Regulation of macrophage phenotype by long-term exposure to IL-10
Introduction
Interleukin 10 (IL-10) is a well known anti-inflammatory and immunosuppressive cytokine that primarily targets antigen-presenting cells (Pestka et al., 2004). IL-10 is produced as part of the homeostatic response to infection and inflammation, and plays a critical role in limiting the duration and intensity of immune and inflammatory reactions. IL-10 suppresses proinflammatory cytokine and chemokine production, and antigen presentation by monocytes or macrophages via down-regulation of MHC II and of co-stimulatory molecules. IL-10 deficient mice show increased Th1 responses, enhanced clearance of bacterial and fungal infections, exaggerated asthmatic and allergic responses and spontaneous enterocolitis, which is prevented by IL-10 administration. IL-10 production is tightly regulated, as excessive IL-10 leads to inability to control infectious pathogens, while insufficient IL-10 leads to pathology secondary to tissue injury. Although the main sources of IL-10 in the body are not well defined, various cell populations, such as T cell subsets (Th2, Tc2, Tr1), monocytes, macrophages, and dendritic cells are able to produce IL-10 upon stimulation by several factors including endotoxins (Asadullah et al., 2003).
Previous work has focused on the effects of short-term (several h up to 1–2 days) exposure of macrophages to IL-10. However, macrophages are chronically exposed to IL-10 under several physiological or pathophysiological conditions. It has been shown that IL-10 is constitutively present in the lungs and has been proposed to regulate the phenotype of alveolar macrophages and restrain the responses of these cells to environmental antigens. (Bonfield et al., 1995; Fernandez et al., 2004). IL-10 is expressed in the gastrointestinal tract and the colitis that develops in IL-10 deficient mice is suppressed by IL-10 (Lindsay and Hodgson, 2001). In pathological settings, IL-10 can be expressed by regulatory T cells during chronic infections (Belkaid et al., 2002), during autoimmune diseases such as systemic lupus erythematosus (SLE) and systemic sclerosis (Llorente et al., 2000), and IL-10 produced by tumor cells has been suggested to induce tumor-associated macrophages (TAM) that promote tumor progression (Mantovani et al., 2002).
The effects of long-term exposure of macrophages to IL-10 have not been elucidated, although IL-10 has been proposed to induce the differentiation of one subtype of alternatively activated macrophage (Goerdt and Orfanos, 1999). Alternatively activated macrophages do not exhibit the classic inflammatory effector functions of macrophages that have been activated by IFNγ or LPS. Alternatively activated macrophages can actually suppress classic activation and exhibit effector functions that include phagocytosis, altered recruitment of cells, clearance of helminths, and promotion of angiogenesis. Alternative activation of macrophages by IL-4 and IL-13 has been the best characterized (Herbert et al., 2004; Pestka et al., 2004) and IL-4/IL-13-activated macrophages have been proposed to represent genuine alternatively activated macrophages, which are distinct from other alternatively activated macrophages, such as those activated by IL-10, TGF-β, and glucocorticoids (Gordon, 2003). To gain further insight into the effects of chronic IL-10 exposure on macrophages and the phenotype of IL-10-induced alternative macrophages, we used microarray analysis to characterize IL-10-induced gene expression in macrophages cultured with IL-10 for 7 days. Long-term exposure to IL-10 induced expression of genes that were not induced after shorter IL-10 stimulation (3–6 h or 2 days) (Antoniv et al., 2005), and the pattern of gene expression suggested a gain of migratory functions and responsiveness to cytokines other than IL-4 that utilize the IL-2 receptor common gamma chain.
Section snippets
Cell culture
Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood from disease-free volunteers by density gradient centrifugation using Ficoll (Invitrogen, Carlsbad, CA). CD14+ monocytes were purified from fresh PBMCs using anti-CD14 magnetic beads (Miltenyi Biotec, Auburn, CA), as recommended by the manufacturer. Purity of monocytes was greater than 97% as verified by FACS. Human control macrophages were derived from CD14+ blood monocytes cultured in RPMI 1640 medium (Invitrogen)
Induction of gene expression by IL-10
To understand the function of macrophages exposed to long-term IL-10 treatment, microarray analysis was performed using macrophages cultured with IL-10 for 7 days (termed 7 day-cells). As controls, we used cells cultured with MCSF or exposed to IL-10 for 2 days after culturing for 5 days with MCSF (termed 2 day-cells). Two days of IL-10 exposure induced expression of 133 genes, whereas 7 days of IL-10 exposure induced expression of 250 genes by >2-fold (, ANOVA t-test) (Fig. 1A). A
Discussion
In this study we used microarray analysis to characterize the effects of long-term exposure to IL-10 on macrophage gene expression and phenotype. Macrophages did not become desensitized to IL-10 after long-term exposure. Instead, IL-10 triggered a differentiation program that was partially apparent after 2 days of IL-10 treatment, and continued to develop over 7 days of culture with IL-10. Macrophages exposed to IL-10 for 7 days not only lost pro-inflammatory functions, such as responsiveness
Acknowledgments
We are grateful to Weill Medical College Microarray Core Facility for technical assistance. This work was supported by a Cancer Research Institute Fellowship and the NIH.
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