macrophages intact. Notably, VAP-1 inhibitors, but not anti–VAP-1 Abs, retarded the growth of melanomas and lymphomas and reduced tumor neoangiogenesis. The VAP-1 inhibitors also reduced the binding of Gr-1+ myeloid cells to the tumor vasculature. We conclude that tumors use the catalytic activity of VAP-1 to recruit myeloid cells into tumors and to support tumor progression. Small-molecule VAP-1 inhibitors therefore might be a potential new tool for immunotherapy of tumors.

Clever-1/Stabilin-1 Controls Cancer Growth and Metastasis

Purpose: Immunosuppressive leukocytes and vasculature are important host cell components regulating tumor progression. Clever-1/Stabilin-1, a multifunctional scavenger and adhesion receptor, is constitutively present on a subset of type II macrophages and lymphatic endothelium, but its functional role in cancer is unknown. Experimental Design: Here, we generated full Clever-1 knockout mice and cell-specific ones lacking Clever-1 either on macrophages or endothelium. We also used anti-Clever-1 antibody therapy to treat B16 melanoma and EL-4 lymphoma. Results: Clever-1–deficient mice had smaller primary and metastatic tumors than wild-type (WT) controls. Growth of primary tumors, but not of metastases, was attenuated also in mice lacking Clever-1 selectively in macrophages or in vascular endothelium. Anti-Clever-1 antibody treatment inhibited tumor progression in WT mice. Both genetically and therapeutically induced absence of functional Clever-1 led to diminished numbers of immunosuppressive leukocyte types in tumors. Functionally Clever-1 mediated binding of immunosuppressive leukocytes to the intratumoral blood vessels aberrantly expressing Clever-1, and tumor cell traffic via the lymphatics. The antibody therapy did not aggravate autoimmunity. Conclusion: This work identifies Clever-1 in type II macrophages and in tumor vasculature as a new immunosuppressive molecule in cancer. Our finding that Clever-1 supports binding of tumor-infiltrating lymphocytes to tumor vasculature increases our understanding of leukocyte immigration to tumors. The ability of anti-Clever-1 antibody treatment to attenuate tumor progression in WT mice in vivo is therapeutically relevant. Thus, Clever-1 may be an emerging new target for modulating immune evasion and lymphatic spread in cancer. Clin Cancer Res; 20(24); 6452–64. ©2014 AACR.

Amine oxidase activity regulates the development of pulmonary fibrosis

In pulmonary fibrosis, an inflammatory reaction and differentiation of myofibroblasts culminate in pathologic deposition of collagen. Amine oxidase copper containing‐3 (AOC3) is a cell‐surface‐expressed oxidase that regulates leukocyte extravasation. Here we analyzed the potential role of AOC3 using gene‐modified and inhibitor‐treated mice in a bleomycin‐induced pulmonary fibrosis model. Inflammation and fibrosis of lungs were assessed by histologic, flow cytometric, and quantitative PCR analysis. AOC3‐deficient mice showed a 30–50% reduction in fibrosis, collagen synthesis, numbers of myofibroblasts, and accumulation of CD4+ lymphocytes, NK T cells, macrophages, and type 2 innate lymphoid cells compared with wild‐type control mice. AOC3‐knock‐in mice, which express a catalytically inactive form of AOC3, were also protected from lung fibrosis. In wild‐type mice, a small‐molecule AOC3 inhibitor treatment reduced leukocyte infiltration, myofibroblast differentiation, and fibrotic injury both in prophylactic and early therapeutic settings by about 50% but was unable to reverse the established fibrosis. AOC3 was also induced in myofibroblasts in human idiopathic pulmonary fibrosis. Thus, the oxidase activity of AOC3 contributes to the development of lung fibrosis mainly by regulating the accumulation of pathogenic leukocyte subtypes, which drive the fibrotic response.—Marttila‐Ichihara, F., Elima, K., Auvinen, K., Veres, T. Z., Rantakari, P., Weston, C., Miyasaka, M., Adams, D., Jalkanen, S., Salmi, M. Amine oxidase activity regulates the development of pulmonary fibrosis. FASEB J. 31, 2477–2491 (2017). www.fasebj.org