Alpha-smooth muscle actin expression identifies subpopulations of mouse lymph node non-hematopoietic cells

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Highlights

  • Transgenic reporter expression of αSMA is examined in the lymph nodes.

  • Non-hematopoietic cells can be fractionated according to αSMA expression.

  • gp38+CD31 and gp38CD31 cells predominantly contain an αSMA-expressing population.

  • αSMA identifies functionally distinct subpopulations in non-hematopoietic cells.

  • αSMA is useful for the further characterization of non-hematopoietic cells in the lymph nodes.

Abstract

Significant attention has been given to the role played by non-hematopoietic cells in the immune organs, including the lymph nodes, in hopes of understanding the development, maintenance, and regulation of the immune system. However, the molecular and cellular characterization of non-hematopoietic cells is still in its infancy. Here we show that non-hematopoietic cells in mouse lymph nodes can be fractionated into previously unidentified subpopulations according to the transgenic reporter expression of alpha-smooth muscle actin (αSMA). αSMA+ non-hematopoietic cells were predominantly detected in gp38+CD31 and gp38CD31 cells. Molecular expression profiles suggest similarities between αSMA+gp38+CD31 and αSMAgp38+CD31 subpopulations and dissimilarities between αSMA+gp38CD31 and αSMAgp38CD31 subpopulations. The results indicate that αSMA is a useful marker for further understanding the molecular and cellular characteristics of non-hematopoietic cells in the lymph nodes.

Introduction

The lymph nodes play an essential role in the orchestration of immune responses in mammalian species, including human. Various non-hematopoietic cells structurally organize the lymph nodes and functionally provide the stromal microenvironments for coordinating the immune responses by hematopoietic cells. It has been shown that the lymph node non-hematopoietic cells can be subdivided into four distinct populations according to the expression of the podocyte marker gp38 (podoplanin) and the endothelial cell marker CD31 (PECAM1). The gp38+CD31 cells include fibroblastic reticular cells (FRCs), which form a three-dimensional network in the T cell zones and regulate the homeostasis and trafficking of T cells [1]. The gp38+CD31+ and gp38CD31+ cells contain lymphatic endothelial cells (LECs) and blood ECs (BECs), respectively, which critically regulate the egress and entry of lymphocytes [2]. However, the molecular and cellular characterization of non-hematopoietic cells in the lymph nodes is still in its infancy.

Alpha-smooth muscle actin (αSMA) is expressed in cells of smooth muscle lineage in various organs. The αSMA expression is often associated with perivascular cells (PVCs), including pericytes surrounding microvessels and vascular smooth muscle cells surrounding large vessels [3], [4]. Although αSMA-expressing cells have been detected also in the lymph nodes [5], the molecular and cellular characteristics of αSMA+ cells in the lymph nodes are unclear, especially in the context of well-studied non-hematopoietic populations that are subdivided according to the expression of gp38 and CD31.

In the present study, we examined αSMA expression in lymph nodes using αSMA-GFP-transgenic mice. Green fluorescent protein (GFP) expression in those mice faithfully reflects the specific expression of αSMA and can identify nonvascular and vascular smooth muscle cells, including renal glomerular mesangial cells, and a small population of bone marrow stromal cells [6]. Our results show that GFP+ cells in the lymph nodes of αSMA-GFP-transgenic mice mostly comprise non-hematopoietic cells and at the same time contain a small population of hematopoietic cells, which represent macrophages that have likely acquired GFP proteins from the non-hematopoietic αSMA+ cells. Interestingly, αSMA-GFP expression in the lymph nodes made possible the identification of subpopulations of gp38+CD31 and gp38CD31 non-hematopoietic cells. The results have enabled the identification of hitherto unknown subpopulations of non-hematopoietic cells in the lymph nodes.

Section snippets

Mice

αSMA-GFP-transgenic mice in C57BL/6 (B6) background, which express GFP under the control of smooth muscle type alpha-actin (αSMA) promoter, were originally from the National Eye Institute, National Institutes of Health, USA [6]. B6 mice were obtained from SLC (Shizuoka, Japan), whereas B6-Ly5.1 mice were maintained in our laboratory. The mice were maintained under specific pathogen-free conditions in our animal facility. The experiments were conducted under the approval of the Institutional

αSMA-GFP expression in lymph nodes

It was reported that GFP expression in αSMA-GFP-transgenic mice faithfully reflects the specific expression of αSMA and can identify vascular and nonvascular smooth muscle cells as well as a small population of bone marrow stromal cells [6]. In order to characterize αSMA-expressing cells in the lymph nodes, we first examined GFP expression in paraformaldehyde-fixed sections of inguinal lymph nodes from αSMA-GFP-transgenic mice. GFP signals, which were detectable in various areas of the lymph

Discussion

The present results indicate that αSMA expression can identify hitherto unknown subpopulations of non-hematopoietic cells in the lymph nodes of αSMA-GFP-transgenic mice. The gp38+CD31 non-hematopoietic lymph node cells, previously known to contain FRCs, can be clearly subdivided into αSMA+ and αSMA subpopulations that are similar in regard to the expression of several cell-surface molecules but different in terms of chemokine gene expression. The gp38CD31 non-hematopoietic lymph node cells,

Acknowledgments

The authors thank Drs. Sanai Sato (National Eye Institute) and Ivo Kalajzic (University of Connecticut Health Center) for providing αSMA-GFP-transgenic mice, and Drs. Mie Sakata and Izumi Ohigashi and other members of the laboratory for support in experiments and discussion. This study was supported by Grants-in-Aid for Scientific Research from MEXT and JSPS (23249025, 24111004, and 25111507), Japan.

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