The adenosine-dependent angiogenic switch of macrophages to an M2-like phenotype is independent of interleukin-4 receptor alpha (IL-4Rα) signaling

Abstract

Murine macrophages are activated by interferon-γ (IFN-γ) and/or Toll-like receptor (TLR) agonists such as bacterial endotoxin (lipopolysaccharide [LPS]) to express an inflammatory (M1) phenotype characterized by the expression of nitric oxide synthase-2 (iNOS) and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-12. In contrast, Th2 cytokines IL-4 and IL-13 activate macrophages by inducing the expression of arginase-1 and the anti-inflammatory cytokine IL-10 in an IL-4 receptor-α (IL-4Rα)-dependent manner. Macrophages activated in this way are designated as "alternatively activated" (M2a) macrophages. We have shown previously that adenosine A2A receptor (A(2A)R) agonists act synergistically with TLR2, TLR4, TLR7, and TLR9 agonists to switch macrophages into an "M2-like" phenotype that we have termed "M2d." Adenosine signaling suppresses the TLR-dependent expression of TNF-α, IL-12, IFN-γ, and several other inflammatory cytokines by macrophages and induces the expression of vascular endothelial growth factor (VEGF) and IL-10. We show here using mice lacking a functional IL-4Rα gene (IL-4Rα(-/-) mice) that this adenosine-mediated switch does not require IL-4Rα-dependent signaling. M2d macrophages express high levels of VEGF, IL-10, and iNOS, low levels of TNF-α and IL-12, and mildly elevated levels of arginase-1. In contrast, M2d macrophages do not express Ym1, Fizz1 (RELM-α), or CD206 at levels greater than those induced by LPS, and dectin-1 expression is suppressed. The use of these markers in vivo to identify "M2" macrophages thus provides an incomplete picture of macrophage functional status and should be viewed with caution.

Figure 1. Comparison of M2a (IL-4) and M2d (LPS/NECA) activation on the production of (A) TNFα and (B) VEGF by murine peritoneal macrophages

Thioglycollate-elicited macrophages from wild type (WT) BALB/c and IL-4Rα−/− mice were treated for 18 hours with LPS (100ng/ml), NECA (1μM), LPS/NECA, IL-4 (10ng/ml) and IL-4 with LPS, NECA and LPS/NECA. Cytokine levels were assayed using ELISA kits as described in Methods. Data indicate the mean ± SD of triplicate samples from a representative experiment. * P<0.01 for WT vs IL-4Rα−/− macrophages treated with IL-4/LPS; # P<0.05 for WT vs IL-4Rα−/− macrophages treated with IL-4/LPS/NECA.

Figure 2. Analysis of cytokine/chemokine production by wild type (WT) BALB/c and IL-4Rα−/− murine peritoneal macrophages

Thioglycollate-elicited macrophages from BALB/c and IL-4Rα−/− mice were treated for 18 hours with LPS (100ng/ml), LPS with NECA (1μM), or IL-4 (10ng/ml). Cytokine/chemokine levels were assayed from three replicate samples from each condition, and each sample was assayed in triplicate using Milliplex^™ MAP immunoassays. Mean ± SD for each sample is presented.

Figure 2. Analysis of cytokine/chemokine production by wild type (WT) BALB/c and IL-4Rα−/− murine peritoneal macrophages

Thioglycollate-elicited macrophages from BALB/c and IL-4Rα−/− mice were treated for 18 hours with LPS (100ng/ml), LPS with NECA (1μM), or IL-4 (10ng/ml). Cytokine/chemokine levels were assayed from three replicate samples from each condition, and each sample was assayed in triplicate using Milliplex^™ MAP immunoassays. Mean ± SD for each sample is presented.

Figure 3. Comparison of canonical M1 and M2 marker expression by M2a and M2d murine peritoneal macrophages

Thioglycollate-elicited macrophages from BALB/c (WT) and IL-4Rα−/− mice were treated for 6 hours with LPS (100ng/ml), NECA (1μM) or IL-4 (10ng/ml), alone and in combination, as indicated. mRNA levels of the M1 marker iNOS (A) and the canonical murine M2 marker Arg1 (B) were determined using qRT-PCR. Data indicate the relative levels of mRNA from three representative experiments.

Figure 4. Flow Cytometry (FACS) comparison of the expression of Ym1, Dectin-1 and IL-4Rα by M2a (IL-4) and M2d (LPS/NECA) murine peritoneal macrophages

A. Representative FACS analysis of thioglycollate-elicited macrophages from BALB/c (WT, left panels) and IL-4Rα−/− (IL-4Rα−/−, right panels) mice treated for 18 hours with LPS (100ng/ml) and NECA (1μM) (black), IL-4 (10ng/ml) (red), or a combination of all three (black dotted), as described in Methods. Cells were analyzed for expression of Ym1 (upper panels), Dectin-1 (middle panels), and IL-4Rα (lower panels). Shaded histograms represent marker expression in untreated controls. B. Analysis of Ym1, dectin-1 and IL-4Rα expression determined by FACS in treated vs untreated macrophages, Results indicate mean fold differences of median fluorescence values by treated macrophages vs controls from 3 independent experiments,

Figure 5. Analysis of the expression of M2 markers (Ym1, Fizz1 and CD206) by M2a and M2d murine peritoneal macrophages

Thioglycollate-elicited macrophages from BALB/c (WT) and IL-4Rα−/− mice were treated for 6 hours with LPS (100ng/ml), NECA (1μM) or IL-4 (10ng/ml), alone and in combination, as indicated. mRNA levels of canonical M2 markers Ym1 (A), Fizz1 (B), and CD206 (C) were evaluated using qRT-PCR. Data indicate the mean relative levels of mRNA from three independent experiments. Each sample was analyzed in duplicate.

Figure 6. Analysis of adenosine A_2A receptor (A_2AR) expression in wild type (Balb-c) and IL-4Rα−/− mouse peritoneal macrophages

Thioglycollate-elicited macrophages from BALB/c (WT) and IL4Rα;−/− mice were treated for 6 hours with LPS (100ng/ml), NECA (1 μM) and IL4 (10ng/ml) alone or in combination. mRNA levels of A_2AR were determined using qRT-PCR. Data presented are the mean relative levels of A_2AR mRNA from three independent experiments. Each sample was analyzed in duplicate.