Elsevier

Human Immunology

Volume 70, Issue 4, April 2009, Pages 211-217
Human Immunology

Expression and function of semaphorin 3A and its receptors in human monocyte-derived macrophages

https://doi.org/10.1016/j.humimm.2009.01.026Get rights and content

Abstract

Semaphorins are a large family of secreted and membrane-bound proteins. Recently, several roles of semaphorins in the immune system have emerged. Several semaphorins and their receptors are expressed in a variety of lymphoid and myeloid cells and affect immune cell functions, including cell proliferation, differentiation, chemotaxis, and cytokine production. However, the roles of class 3 semaphorins in human myeloid cells are not well known. Here we examined the regulation of expression of class 3 semaphorins and their receptors by inflammatory stimuli and their function in human macrophages. We show that the expression of Sema3A receptors (neuropilin-1 (NRP-1), NRP-2, plexin A1, plexin A2, and plexin A3) significantly increased during M-CSF–mediated differentiation of monocytes into macrophages under conditions that promote an M2 alternatively activated macrophage phenotype. Consistent with increased NRP-1 expression, cell surface binding of Sema3A increased during M2 differentiation. Interferon (IFN)–γ and lipopolysaccharide, which promote classical M1 macrophage activation affected expression of NRP-1, NRP-2 and plexin A1. IFN-γ decreased NRP-1 expression and LPS suppressed NRP-2 and plexin A1 expression. Furthermore we show that Sema3A induced apoptosis in monocyte-derived macrophages and cooperated with anti-Fas CH11 antibody to augment apoptosis. Our results suggest that Sema3A plays a role in induction of apoptosis in monocyte-derived macrophages that are resistant to Fas-induced apoptosis, and that its function can be modulated in inflammatory conditions.

Introduction

Semaphorins are a large family of secreted and membrane-bound proteins, characterized by a distinct cysteine-rich domain of about 500 amino acids called the sema domain. These proteins were originally discovered in the nervous system, where they have been implicated in repulsive axon guidance during the development of the nervous system [1], [2]. Class 3 semaphorins are secreted proteins and have six members, Sema3A–3F. Class 3 semaphorins bind to signaling receptors of the plexinA family but also require neuropilins as binding co-receptors. Sema3A binds only neuropilin-1 (NRP-1), whereas Sema3B, Sema3C, and Sema3F bind both NRP-1 and NRP-2. In contrast to other class 3 semaphorins, Sema3E does not require neuropilins for signaling. Neuropilins have a very short cytoplasmic domain that cannot transduce intracellular signals, and plexins form complexes with neuropilins and act as the signal-transducing components of the Semaphorin–neuropilin–plexin ligand-receptor complex [3]. Recently, several roles of semaphorins in the immune system have emerged. Several semaphorins and their receptors are expressed in a variety of lymphoid and myeloid cells and affect immune cell functions, including cell proliferation, differentiation, chemotaxis, and cytokine production [4]. However, the expression and the roles of class 3 semaphorins in human myeloid cells are not well known.

In this report, we examined the expression and function of class 3 semaphorins and their receptors in human peripheral blood monocytes and monocyte-derived M2-like macrophages. M2 macrophages are alternatively activated macrophages that produce low amounts of inflammatory cytokins such as tumor necrosis factor (TNF) or interleukin (IL)–12 and are involved in immunoregulation and tissue remodeling [5]. We show that expression of NRP-1, NRP-2, plexin A1, plexin A2, and plexin A3 increased during M-CSF-driven differentiation of human monocytes into M2-like macrophages. In monocyte-derived macrophages, Sema3A significantly induced apoptosis. These results describe a new role for class 3 semaphorins in human myeloid cells.

Section snippets

Cell isolation and culture

Monocytes were obtained from peripheral blood mononuclear cells by positive selection using anti-CD14 magnetic beads, as recommended by the manufacturer (Miltenyi Biotech, Aubuon, CA). Monocytes were used fresh or differentiated to macrophages during 2 days of culture in αMEM medium supplemented with 10% fetal bovine serum and 20 ng/ml macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN). THP-1 cells were cultured at 37°C in 5% CO2 using RPMI medium with 10% fetal bovine serum.

Flow cytometry

Expression of Class 3 semaphorins and their receptors

Semaphorins are widely expressed in most tissues and organ systems such as the nervous, cardiovascular, musculoskeletal, renal, reproductive, and immune systems [2]. Because there is no previous report about expression of class 3 semaphorins in human monocytes and monocyte-derived macrophages, we examined expression of class 3 semaphorins in human monocytes, monocyte-derived macrophages, and THP-1 cells. For excluding the contamination of genomic DNA, PCR product signal was examined using RT

Discussion

In this report, we show that the expression of Sema3A receptors (NRP-1, NRP-2, plexin A1, plexin A2, and plexin A3) significantly increased during M-CSF-mediated differentiation of monocytes into macrophages. Consistent with increased expression of NRP-1, cell surface binding of Sema3A increased during differentiation. We first observed that Sema3A and its receptors are co-expressed in human monocyte–derived macrophages during M-CSF–mediated differentiation, and these results suggest that

Acknowledgements

This work was supported by grants from the National Institutes of Health (to L.B.I.) and the Arthritis Foundation (to K.-H.P.-M).

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