The study generated and characterized three distinct populations of macrophages (Mφ) in vitro: classically activated Mφ (Ca-Mφ), type II-activated Mφ (Mφ-II), and alternatively activated Mφ (AA-Mφ). Ca-Mφ were primed with IFN-γ and stimulated with LPS, Mφ-II were similarly primed but stimulated with LPS plus immune complexes (IC), and AA-Mφ were primed with IL-4. The authors compared the cytokine production, arginine metabolism, mRNA expression, antigen presentation, and T cell activation properties of these three Mφ populations. They found that Mφ-II more closely resembled Ca-Mφ than AA-Mφ in terms of cytokine production, arginine metabolism, and costimulatory molecule expression. Mφ-II and Ca-Mφ produced high levels of NO and had low arginase activity, while AA-Mφ expressed FIZZ1. Mφ-II and Ca-Mφ expressed high levels of CD86, whereas AA-Mφ did not. Ca-Mφ and Mφ-II were efficient antigen-presenting cells (APCs) that induced T cell proliferation, whereas AA-Mφ failed to do so. The differences between Ca-Mφ and Mφ-II were more subtle, with Ca-Mφ producing IL-12 and inducing Th1 cells, while Mφ-II produced IL-10 and induced Th2 cells. Mφ-II also expressed sphingosine kinase-1 (SPHK1) and LIGHT (TNFSF14), which may serve as markers for this population. The study concludes that each of the three Mφ populations has distinct characteristics and functions, challenging the oversimplification of classifying all non-Ca-Mφ as M2.The study generated and characterized three distinct populations of macrophages (Mφ) in vitro: classically activated Mφ (Ca-Mφ), type II-activated Mφ (Mφ-II), and alternatively activated Mφ (AA-Mφ). Ca-Mφ were primed with IFN-γ and stimulated with LPS, Mφ-II were similarly primed but stimulated with LPS plus immune complexes (IC), and AA-Mφ were primed with IL-4. The authors compared the cytokine production, arginine metabolism, mRNA expression, antigen presentation, and T cell activation properties of these three Mφ populations. They found that Mφ-II more closely resembled Ca-Mφ than AA-Mφ in terms of cytokine production, arginine metabolism, and costimulatory molecule expression. Mφ-II and Ca-Mφ produced high levels of NO and had low arginase activity, while AA-Mφ expressed FIZZ1. Mφ-II and Ca-Mφ expressed high levels of CD86, whereas AA-Mφ did not. Ca-Mφ and Mφ-II were efficient antigen-presenting cells (APCs) that induced T cell proliferation, whereas AA-Mφ failed to do so. The differences between Ca-Mφ and Mφ-II were more subtle, with Ca-Mφ producing IL-12 and inducing Th1 cells, while Mφ-II produced IL-10 and induced Th2 cells. Mφ-II also expressed sphingosine kinase-1 (SPHK1) and LIGHT (TNFSF14), which may serve as markers for this population. The study concludes that each of the three Mφ populations has distinct characteristics and functions, challenging the oversimplification of classifying all non-Ca-Mφ as M2.