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Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages
Published on: June 23, 2013
Biochemical and functional characterization of three activated macrophage populations
Justin P Edwards1, Xia Zhang, Kenneth A Frauwirth
1Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.
Abstract:
We generated three populations of macrophages (Mphi) in vitro and characterized each. Classically activated Mphi (Ca-Mphi) were primed with IFN-gamma and stimulated with LPS. Type II-activated Mphi (Mphi-II) were similarly primed but stimulated with LPS plus immune complexes. Alternatively activated Mphi (AA-Mphi) were primed overnight with IL-4. Here, we present a side-by-side comparison of the three cell types. We focus primarily on differences between Mphi-II and AA-Mphi, as both have been classified as M2 Mphi, distinct from Ca-Mphi. We show that Mphi-II more closely resemble Ca-Mphi than they are to AA-Mphi. Mphi-II and Ca-Mphi, but not AA-Mphi, produce high levels of NO and have low arginase activity. AA-Mphi express FIZZ1, whereas neither Mphi-II nor Ca-Mphi do. Mphi-II and Ca-Mphi express relatively high levels of CD86, whereas AA-Mphi are virtually devoid of this costimulatory molecule. Ca-Mphi and Mphi-II are efficient APC, whereas AA-Mphi fail to stimulate efficient T cell proliferation. The differences between Ca-Mphi and Mphi-II are more subtle. Ca-Mphi produce IL-12 and give rise to Th1 cells, whereas Mphi-II produce high levels of IL-10 and thus, give rise to Th2 cells secreting IL-4 and IL-10. Mphi-II express two markers that may be used to identify them in tissue. These are sphingosine kinase-1 and LIGHT (TNF superfamily 14). Thus, Ca-Mphi, Mphi-II, and AA-Mphi represent three populations of cells with different biological functions.
Insights
This study compares three macrophage populations: classically activated (Ca-Mphi), type II-activated (Mphi-II), and alternatively activated (AA-Mphi). Mphi-II share more characteristics with Ca-Mphi than AA-Mphi, highlighting distinct macrophage functions.
Area of Science:
- Immunology
- Cell Biology
Background:
- Macrophages are key immune cells with diverse activation states.
- Distinguishing between M2 macrophage subtypes, like Type II-activated (Mphi-II) and alternatively activated (AA-Mphi), is crucial for understanding their distinct roles.
Purpose of the Study:
- To provide a comprehensive side-by-side comparison of three distinct macrophage populations generated in vitro: classically activated (Ca-Mphi), Type II-activated (Mphi-II), and alternatively activated (AA-Mphi).
- To elucidate the functional and molecular differences between Mphi-II and AA-Mphi, both classified as M2 macrophages, and contrast them with Ca-Mphi (M1 macrophages).
Main Methods:
- In vitro generation of three macrophage populations: Ca-Mphi (IFN-gamma/LPS), Mphi-II (IFN-gamma/LPS/immune complexes), and AA-Mphi (IL-4).
- Characterization of macrophage populations through analysis of nitric oxide (NO) production, arginase activity, FIZZ1 expression, CD86 levels, and antigen-presenting cell (APC) capacity.
- Assessment of cytokine production (IL-12, IL-10) and T helper cell polarization (Th1, Th2).
Main Results:
- Mphi-II exhibit greater similarity to Ca-Mphi than to AA-Mphi.
- Mphi-II and Ca-Mphi produce high levels of NO and have low arginase activity, unlike AA-Mphi.
- AA-Mphi express FIZZ1 and are poor APCs, while Mphi-II and Ca-Mphi express CD86 and are efficient APCs.
- Ca-Mphi induce Th1 responses, whereas Mphi-II promote Th2 responses via IL-10 production.
- Mphi-II can be identified by sphingosine kinase-1 and LIGHT expression.
Conclusions:
- Classically activated Mphi (Ca-Mphi), Type II-activated Mphi (Mphi-II), and alternatively activated Mphi (AA-Mphi) represent distinct functional macrophage subsets.
- Mphi-II, despite being M2-like, share more functional and molecular characteristics with Ca-Mphi than with AA-Mphi.
- The findings highlight unique biological functions and potential tissue markers for Mphi-II, aiding in their identification and understanding of their roles in immunity.
