Galectin-1 and galectin-3 expression profiles in classically and alternatively activated human macrophages

https://doi.org/10.1016/j.bbagen.2011.11.014Get rights and content

Abstract

Background

Galectins have been identified as modulators of many monocyte/macrophage functions. In the response to a wide range of environmental cues macrophages may exhibit different biochemical and biological characteristics, but two main subtypes, classically (M1) and alternatively (M2) activated macrophages have been recognized. To contribute to elucidation of role and regulation of galectin-1 and galectin-3 in differently activated macrophages we explored their expression profiles in these cells.

Methods

Human monocytes obtained from blood donors were differentiated into classically (M1) and alternatively (M2a/M2c) activated macrophages. Gene and protein expression levels of intra- and extracellular galectins were investigated by qRT-PCR, Western-blot, flow cytometry, and ELISA while cytokine and surface receptor expression profiling was performed by flow cytometry.

Results

Differentiation/polarization of human monocytes into classically (M1) and alternatively (M2a/M2c) activated macrophages was followed by profound changes of galectin-3 expression and its proteolytic cleavage. Expression and secretion of Gal-3 was tightly regulated and significantly differed among classically (M1) and alternatively (M2a/M2c) activated macrophages, while the differences of galectin-1 expression profiles were not as pronounced. Human monocytes exhibited high amount of free galectin-3 receptors, while on both types of activated macrophages were fully saturated.

Conclusions

Galectin-3 is more distinctive descriptor of macrophages differentiation/activation than galectin-1. Its specific expression and secretion pattern in M1 vs. M2a/M2c macrophages contributes to better understanding of its role and regulation in these cells.

General significance

Recognition of distinct galectin-1 and galectin-3 expression profiles in differently activated macrophages provides a new insight on biological characteristics of these cells and sheds a new light of galectin-3 as a modulator of individual macrophage subset. This article is part of a Special Issue entitled Glycoproteomics.

Highlights

► Similar gene and protein expression of galectin-1 in monocytes (Mo) and macrophages (M1/M2). ► Cellular and membrane galectin-3 expression patterns follow the scheme Mo << M1 < M2. ► Up to 5 galectin-3 truncated forms detected in activated macrophages. ► M1 macrophages secrete double amount of galectin-3 in comparison to M2 macrophages. ► Free Gal-3 receptors present on monocyte surface, on macrophages they are fully saturated

Introduction

Macrophages are crucial regulatory cells of host defense and tissue homeostasis. Characterized by a high level of plasticity, macrophages are able to modulate their biological functions in the response to a wide range of microenvironmental stimuli. In general, two main subtypes of macrophages have been recognized according to the cytokine production profile and the expression of surface receptors; macrophages differentiated from monocytes by the exposure to granulocyte macrophage colony-stimulating factor (GM-CSF) named M1 and those exposed to macrophage colony-stimulating factor (M-CSF), named M2. M1 macrophages could be additionally activated by the exposure to IFN-γ and/or LPS yielding so-called classically activated macrophages, while activated M2 macrophages were consequently named alternatively activated macrophages [1], [2], [3]. While M1 macrophages mediate resistance to intracellular pathogens, tissue destruction, and anti-tumor resistance, M2 macrophages are generally oriented to tissue remodeling and repair, resistance to parasites, immunoregulation, and tumor promotion [4]. By more precise distinction, based on the biochemical characteristics and biological functions, three different subtypes of activated M2 macrophages have been recognized: (i) M2a or alternatively activated macrophages by IL-4 or IL-13, (ii) M2b or Type II, activated by glucocorticoids or immune complexes and (iii) M2c activated by IL-10, also called true deactivated macrophages [5].

Galectin-1 (Gal-1) (~ 14.5 kDa) and galectin-3 (Gal-3) (~ 32 kDa), most studied members of the β-galactoside binding lectins, have been implicated in numerous biological processes and play a significant role in many aspects of monocyte-macrophage cell biology. Though structurally distinct (Gal-1 has one carbohydrate recognition domain (CRD), while Gal-3 besides CRD possesses additional, N-terminal non-lectin domain), both galectins can be present intracellularly, secreted by an unknown mechanism outside of the cells or bound on cell-surface receptors or extracellular matrix ligands. Inside the cells they bind their ligands presumably through protein–protein interaction, while binding of cell-surface ligands initiates a dimerization of Gal-1 and oligomerization of Gal-3 and triggering a cascade of, so far mostly known, transmembrane signaling events [6], [7].

Gal-1 inhibits arachidonic acid release [8], blocks nitric oxide synthesis and increases arginase activity, suggesting a role for this protein in triggering a state of alternative activation in cells of the monocyte/macrophage lineage [9]. Accordingly, Gal-1 treatment inhibits IFN-γ-induced FcγRI-dependent phagocytosis and major histocompatibility complex (MHC) II expression in human monocytes and macrophages [10] and blocks IL-12 secretion from parasite-infected macrophages [11]. It was also suggested that Gal-1-glycan lattices may have evolved to negatively regulate the antigen-presenting function and activation of monocytes/macrophages [12].

On the other hand, Gal-3 was shown to modulate the production of some cytokines, such as IL-1 [13], IL-5 [14] and IL-8 [15] in the cells of monocyte-macrophage lineage, as well as in some other cell types [16], [17], [18]. Gal-3 also induces Ca2 + influx in monocytes [19], acts as a chemoattractant for monocytes, macrophages and endothelial cells [20], enhances macrophage clearance of apoptotic neutrophils [21], regulates alternative macrophage activation [22] and participates in many other processes (reviewed in [23], [24], [25]).

However, the role and regulation of Gal-1 and Gal-3 expression during differentiation of human monocytes into particular macrophage subsets and their activation is mainly unknown. To provide better insight into biology of differently activated macrophages on one hand, and Gal-1 and Gal-3 on the other. In this study we determined the intra- and extracellular expression profiles of Gal-1 and Gal-3 in classically (M1) and alternatively (M2a/M2c) activated macrophages obtained by differentiation/activation of human blood monocytes in vitro.

Section snippets

Material

All chemicals were of analytical grade, and if not stated otherwise were purchased from Sigma (St. Louis, MO, USA). Isopropyl-β-D-thio-galactoside (IPTG) was from Fermentas (St. Leon-Rot, Germany), complete EDTA-free Protease Inhibitor Cocktail Tablets from Roche (Basel, Switzerland), Immobilon PVDF-membranes from Millipore Corp. (Bedford, MA, USA), Ficoll-Paque Plus and enhanced chemiluminescence (ECL) Western blot detection kit from Amersham Biosciences (Cardiff, UK), while RPMI 1640 medium,

Monocyte differentiation and activation of macrophages

To analyze the regulation of expression of Gal-1 and -3 in human monocyte-derived cells, freshly isolated monocytes were exposed either to GM-CSF and IFN-γ/LPS to obtain classically activated macrophages (M1) or to M-CSF and IL-4 or IL-10 to obtain alternatively activated macrophages M2a or M2c, respectively. Harvested cells were shown to be > 98% viable. To assess whether the expected cell types were obtained, the cells were analyzed for their cytokine production and specific surface receptor

Discussion

It is known for more than fifteen years that human blood monocytes express Gal-3 [19]. Yet, the knowledge on the role and regulation of Gal-3 expression during differentiation of human monocytes into distinct macrophage subtypes is still limited. Here we provide an insight into the expression and secretion of Gal-3 in classically activated (M1) and alternatively activated macrophages (M2a/M2c).

The expression of Gal-3 on the monocyte surface was previously shown by immunogold/electron

Conclusions

In addition to already recognized uniquely expressed biochemical markers, classically and alternatively activated macrophages also differ by the expression profiles of Gal-3, and to some lesser extent of Gal-1. Since these cells exhibit remarkable plasticity and can change their phenotype in response to different environmental signals, understanding of the role and regulation of Gal-1 and Gal-3 expression in macrophage biology is of utmost interest. Gal-3 has been previously recognized as an

Acknowledgement

This work was supported by grant #006-006-1194-1218 from the Ministry of Science, Education and Sports of the Republic of Croatia.

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