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. 2016 Sep;16(9):2563-73.
doi: 10.1111/ajt.13808. Epub 2016 May 2.

Graft-Infiltrating Macrophages Adopt an M2 Phenotype and Are Inhibited by Purinergic Receptor P2X7 Antagonist in Chronic Rejection

C Wu  1   2 , Y Zhao  1 , X Xiao  1 , Y Fan  1 , M Kloc  1 , W Liu  1 , R M Ghobrial  1 , P Lan  1 , X He  2 , X C Li  1
Affiliations

Graft-Infiltrating Macrophages Adopt an M2 Phenotype and Are Inhibited by Purinergic Receptor P2X7 Antagonist in Chronic Rejection

C Wu et al. Am J Transplant. 2016 Sep.

Abstract

Macrophages exhibit diverse phenotypes and functions; they are also a major cell type infiltrating chronically rejected allografts. The exact phenotypes and roles of macrophages in chronic graft loss remain poorly defined. In the present study, we used a mouse heart transplant model to examine macrophages in chronic allograft rejection. We found that treatment of C57BL/6 mice with CTLA4 immunoglobulin fusion protein (CTLA4-Ig) prevented acute rejection of a Balb/c heart allograft but allowed chronic rejection to develop over time, characterized by prominent neointima formation in the graft. There was extensive macrophage infiltration in the chronically rejected allografts, and the graft-infiltrating macrophages expressed markers associated with M2 cells but not M1 cells. In an in vitro system in which macrophages were polarized into either M1 or M2 cells, we screened phenotypic differences between M1 and M2 cells and identified purinergic receptor P2X7 (P2x7r), an adenosine triphosphate (ATP)-gated ion channel protein that was preferentially expressed by M2 cells. We further showed that blocking the P2x7r using oxidized ATP (oATP) inhibited M2 induction in a dose-dependent fashion in vitro. Moreover, treatment of C57BL/6 recipients with the P2x7r antagonist oATP, in addition to CTLA4-Ig treatment, inhibited graft-infiltrating M2 cells, prevented transplant vasculopathy, and induced long-term heart allografts survival. These findings highlight the importance of the P2x7r-M2 axis in chronic rejection and establish P2x7r as a potential therapeutic target in suppression of chronic rejection.

Keywords: basic (laboratory) research/science; heart (allograft) function/dysfunction; heart transplantation/cardiology; macrophage/monocyte biology; rejection: chronic; vasculopathy.

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Conflict of interest statement

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Figures

Figure 1
Figure 1. CTLA4-Ig treatment prevents acute heart allograft rejection but induces chronic allograft rejection (transplant vasculopathy)
(A) Survival curves of heart grafts transplanted into syngeneic and allogeneic C57BL/6 hosts with or without CTLA4-Ig treatment. The MST of Balb/c heart allografts in CTLA4-Ig-treated mice was ≈31 days, and the MST of the control group was 8 days. The syngeneic heart grafts survived indefinitely. (B) Hematoxylin and eosin staining of syngeneic and CTLA4-Ig-treated grafts at 30 days after transplantation showing tissue and vascular structure in the grafts as well as cellular infiltration. (C) Masson’s trichrome staining of heart transplants at 30 days after transplantation showing tissue and vascular fibrosis. Representative pictures of one of three grafts examined in each group are shown (*p < 0.05). CTLA4-Ig, CTLA4 immunoglobulin; MST, mean survival time.
Figure 2
Figure 2. Assessment of graft-infiltrating macrophages in heart allograft at 30 days after transplantation
(A) Immunofluorescence staining of graft-infiltrating macrophages in CTLA4-Ig–treated mice was shown. Syngeneic heart grafts were used as controls. The arrows indicate arteries in the grafts (upper panel). Expression of CD206 or iNOS (red) by infiltrating macrophages was determined by double staining for DAPI (blue), CD11b (green) and CD206 (red) or iNOS (red). The merged fluorescence (yellow) identifies cells staining for both CD11b and CD206 or CD11b and CD206. Scale bar = 100 μm. (B) Cell counts from immunofluorescence staining in (A) showing CD11b+CD206+ (M2) and CD11b+iNOS+ (M1) cells per microscopic view. Data shown are mean cell number ± SE of five random views per sample for a total of three samples. (C) FACS plot showing gating strategy of graft infiltrating cells among CD45+ cells obtained from syngeneic and CTLA4-Ig–treated allografts. (D) Relative percentage of CD11b+ macrophages retrieved from each heart graft. Data shown are mean ± SE of four transplants in each group. (E) The absolute number of CD11b+ cells from each heart transplant. Data shown are mean ± SE of four transplants in each group. (F) FACS plot showing gating strategy for CD206+ cells among the total CD11b+ population. (G) Relative percentage of CD206+ cells among the total CD11b+ cells from syngeneic and CTLA4-Ig–treated allografts. Data shown are mean ± SE of four transplants in each group. (H) The absolute number of CD11b+CD206+ cells in each graft. Data shown are mean ± SE of four transplants in each group (*p < 0.05). CTLA4-Ig, CTLA4 immunoglobulin; DAPI, 4′,6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; iNOS, inducible nitric oxide synthase; SE, standard error.
Figure 3
Figure 3. Polarization of bone marrow–derived macrophages into M1 and M2 cells in vitro.
(A) Mouse bone marrow–derived macrophages were polarized into M1 cells with LPS and IFN-γ or to M2 cells with IL-4 and IL-13. Expression of M1- and M2-associated molecules were assessed by quantitative reverse transcriptase polymerase chain reaction 24 h later. Bone marrow–derived macrophages without polarizing cytokines were included as controls and referred to as M0 cells. The relative levels for Arg-1, CD206, Fizz1, iNOS, TNF-α, and IL-6 gene transcripts were normalized against HPRT and shown as mean ± SE of four independent experiments. (B) Western blot showing expression of CD206, Arg-1 and iNOS proteins in polarized M1 and M2 cells. The blot shown is representative of four independent experiments. Arg-1, arginase 1; IFN-γ, interferon γ; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; SE, standard error; TNF-α, tumor necrosis factor α.
Figure 4
Figure 4. Analysis of cell surface markers expressed by polarized M1 and M2 cells
(A) Mouse bone marrow–derived macrophages were polarized into M1 cells (cultured in LPS and IFN-γ) or M2 cells (cultured in IL4- and IL-13), and macrophages cultured without polarizing cytokines were referred as M0 cells. The polarized macrophages were analyzed for expression of CD11b, F4/80, CD80, CD86, CD206, and Dectin-1 expression by FACS. (B) The FACS plot showing the expression of P2x7r by polarized M1 and M2 cells. (C) Western blot showing P2x7r protein expression in polarized M1 and M2 cells. Representative data of one of three experiments are shown. FACS, fluorescence-activated cell sorting; P2x7r, purinergic receptor P2X7.
Figure 5
Figure 5. The purinergic receptor P2X7 antagonist oATP inhibits macrophage polarization to M2 cells in vitro.
(A) Mouse bone marrow–derived macrophages were polarized toward M1 or M2 macrophages in the presence of different concentrations of oATP for 24 h. Cells were harvested; induction of Arg-1, CD206, Fizz1, iNOS, TNF-α, and IL-6 mRNA was analyzed by quantitative reverse transcriptase polymerase chain reaction; and relative levels normalized against HPRT were shown. (B) Western blot analysis of CD206 and Arg-1 protein expression by M2 cells with or without oATP. Results shown are from one of three independent experiments. (C) FACS plot showing viability of M2 cells cultured with oATP in vitro. Live bone marrow–derived macrophages were cultured under M2 conditions with or without oATP for 24 h, stained with PI and annexin V, and assessed by FACS (*p < 0.05). Arg-1, arginase 1; Ctrl, control; FACS, fluorescence-activated cell sorting; iNOS, inducible nitric oxide synthase; oATP, oxidized adenosine triphosphate; TNF-α, tumor necrosis factor α.
Figure 6
Figure 6. Treatment with CTLA4-Ig and oATP inhibited chronic rejection and induced long-term heart allograft survival
(A) Survival curves for Balb/c heart allografts in B6 recipients treated with CTLA4-Ig, oATP or a combination of CTLA4-Ig and oATP. (B) Sections of hematoxylin and eosin staining of heart transplants from recipients treated with CTLA4-Ig alone or combined CTLA4-Ig and oATP showing vascular changes and tissue damage. Arrows indicate small arteries in the graft. Syngeneic heart transplants were used as controls for comparison. (C) Representative fluorescence-activated cell sorting plots showing CD206+ cells among CD11b+ graft-infiltrating cells in recipient mice treated with CTLA4-Ig or combined CTLA4-Ig and oATP 30 days after transplantation. (D) The number of CD11b+ cells retrieved from heart allografts. Data shown are mean ± SE of three transplants in each group. (E) The number of CD11b+CD206+ cells from heart allografts is shown. Data shown are mean ± SE of three transplants in each group. (*p < 0.05). CTLA4-Ig, CTLA4 immunoglobulin fusion protein; oATP, oxidized adenosine triphosphate; SE, standard error.
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