Differentiation of human monocytes in vitro with granulocyte–macrophage colony-stimulating factor and macrophage colony-stimulating factor produces distinct changes in cGMP phosphodiesterase expression
Introduction
Macrophages perform many functions including phagocytosis and killing of invading pathogens, release of cytokines and inflammatory mediators, and presentation of antigen (for review, see [1], [2], [3]). Macrophages can be derived from monocytes that normally circulate in peripheral blood. In response to cytokines or other chemotactic signals, monocytes will leave the circulation and enter a tissue where they can differentiate into macrophages. The two cytokines previously identified as predominant promoters of monocyte differentiation are macrophage colony-stimulating factor (M-CSF) and granulocyte–macrophage colony-stimulating factor (GM-CSF) [4].
One of the means by which macrophage function is regulated is by alterations in the levels of the second messenger cyclic nucleotide, cAMP. cAMP is synthesized by adenylyl cyclase and has been found generally to have anti-inflammatory effects in macrophages. cGMP is synthesized by guanylyl cyclase (GC), which has three membrane-bound isoforms (GC-A, GC-B, and GC-C) that are activated by peptide ligands, and one soluble isoform (soluble guanylyl cyclase, sGC), which is activated by nitric oxide (NO) [5]. Compared to cAMP, significantly less is known about how cGMP regulates macrophage function. Although its in vivo importance is not universally agreed upon, it has been implicated to have effects on several monocyte and macrophage processes, such as inhibition of iNOS induction [6], [7], stimulation [8], [9], [10] or inhibition [11] of TNF-α release, and stimulation [12] or inhibition [13] of phagocytosis. Given that exquisite control of cyclic nucleotide levels is needed for proper function of cells, it is likely that a delicate balance between cGMP synthesis by cyclases and degradation by phosphodiesterases (PDEs) must be maintained. For example, in neutrophils, immune cells that perform some of the same functions as macrophages, cGMP can have either stimulatory or inhibitory effects on neutrophil migration depending on its concentration (for review, see Ref. [14]). It is quite possible that the same biphasic effect may be present in macrophages. Thus, the expression of specific GCs or PDEs will determine how macrophages regulate functions controlled by cGMP and respond to stimuli that trigger cGMP production. An important step in beginning to understand how cGMP may be regulated in macrophages is the determination of which cGMP PDEs and GCs are expressed in various types of macrophages.
Cellular cyclic nucleotide degradation is controlled by PDEs—enzymes that hydrolyze cAMP and cGMP. Eleven different PDE families are currently known, with many families containing multiple isoforms [15], [16]. The PDE families are distinguished by differing primary structures, regulation by allosteric activators and inhibitors, enzymatic characteristics, pharmacological inhibitor profiles, and tissue as well as cellular distribution [15]. Several reports have begun to characterize which PDEs hydrolyze cAMP in monocytes and macrophages [17], [18], [19]. These initial studies have found high levels of PDE4 in monocytes and significant increases in PDE1 and PDE3 activity upon differentiation to macrophages. Most studies concerning functional roles for PDEs in macrophages have primarily identified effects resulting from PDE4 inhibition and some synergistic effects of PDE3 and PDE4 inhibition [20]. However, the observation that the expression of PDE1 is increased upon differentiation suggests that it may serve an important role in macrophage function. Furthermore, as most of the PDE1 isozymes are known to be most proficient at hydrolyzing cGMP, it implies that the regulation of cGMP in these cells is likely to be important. However, the metabolism of this cyclic nucleotide by these cells has not been explored previously in any detail.
In this report, we have characterized the expression profile of cGMP PDEs and GCs in primary human monocytes and monocyte-derived macrophages. As many different macrophage phenotypes and morphologies exist, we attempted to produce two distinct and homogeneous types of macrophage by isolating monocytes from the peripheral blood of human donors and by subsequently differentiating them in vitro with two different cytokines, M-CSF or GM-CSF. We have found that PDE1B, PDE2A, and PDE5A are the primary cGMP-metabolizing PDEs expressed in these cells. Interestingly, the expression of these PDEs varies greatly with the differentiation signal and subsequent cellular phenotype. We also identified THP-1 and U937 cell lines as potential models to study the roles of PDE1 and PDE2, respectively, in macrophage function. To try and gain further insight into the regulation of cGMP in monocytes and monocyte-derived macrophages, we have also determined the expression of GC isoforms in these cells. Like the PDEs, the expression of GC isoforms was also found to vary with the cellular phenotype, with sGC expression decreasing and membrane-bound GC-A expression increasing with differentiation. This study describes how pathways for cGMP synthesis and breakdown can vary in monocytes and monocytes differentiated with different cytokines. By characterizing the expression of PDEs and GCs, our studies provide new insights into how cGMP can be differentially regulated in monocytes and monocyte-derived macrophages and present a mechanistic basis for dissecting its function. Moreover, the identification of unique PDEs may provide an approach that allows differential modulation of cGMP in these various cell types by isoform-selective PDE inhibitors. Such modulation may have applications in the treatment of macrophage-mediated disorders.
Section snippets
Materials
Magnetic CD14 microbeads were purchased from Miltenyi Biotech (Auburn, CA). Recombinant human GM-CSF was from Biosource International (Camarillo, CA) and recombinant human M-CSF and IL-10 were from Research Diagnostics (Flanders, NJ). The PDE inhibitors milrinone, rolipram, and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) were obtained from Calbiochem (San Diego, CA). Sildenafil was a generous gift from Pfizer. Buffy coats from human donors were obtained from the American Red Cross Blood Bank
Monocyte to macrophage differentiation
Treatment with either of the cytokines GM-CSF or M-CSF effectively differentiated monocytes to a more macrophage-like phenotype as shown by the cell surface marker expression depicted in Fig. 1. With M-CSF or GM-CSF treatment, CCR2 (the receptor for monocyte chemotactic protein 1) expression was decreased to undetectable levels as has previously been demonstrated [28], [29]. Conversely, mannose receptor expression was upregulated, as has been seen with monocyte-to-macrophage differentiation [29]
Discussion
Macrophages are needed for a variety of physiological functions, ranging from fighting invading microbes to eliminating cancerous cells to assisting in wound repair and tissue remodeling. Consequently, it is important to maintain populations of macrophages with different phenotypes throughout the body. Macrophages derived in culture from M-CSF differentiated monocytes appear to be very similar to naturally occurring peritoneal macrophages [21], [47], [48], while GM-CSF differentiated cells
Acknowledgements
This work was supported by NIH grant DK21723 to J.A. Beavo.
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