Strategically altering the balance of macrophage subpopulations to inhibit chronic rejection

In 1970 the Stanford cardiac transplant team analyzed the pathology from their first 18 patients and thoroughly described the features of chronic rejection (1). They summarized that chronic rejection “manifested primarily by obliterative intimal proliferation in coronary arteries, was present in most allografts obtained from patients surviving at least 1 month.” Furthermore, they astutely predicted: “This intimal thickening may limit long survival of patients undergoing cardiac transplantation.” Even as advances in immunosuppression have progressively decreased the incidence of acute rejection, the incidence of chronic rejection has remained at about 5% per year and is the major limitation of graft survival. Better suppression of T-cell responses has also uncovered a more central role for macrophages in the pathogenesis of arterial injury. In 1991, pathologists in the Boston Heart Transplant Consortium proposed that macrophages intermingled with T cells and HLA-DR expressing endothelial cells in neointimal lesions could produce a cascade of inflammatory cytokines including interleukin (IL)-1, Tumor Necrosis Factor (TNF), Platelet-derived Growth Factor (PDGF), and Transforming Growth Factor (TGF). They hypothesized that macrophage-derived inflammatory cytokines induced smooth muscle proliferation and expanded the neointima (2).

In the 25 years since these observations, knowledge about the multiplicity and plasticity of macrophages has progressed substantially (3). IL-1 and TNFα are now considered to be signature products of classically activated macrophages (also referred to as M1 or inflammatory macrophages), whereas, PDGF and TGFβ are associated with alternatively activated (or M2) macrophages. Depending on the microenvironment, M2 macrophages can produce anti-inflammatory mediators such as IL-10 or pro-fibrotic mediators such as Connective Tissue Growth Factor (CTGF) and TGFβ (4). Recently, Kaul and colleagues (5) demonstrated a differential distribution of macrophages in the interstitial and arterial compartments of murine cardiac allografts with M2 macrophages accumulating in the arterial compartment as chronic rejection progressed.

In the current issue of American Journal of Transplantation, Wu and colleagues (6) report a protocol designed to inhibit M2 macrophages and diminish intimal hyperplasia in a cardiac transplant model in mice. They treated C57BL/6 mice with a single dose of CTLA4-Ig one day after transplantation to inhibit acute rejection of BALB/c cardiac allografts. These allografts rejected 20 to 50 days after transplantation with occlusive arterial pathology. The neointimal lesions contained increased numbers of macrophages that expressed macrophage mannose receptor (CD206), a marker of M2 macrophages. To identify potential therapeutically useful targets on macrophage subpopulations, the investigators screened for surface markers that are differentially expressed on macrophages following in vitro polarization into either M1 or M2 phenotypes. M2 macrophages were found to preferentially express P2X7 receptors (P2X7R), an ATP gated ion channel that promotes inflammasome formation and release of IL-1β in LPS-stimulated macrophages/monocytes. After confirming the functional relevance of P2X7R to M2 induction by blocking the receptor with oxidized ATP (oATP) in vitro, the therapeutic potential of oATP was tested by treating C57BL/6 recipients of BALB/c hearts with CTLA4-Ig followed by oATP on days 14, 16, 18, 20 and 22 after transplantation. The combined treatment decreased arterial pathology and prolonged graft survival. Analysis of the macrophage infiltrates indicated that the combined treatment decreased the total number of macrophages infiltrating the allografts and that the M2 macrophages were decreased preferentially. This skewing of macrophage polarization in oATP treated mice was expected from the inhibition of M2 induction in vitro.

Although the effects of oATP are demonstrated on isolated macrophages in this paper, P2X7R is expressed on most cells in the innate and adaptive immune system including granulocytes, NK cells, T cells, B cells, and dendritic cells (7). In addition, oATP is a non-selective P2X7R antagonist that can reduce inflammatory responses independently of P2 receptor blockade (8).

Others have shown that treatment with oATP for the first 14 days after transplantation decreases T-cell responses and prolongs BALB/c cardiac allograft survival in C57BL/6 recipients (9). In their experimental design, Wu et al used CTLA4-Ig to inhibit the acute T-cell response, and a delayed treatment with oATP to block subsequent M2 macrophage responses. The potential effects of oATP on antibody production to allografts was not tested. It is possible that oATP could modulate multiple aspects of antibody-mediated rejection, including production of antibody by B cells and responses to antibodies by macrophages and NK cells.

Wu et al acknowledge that the macrophages localized in the neointima and adventitia of arteries of chronically rejected allografts do not fit neatly into the simplified paradigm of two subsets of macrophages. A spectrum of macrophage phenotypes is now recognized (3, 10). Of particular relevance to arterial pathology is the recently described pro-atherogenic M4 subpopulation of macrophages that is induced by CXCL4 from platelets (11, 12). Platelets are also a critical source of ATP in the development of intimal hyperplasia in atherosclerosis (13).

Although many unknowns remain, methods to strategically alter the balance of macrophage subpopulations would be a valuable addition to current immunosuppressive protocols. Strategies based on the findings presented by Wu et al (6) could also be used to stabilize the M2 polarization of passively transferred macrophages (14). P2X7R antagonists more selective than oATP have been or are currently being tested in phase 1 and phase 2 clinical trials designed to develop therapies against chronic inflammatory diseases and autoimmune disorders (8). In most cases, the P2X7R antagonists were well tolerated with side effects that included gastrointestinal upset, dizziness and headaches (8). Differential binding of P2X7R antagonists with polymorphic variants of the receptor, and redundancy in the function of P2X7R in the inflammatory pathway, are factors to consider for the efficacy of therapies based on P2X7R inhibition.