The atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory potential due to its inhibitory action on the production of inflammatory mediators, such as tumor necrosis factor-α (TNF-α). The aim of this study was to determine whether ANP is able to attenuate inflammatory effects of TNF-α on target cells. Human umbilical vein endothelial cells (HUVECs) were treated with TNF-α in the presence or absence of ANP. Changes in permeability, cytoskeletal alterations, phosphorylation of p38 MAPK and HSP27, and expression of MKP-1 were determined by macromolecule permeability assay, fluorescence labeling, RT-PCR, and immunoblotting. Antisense studies were done by transfecting cells with MKP-1 antisense oligonucleotides. Activation of HUVECs with TNF-α lead to a significant increase of macromolecule permeability and formation of stress fibers. Treatment of cells with ANP (10 ⁻⁸ to 10 ⁻⁶ mol/L) significantly reduced the formation of stress fibers and elevated permeability. Both TNF-α–induced effects were shown to be mediated via the activation of p38 using SB203580, a specific inhibitor of p38. ANP significantly reduced the TNF-α–induced activation of p38 and attenuated the phosphorylation of HSP27, a central target downstream of p38. ANP showed no effect on p38 upstream kinases MKK3/6. However, a significant induction of the MAPK phosphatase MKP-1 mRNA and protein could be observed in ANP-treated cells. Antisense experiments proved a causal role for MKP-1 induction in the ANP-mediated inhibition of p38. These data show the inhibitory action of ANP on TNF-α–induced changes in endothelial cytoskeleton and macromolecule permeability involving an MKP-1–induced inactivation of p38 MAPK. These effects point to an antiinflammatory and antiatherogenic potential of this cardiovascular hormone.

While silica nanoparticles have enabled numerous industrial and medical applications, their toxicological safety requires further evaluation. Macrophages are the major cell population responsible for nanoparticle clearance in vivo. The prevailing macrophage phenotype largely depends on the local immune status of the host. Whereas M1-polarized macrophages are considered as pro-inflammatory macrophages involved in host defense, M2 macrophages exhibit anti-inflammatory and wound-healing properties, but also promote tumor growth. We employed different models of M1 and M2 polarization: GM-CSF/LPS/IFN-gamma was used to generate primary human M1 cells and M-CSF/IL-10 to differentiate M2 monocyte-derived macrophages. PMA-differentiated THP-1 cells were polarized towards an M1 type by LPS/IFN-gamma and towards M2 by IL-10. Uptake of fluorescent silica nanoparticles (Ø 26 and 41 nm) and microparticles (Ø 1.75 µm) was quantified. At the concentration used (50 µg/ml), silica nanoparticles did not influence cell viability as assessed by MTT assay. Nanoparticle uptake was enhanced in M2-polarized primary human monocyte-derived macrophages compared with M1 cells, as shown by flow cytometric and microscopic approaches. In contrast, the uptake of microparticles did not differ between M1 and M2 phenotypes. M2 polarization was also associated with increased nanoparticle uptake in the macrophage-like THP-1 cell line. In accordance, in vivo polarized M2-like primary human tumor-associated macrophages (TAM) obtained from lung tumors took up more nanoparticles than M1-like alveolar macrophages isolated from the surrounding lung tissue. In summary, our data indicate that the M2 polarization of macrophages promotes nanoparticle internalization. Therefore, the phenotypical differences between macrophage subsets should be taken into consideration in future investigations on nanosafety, but might also open up therapeutic perspectives allowing to specifically target M2 polarized macrophages.

The atrial natriuretic peptide (ANP) is suggested to regulate inflammatory response by alteration of macrophage functions. The aim of this study was to investigate whether ANP influences production of TNF-α. TNF-α production in murine bone marrow-derived macrophages was induced by LPS, and TNF-α secretion (±ANP) was determined by L929 bioassay. ANP dose dependently (10−8–10−6 M) inhibited TNF-α release by up to 95%. The effect was mediated via the guanylate cyclase-coupled A receptor, as was shown by employing dibutyryl-cGMP, the cGMP-inhibitory compound Ly-83583, and the A receptor antagonist HS-142-1. A specific ligand of the natriuretic peptide “clearance” receptor inhibited TNF-α production only at 10−7 and 10−8 M, but not at 10−6 M. The B receptor ligand C-type natriuretic peptide showed no TNF-α-inhibitory effect. To investigate the underlying mechanism of ANP-mediated TNF-α inhibition, Northern blot was performed. ANP-treated macrophages displayed decreased TNF-α-mRNA levels. Besides the known inhibition of NF-κB activation, in this study we demonstrated that ANP also attenuates the activation of the proinflammatory transcription factor AP-1 (gel shift assay). ANP did not alter subunit composition of AP-1 complexes, as was shown by supershift assays applying anti-c-jun and anti-c-fos Abs. To get information on the ANP effect for human inflammatory processes, we investigated cytokine production in human LPS-activated blood. ANP significantly attenuated production of TNF-α and IL-1β without affecting production of IL-10 and IL-1ra. In summary, ANP was shown to attenuate TNF-α production of LPS-activated macrophages via cGMP. The inhibition is suggested to involve transcriptional processes that are the result of reduced activation of responsible transcription factors.