Classically and alternatively activated bone marrow derived macrophages differ in cytoskeletal functions and migration towards specific CNS cell types

Abstract

BackgroundMacrophages play an important role in neuroinflammatory diseases such as multiple sclerosis (MS) and spinal cord injury (SCI), being involved in both damage and repair. The divergent effects of macrophages might be explained by their different activation status: classically activated (CA/M1), pro-inflammatory, macrophages and alternatively activated (AA/M2), growth promoting, macrophages. Little is known about the effect of macrophages with these phenotypes in the central nervous system (CNS) and how they influence pathogenesis. The aim of this study was therefore to determine the characteristics of these phenotypically different macrophages in the context of the CNS in an in vitro setting.ResultsHere we show that bone marrow derived CA and AA macrophages have a distinct migratory capacity towards medium conditioned by various cell types of the CNS. AA macrophages were preferentially attracted by the low weight (< 10 kD) fraction of neuronal conditioned medium, while CA macrophages were attracted in higher numbers by astrocyte- and oligodendrocyte conditioned medium. Intrinsic motility was twice as high in AA macrophages compared to CA macrophages. The adhesion to extracellular matrix molecules (ECM) was significantly enhanced in CA macrophages compared to control and AA macrophages. The actin cytoskeleton was differentially organized between CA and AA macrophages, possibly due to greater activity of the GTPases RhoA and Rac in CA macrophages. Phagocytosis of myelin and neuronal fragments was increased in CA macrophages compared to AA macrophages. The increase in myelin phagocytosis was associated with higher expression of CR3/MAC-1 in CA macrophages.ConclusionIn conclusion, since AA macrophages are more motile and are attracted by NCM, they are prone to migrate towards neurons in the CNS. CA macrophages have a lower motility and a stronger adhesion to ECM. In neuroinflammatory diseases the restricted migration and motility of CA macrophages might limit lesion size due to bystander damage.

Modelling Multiple Sclerosis In Vitro and the Influence of Activated Macrophages

“…A larger number of spheroids contained one or more macrophages when exposed to either AA or US macrophages compared to CA macrophages ( Figure 4D). These results were in agreement with our previous observations showing that AA macrophages have a higher intrinsic motility compared to CA macrophages [Vereyken et al, 2011]. Furthermore, CA macrophages had been observed to express higher levels of adhesion receptors compared to both AA and US macrophages [Vereyken et al, 2011].…”

“…These results were in agreement with our previous observations showing that AA macrophages have a higher intrinsic motility compared to CA macrophages [Vereyken et al, 2011]. Furthermore, CA macrophages had been observed to express higher levels of adhesion receptors compared to both AA and US macrophages [Vereyken et al, 2011]. Thus, the lower motility and higher adhesion could contribute to the lower migration of CA macrophages into spheroids.…”

“…Clinical EAE was reversed by the adoptive transfer of AA macrophages, which were induced in vitro with glatiramer acetate [Weber et al, 2007]. Our studies have shown that macrophage migration towards CNS cell types differs upon skewing [Vereyken et al, 2011] and that migration of macrophages in spheroids is limited and varies with skewing of the macrophages ( Figure 4E). Microinjection of skewed macrophages into spheroids or co-culture of differently activated macrophages would allow more in depth analysis into the cellular mechanisms behind the effects reported in these studies.…”

“…For this, we cultured rat whole brain spheroids during 4 weeks, to reach optimal myelin development, and exposed them to differently activated macrophages, that were fluorescently labeled, [method of macrophage activation: Vereyken et al, 2011] during maximal 1 week. At several time points, cryostat sections were cut of the spheroids and the number of spheroids containing macrophages were counted.…”

Mechanisms Involved in the Remyelinating Effect of Sildenafil

“…These cells are readily polarized to a M1 phenotype by incubation 71 with LPS (10-100 ng/ml)+IFNγ (20 ng/ml) and to a M2 phenotype by incubation with IL-4 (10-20 ng/ml) (Classen et al 2009). These conditions were used as controls of phenotypic changes for 24h the morphology of cells changed from a more rounded/amoeboid type to an elongated shape similar to that acquired when BMDM are treated with IL-4 (Durafourt et al 2012, McWhorter et al 2013, Vereyken et al 2011). …”