Increased Hydrostatic Pressure Promotes Primary M1 Reaction and Secondary M2 Polarization in Macrophages.

Patients with chronic anterior uveitis are at particularly high risk of developing secondary glaucoma when corticosteroids [e.g., dexamethasone (Dex)] are used or when inflammatory activity has regressed. Macrophage migration into the eye increases when secondary glaucoma develops and may play an important role in the development of secondary glaucoma. Our aim was to evaluate in vitro if increased hydrostatic pressure and corticosteroids could induce changes in macrophages phenotype. By using a pressure chamber cell culture system, we assessed the effect of increased hydrostatic pressure (HP), inflammation, and immunosuppression (Dex) on the M1/M2 phenotype of macrophages. Bone marrow-derived macrophages (BMDMs) were stimulated with medium, lipopolysaccharide (LPS, 100 ng/ml), Dex (200 ng/ml), or LPS + Dex and incubated with different HP (0, 20, or 60 mmHg) for 2 or 7 days. The numbers of CD86+/CD206- (M1 phenotype), CD86-/CD206+ (M2 phenotype), CD86+/CD206+ (intermediate phenotype), F4/80+/TNF-α+, and F4/80+/IL-10+ macrophages were determined by flow cytometry. TNF-α and IL-10 levels in cell culture supernatants were quantified by ELISA. TNF-α, IL-10, fibronectin, and collagen IV expression in BMDMs were detected by immunofluorescence microscopy. Higher HP polarizes macrophages primarily to an M1 phenotype (LPS, 60 vs. 0 mmHg, d2: p = 0.0034) with less extra cellular matrix (ECM) production and secondary to an M2 phenotype (medium, 60 vs. 0 mmHg, d7: p = 0.0089) (medium, 60 vs. 20 mmHg, d7: p = 0.0433) with enhanced ECM production. Dex induces an M2 phenotype (Dex, medium vs. Dex, d2: p < 0.0001; d7: p < 0.0001) with more ECM production. Higher HP further increased M2 polarization of Dex-treated macrophages (Dex, 60 vs. 0 mmHg, d2: p = 0.0417; d7: p = 0.0454). These changes in the M1/M2 phenotype by high HP or Dex treatment may play a role in the pathogenesis of secondary uveitic glaucoma- or glucocorticoid (GC)-induced glaucoma.

Phenotypic Differences in Primary Murine Microglia Treated with NOD1, NOD2, and NOD1/2 Agonists.

The purpose of the study was studying the influence of different NOD agonists on the morphological phenotype of primary murine microglia and to examine their influence on characteristic cytokines. Primary CD11b-positive cells were isolated from the brain of neonatal mice. The microglial phenotype of the cells was examined by ionized calcium-binding adapter molecule (Iba)1 staining. After14 days in culture, these cells were stimulated by iE-DAP, L18-MDP, or M-TriDAP as NOD1, NOD2, and NOD1/2 agonists, respectively. The cellular morphology was recorded and compared to the phenotype of cells cultured in medium alone or after LPS stimulation. The cells developed a specific phenotype only after treatment with the NOD2 agonist L18-MDP. These cells were characterized by straight extensions carrying tiny spikes and had a high ramification index. This was in sharp contrast to all other treatments, which always resulted in an amoeboid phenotype typically shown by activated microglia in vivo and by cultured microglia in vitro. The staining intensity of IL-6 and TNF-α did not reveal any clear difference independent of the NOD agonist treatment. In contrast, an increased staining intensity was observed for IL-10 after L18-MDP treatment. The NOD2 agonist L18-MDP induced a morphologically distinct phenotype characterized by microspike-decorated dendritiform extensions and a high degree of ramification in primary murine microglia. Increased ramification index and elevated staining intensity of anti-inflammatory IL-10 as hallmarks suggest that a M2-like phenotype of microglia was induced.