Research ArticleIL4/PGE2 induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent
Introduction
The endosomal apparatus is a collection of vesicles and tubules that mediate and orchestrate transport of internalized fluid, receptor-associated ligands and membrane proteins to various intracellular destinations including the lysosomal compartment, trans-Golgi network, endoplasmic reticulum and plasma membrane [1]. In polarized cells, the endosomal apparatus plays a role in transcellular transport and in professional phagocytes, endosomes are the source of membrane as well as an entry point for access to lysosomes [2]. Rab GTPases regulate vesicular transport in endocytosis and exocytosis, and they have been associated with the control of vesicle docking and fusion [3], [4]. Particularly, Rab5 plays a central role in both cargo receptor internalization and in receptor tyrosine kinase internalization [5], [6]. Little is known about the dynamic relationships among the tubules and vesicles that make up the early endosomal compartment or the factors/structural elements that relate early endosomes to late endosomes. Although recent work by Zerial and colleagues [7] has offered a first glimpse of how these two major endocytic compartments, Rab5-positive early endosomes and Rab7-positive late endosomes, relate to each other. Work from Gordon and colleagues, has shown that differentiation of macrophages along the traditional Th-1 and Th-2 pathways is accompanied by alterations in the apparent activity of the endocytic pathway [8], [9]. Th-1 cytokines enhance late endosome/TGN membrane accumulation, whereas Th-2 cytokines enhance the early endocytic pathway. The latter is accompanied by the enhanced expression of the macrophage mannose receptor. The mannose receptor (MR) (CD206), which was first identified via in vivo plasma clearance studies [10] and endocytosis experiments [11], is a specialized pattern recognition receptor expressed by most macrophage populations and selected endothelial cells. MR efficiently endocytoses and phagocytoses mannosylated glycoproteins, particles and microorganisms [12], [13], [14]. MR is the prototype of a family of innate immune receptors (the other family members include Endo180 [15], PLA2R [16], [17] and DEC205 [18]) that are characterized by a short (43AA) C-terminal cytoplasmic tail, a tandem series of 8–10 carbohydrate recognition domains, a fibronectin type II repeat and an N-terminal cysteine-rich domain. Interestingly, the extracellular portion of the MR is selectively cleaved by proteases [19], [20] and may function as a delivery vehicle for antigens; therefore, this extracellular component may provide a humoral link between innate and acquired immunity [21].
In this study, we have explored the induction and localization of MR in murine macrophages by IL4/PGE2. Over-expressed MR in IL4/PGE2-treated cells behaves normally with respect to ligand binding and endocytosis. However, the induced MR accumulates in an enlarged early endosomal compartment. Light microscopy and fractionation studies indicate that the enlarged early endosomal (EEE) compartment is not only enriched with MR but also with Rab5a and other factors that regulate Rab5 function. Expression of dominant negative Rab5, intracellular delivery of anti-Rab5 monoclonal antibody, and Rab5 gene silencing reverses the IL4/PGE2 induction of EEE and increases MR localization in the plasma membrane. Finally, we report on some of the proteins enhanced in the EEE in an effort to elucidate the cellular and physiological functions of IL4/PGE2-induced endosomal compartments.
Section snippets
Reagents
125I was purchased from Amersham (Arlington Heights, IL). Three- to 5-week-old male C57Bl/6J mice were obtained from Jackson Laboratory (Bar Harbor, Maine). Recombinant mouse M-CSF was generously provided by P. Ross, Washington University School of Medicine (St. Louis, MO). Recombinant mouse IL4 was purchased from Calbiochem (San Diego, CA). PGE2 and monoclonal anti-α-tubulin (clone DM1A) were purchased from Sigma-Aldrich (St. Louis, MO). Polyclonal anti-murine mannose receptor was raised
IL4/PGE2 synergistically induce MR expression in murine macrophages
Differentiation of macrophages along the “alternative” activation pathway via Th-2 cytokines involves the activation of many genes whose expression results in various cellular phenotypes including the induction of the mannose receptor and the activation of the endocytic pathway [8]. To explore the induction of the endocytic pathway in response to Th-2 cytokines, we characterized the mannose receptor response to IL4 as well as the expression of early endosome markers in primary mouse macrophages
Discussion
In this paper, we set out to characterize more fully the effect of IL4 treatment on the endocytic pathway in macrophages using mannose receptor as a marker. Extending initial observations with human macrophages to mouse macrophages, we found that the MR response to IL4 was less robust in mouse cells [27]. Earlier work from our laboratory showed that treatment of mouse macrophages with PGE2 enhanced expression of MR [26]. This led us to examine the additive effects of IL4 and PGE2 on bone marrow
Acknowledgments
We are grateful to Dr. Xiong Su for sharing unpublished results on RNA silencing of Rab5 and its isoforms. We thank E. Peters, M. Levy and T.P. Ramkumar for their excellent technical assistance and D. Owyoung for editing assistance. This work was supported in part by National Institutes of Health grants 5 R01 GM42259 and 1 R01 DK065844 (to P. D. S.) and by National Institutes of Health Training grants IRG5801045-1 and 2P60DK20579 (to M. A. B.) from the Washington University Siteman Cancer
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- 1
Both authors contributed equally to this work.
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Present address: Department of Biological Sciences, Florida International University, Miami, FL 33199, USA.