Alveolar macrophages of GM-CSF knockout mice exhibit mixed M1 and M2 phenotypes

Activin A, a pleiotrophic cytokine belonging to the transforming growth factor-beta (TGF-β) superfamily, is synthesized by many cell types throughout the body [1, 2]. The molecular structure is a disulphide-linked, homodimeric glycoprotein composed of two inhibin βA chains. Activin A was first recognized as an endocrine factor, but is now known to be essential to developmental and repair processes, and total ablation is neonatal lethal [3]. Contrasting regulatory roles have been cited for Activin A in inflammation [4]. Human monocytes synthesize activin A upon stimulation with classical M1 macrophage activation inducers such as GM-CSF, LPS, and IFNγ [5, 6]. Exposure of GM-CSF treated macrophages to anti-Activin A reduces M1 markers and enhances alternative M2 phenotype markers such as IL-10 [7]. Activin A also inhibits monocyte production of IL-1β and enhances IL-1 receptor antagonist production [8]. Interestingly, in severe asthma, activin A may be elevated in serum, and data from animal models suggests that activin A may suppress T helper 2 (Th2) mediated allergic responses [9]. Collectively these observations suggest multifunctional roles for activin A in inflammatory processes.

Maintenance of lung homeostasis is a complex process dependent upon a network of interacting cells and cytokines. GM-CSF is required for alveolar macrophage (AM) function and pulmonary homeostasis [10]. In genetically altered mice homozygous for a disrupted GM-CSF gene (GM-CSF knockout), hematopoiesis is normal but there is accumulation of excess lung surfactant [11, 12]. This surfactant pathology mirrors that of human PAP, an autoimmune disease characterized by high levels of autoantibody to GM-CSF [12‐14]. Aerosolized GM-CSF resolves the pulmonary pathology of GM-CSF knockout mice, thus demonstrating that surfactant homeostasis can be influenced by local administration of GM-CSF to the respiratory tract [15].

Previously we reported that healthy human AMs synthesize activin A in response to GM-CSF but AMs of patients with PAP are deficient in activin A [16]. In addition, PAP AMs are deficient in the nuclear transcription factor, Peroxisome Proliferator-activated Receptor, (PPARγ), a regulator of lipid and glucose metabolism that is restored by GM-CSF treatment [17]. PPARγ has also been shown to be a negative regulator of inflammation [18, 19]. Interestingly, alveolar macrophages of GM-CSF knockout mice are also deficient in PPARγ [20]. The role of activin A in the lung has not been established. Because of the phenotypic similarities between human PAP and the GM-CSF knockout mouse, this study was undertaken to investigate activin A regulation in the lung. Initially, it was hypothesized that activin A might be impaired in GM-CSF knockout mice based upon previous data from PAP studies [16].

The current findings suggest that IFNγ is a major contributory factor to the intrinsic elevation of activin A in AMs. Findings also point out a striking difference in activin A expression in human PAP and GM-CSF knockout mice despite common deficiencies of GM-CSF and PPARγ (summarized in Table 1). In parallel with activin A, GM-CSF knockout mice displayed over-expression of IFNγ [23], a positive regulator of activin A [5]. In contrast, BAL cells of PAP patients do not exhibit elevated IFNγ and activin A is deficient [16].

Elevated IFNγ has been reported previously in the BAL fluids of GM-CSF knockout mice [23]. Our previous studies also found elevated IFNγ expression in macrophage-specific PPARγ knockout mice [25]. Restoration of PPARγ via lentivirus vector in these mice greatly diminished IFNγ expression [25]. In the current study, similar results were seen after PPARγ–lentivirus treatment of GM-CSF knockout mice. Such findings suggest that the PPARγ deficiency present in GM-CSF knockout mice may contribute to elevated IFNγ. GM-CSF has been shown to be a critical upregulator of PPARγ [31, 34]. The total lack of GM-CSF in knockout mice may maintain an extreme PPARγ deficiency which is ineffective at repressing inflammatory mediators such as IFNγ. In human PAP, IFNγ levels are not increased despite PPARγ deficiency, furthermore, GM-CSF is not totally absent [29]. The primary etiology of PAP is considered to be an autoimmune response to GM-CSF in the form of high levels of circulating, neutralizing autoantibody to GM-CSF [13]. It is also possible that additional regulatory mechanisms are present in human lung to help prevent IFNγ buildup in PAP.

The varying characteristics of activated macrophages have led to attempts to categorize activation phenotypes [35‐39]. The M1 phenotype is characterized by production of microbial or IFNγ-triggered molecules such as iNOS and IL-12. GM-CSF has been cited as an inducer of M1 phenotypes while M-CSF has been shown to induce the M2 alternative activation phenotype in which IL-10 or TGFβ may be produced [7, 40]. We have shown that M-CSF is elevated in GM-CSF knockout mice [22] and in human PAP [33] which might suggest the presence of an M2 macrophage phenotype (see Table 1). Interestingly, PPARγ, which is deficient in GM-CSF knockout mice, is also a major driver of the M2 phenotype [41]. It has been pointed out however, that macrophage phenotypes were defined by carefully controlled in vitro conditions which may be vastly different from the in vivo milieu [42]. Thus the juxtaposition of both IFNγ and M-CSF in the lungs of GM-CSF knockout mice could produce the novel combination of macrophage activation phenotypes illustrated by elevated M1 (iNOS, CCL5, IL-6) and M2 (IL-10, CCL2) markers (Table 1). Other IFNγ-inducible pro-inflammatory mediators (chemokines CXCL9, CXCL10, and CXCL11) have been noted in the lungs of GM-CSF knockout mice [23]. Previously, we found that MMP-2, a matrix metalloproteinase associated with M-CSF and alternative M2 activation, is also elevated in GM-CSF knockout BAL cells [33].

The current findings extend our previous studies examining pulmonary mechanisms operative in human PAP and the GM-CSF knockout mouse. It is clear that pathways of activin A regulation may utilize GM-CSF or IFNγ as stimulatory factors. In the GM-CSF knockout mouse, lack of GM-CSF may restrict production of sufficient PPARγ to control inflammation. The persistent elevation of both M-CSF and IFNγ may influence AMs to express characteristics of both M1 and M2 phenotypes. The current data emphasize the plasticity of alveolar macrophages in assuming a unique activation phenotype when regulatory pathways become dysfunctional.