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Protective effects of the galectin-1 protein on in vivo and in vitro models of ocular inflammation.

Zanon Cde F, Sonehara NM, Girol AP, Gil CD, Oliani SM - Mol. Vis. (2015)

Bottom Line: To better understand the effects of Gal-1 in the retina, in vitro studies were performed using ARPE-19 cells.Ultrastructural immunocytochemical analyses showed decreased levels of endogenous Gal-1 in LPS-stimulated cells (24 h), while Dex treatment upregulated this protein.The protective effects of rGal-1 on LPS-stimulated cells were associated with the significant reduction of the release of cytokines (IL-8 and IL-6), similar to Dex treatment.

View Article: PubMed Central - PubMed

Affiliation: Departament of Biology, Instituto de Biociências, Letras e Ciências Exatas; São Paulo State University (UNESP), São José do Rio Preto, SP, Brazil.

ABSTRACT

Purpose: Galectin-1 (Gal-1) is a β-galactoside-binding protein with diverse biological activities in the pathogenesis of inflammation but has been poorly investigated in terms of ocular inflammation. In the present study, we monitored the anti-inflammatory effects of Gal-1 using the in vivo rodent model of endotoxin-induced uveitis (EIU) and in vitro assays with human RPE (ARPE-19) cells.

Methods: For this purpose, EIU was induced by subcutaneous sterile saline injection of 0.1 ml of lipopolysaccharide (LPS, 1 mg/Kg) in the rat paw, which was maintained under these conditions for 24 h. The therapeutic efficacy of recombinant Gal-1 (rGal-1) was tested in the EIU animals by intraperitoneal inoculation (3 µg/100 µl per animal) 15 min after the LPS injection. In vitro studies were performed using LPS-stimulated ARPE-19 cells (10 μg/ml) for 2, 8, 24 and 48 h, treated or not with rGal-1 (4 μg/ml) or dexamethasone (Dex, 1.0 μM).

Results: Gal-1 treatment attenuated the histopathological manifestation of EIU via the inhibition of polymorphonuclear cells (PMN) infiltration in the eye and by causing an imbalance in adhesion molecule expression and suppressing interleukin (IL)-1β, IL-6, and monocyte chemotactic protein-1 (MCP-1) productions. Immunohistochemical and western blotting analyses revealed significant upregulation of Gal-1 in the eyes induced by EIU after 24 h. In the retina, there was no difference in the Gal-1 expression, which was high in all groups, demonstrating its structural role in this region. To better understand the effects of Gal-1 in the retina, in vitro studies were performed using ARPE-19 cells. Ultrastructural immunocytochemical analyses showed decreased levels of endogenous Gal-1 in LPS-stimulated cells (24 h), while Dex treatment upregulated this protein. The protective effects of rGal-1 on LPS-stimulated cells were associated with the significant reduction of the release of cytokines (IL-8 and IL-6), similar to Dex treatment. Furthermore, rGal-1 and Dex inhibited cyclooxygenase-2 (COX-2) expression in LPS-stimulated cells, as shown by immunofluorescence.

Conclusions: Overall, this study identified potential roles for Gal-1 in ocular inflammation, especially uveitis, and may lead to future therapeutic approaches.

No MeSH data available.


Related in: MedlinePlus

In vivo and in vitro administration of rGal-1 modulates proinflammatory mediators release during ocular inflammation: IL-1β (A), IL-6 (B), and MCP-1 (C) in ocular tissues (in vivo). Interleukin-8 (IL-8) (D), IL-6 (E), and monocyte chemotactic protein-1 (MCP-1; F) in ARPE-19 cells (in vitro). Data are expressed as the mean ± SEM (pg/ml) from the eye homogenates (n = 5) or cell supernatants. * p<0.05, ** p<0.01, *** p<0.001 versus control; #p<0.05, ##p<0.01, ###p<0.001 versus lipopolysaccharide (LPS) or LPS-stimulated cells. (G) Cyclooxygenase-2 (COX-2) immunofluorescence in ARPE-19 cells showed decreased COX-2 immunoreactivity in the cytoplasm after recombinant galectin-1 (rGal-1) treatment in LPS-stimulated cells after 24 h. Bars: 10 µm. H: Densitometric analysis of COX-2. The data are presented as the mean ± SEM of the densitometric index (arbitrary units [a.u.]) of cells from three independent experiments. ***p<0.001 versus control; ###p<0.001 versus LPS.
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f4: In vivo and in vitro administration of rGal-1 modulates proinflammatory mediators release during ocular inflammation: IL-1β (A), IL-6 (B), and MCP-1 (C) in ocular tissues (in vivo). Interleukin-8 (IL-8) (D), IL-6 (E), and monocyte chemotactic protein-1 (MCP-1; F) in ARPE-19 cells (in vitro). Data are expressed as the mean ± SEM (pg/ml) from the eye homogenates (n = 5) or cell supernatants. * p<0.05, ** p<0.01, *** p<0.001 versus control; #p<0.05, ##p<0.01, ###p<0.001 versus lipopolysaccharide (LPS) or LPS-stimulated cells. (G) Cyclooxygenase-2 (COX-2) immunofluorescence in ARPE-19 cells showed decreased COX-2 immunoreactivity in the cytoplasm after recombinant galectin-1 (rGal-1) treatment in LPS-stimulated cells after 24 h. Bars: 10 µm. H: Densitometric analysis of COX-2. The data are presented as the mean ± SEM of the densitometric index (arbitrary units [a.u.]) of cells from three independent experiments. ***p<0.001 versus control; ###p<0.001 versus LPS.

Mentions: As expected, the levels of IL-1β, IL-6, and MCP-1 increased significantly in the eyes 24 h after LPS injection (Figure 4A-C; p<0.001, p<0.05, and p<0.01, respectively). Administration of rGal-1 decreased the levels of IL-1β (p<0.05) and IL-6 (p<0.05) after 24 h compared with the untreated animals. In addition, the administration of rGal-1 induced a significant increase of chemokine MCP-1 compared to the control group (Figure 4C; p<0.05).


Protective effects of the galectin-1 protein on in vivo and in vitro models of ocular inflammation.

Zanon Cde F, Sonehara NM, Girol AP, Gil CD, Oliani SM - Mol. Vis. (2015)

In vivo and in vitro administration of rGal-1 modulates proinflammatory mediators release during ocular inflammation: IL-1β (A), IL-6 (B), and MCP-1 (C) in ocular tissues (in vivo). Interleukin-8 (IL-8) (D), IL-6 (E), and monocyte chemotactic protein-1 (MCP-1; F) in ARPE-19 cells (in vitro). Data are expressed as the mean ± SEM (pg/ml) from the eye homogenates (n = 5) or cell supernatants. * p<0.05, ** p<0.01, *** p<0.001 versus control; #p<0.05, ##p<0.01, ###p<0.001 versus lipopolysaccharide (LPS) or LPS-stimulated cells. (G) Cyclooxygenase-2 (COX-2) immunofluorescence in ARPE-19 cells showed decreased COX-2 immunoreactivity in the cytoplasm after recombinant galectin-1 (rGal-1) treatment in LPS-stimulated cells after 24 h. Bars: 10 µm. H: Densitometric analysis of COX-2. The data are presented as the mean ± SEM of the densitometric index (arbitrary units [a.u.]) of cells from three independent experiments. ***p<0.001 versus control; ###p<0.001 versus LPS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4556161&req=5

f4: In vivo and in vitro administration of rGal-1 modulates proinflammatory mediators release during ocular inflammation: IL-1β (A), IL-6 (B), and MCP-1 (C) in ocular tissues (in vivo). Interleukin-8 (IL-8) (D), IL-6 (E), and monocyte chemotactic protein-1 (MCP-1; F) in ARPE-19 cells (in vitro). Data are expressed as the mean ± SEM (pg/ml) from the eye homogenates (n = 5) or cell supernatants. * p<0.05, ** p<0.01, *** p<0.001 versus control; #p<0.05, ##p<0.01, ###p<0.001 versus lipopolysaccharide (LPS) or LPS-stimulated cells. (G) Cyclooxygenase-2 (COX-2) immunofluorescence in ARPE-19 cells showed decreased COX-2 immunoreactivity in the cytoplasm after recombinant galectin-1 (rGal-1) treatment in LPS-stimulated cells after 24 h. Bars: 10 µm. H: Densitometric analysis of COX-2. The data are presented as the mean ± SEM of the densitometric index (arbitrary units [a.u.]) of cells from three independent experiments. ***p<0.001 versus control; ###p<0.001 versus LPS.
Mentions: As expected, the levels of IL-1β, IL-6, and MCP-1 increased significantly in the eyes 24 h after LPS injection (Figure 4A-C; p<0.001, p<0.05, and p<0.01, respectively). Administration of rGal-1 decreased the levels of IL-1β (p<0.05) and IL-6 (p<0.05) after 24 h compared with the untreated animals. In addition, the administration of rGal-1 induced a significant increase of chemokine MCP-1 compared to the control group (Figure 4C; p<0.05).

Bottom Line: To better understand the effects of Gal-1 in the retina, in vitro studies were performed using ARPE-19 cells.Ultrastructural immunocytochemical analyses showed decreased levels of endogenous Gal-1 in LPS-stimulated cells (24 h), while Dex treatment upregulated this protein.The protective effects of rGal-1 on LPS-stimulated cells were associated with the significant reduction of the release of cytokines (IL-8 and IL-6), similar to Dex treatment.

View Article: PubMed Central - PubMed

Affiliation: Departament of Biology, Instituto de Biociências, Letras e Ciências Exatas; São Paulo State University (UNESP), São José do Rio Preto, SP, Brazil.

ABSTRACT

Purpose: Galectin-1 (Gal-1) is a β-galactoside-binding protein with diverse biological activities in the pathogenesis of inflammation but has been poorly investigated in terms of ocular inflammation. In the present study, we monitored the anti-inflammatory effects of Gal-1 using the in vivo rodent model of endotoxin-induced uveitis (EIU) and in vitro assays with human RPE (ARPE-19) cells.

Methods: For this purpose, EIU was induced by subcutaneous sterile saline injection of 0.1 ml of lipopolysaccharide (LPS, 1 mg/Kg) in the rat paw, which was maintained under these conditions for 24 h. The therapeutic efficacy of recombinant Gal-1 (rGal-1) was tested in the EIU animals by intraperitoneal inoculation (3 µg/100 µl per animal) 15 min after the LPS injection. In vitro studies were performed using LPS-stimulated ARPE-19 cells (10 μg/ml) for 2, 8, 24 and 48 h, treated or not with rGal-1 (4 μg/ml) or dexamethasone (Dex, 1.0 μM).

Results: Gal-1 treatment attenuated the histopathological manifestation of EIU via the inhibition of polymorphonuclear cells (PMN) infiltration in the eye and by causing an imbalance in adhesion molecule expression and suppressing interleukin (IL)-1β, IL-6, and monocyte chemotactic protein-1 (MCP-1) productions. Immunohistochemical and western blotting analyses revealed significant upregulation of Gal-1 in the eyes induced by EIU after 24 h. In the retina, there was no difference in the Gal-1 expression, which was high in all groups, demonstrating its structural role in this region. To better understand the effects of Gal-1 in the retina, in vitro studies were performed using ARPE-19 cells. Ultrastructural immunocytochemical analyses showed decreased levels of endogenous Gal-1 in LPS-stimulated cells (24 h), while Dex treatment upregulated this protein. The protective effects of rGal-1 on LPS-stimulated cells were associated with the significant reduction of the release of cytokines (IL-8 and IL-6), similar to Dex treatment. Furthermore, rGal-1 and Dex inhibited cyclooxygenase-2 (COX-2) expression in LPS-stimulated cells, as shown by immunofluorescence.

Conclusions: Overall, this study identified potential roles for Gal-1 in ocular inflammation, especially uveitis, and may lead to future therapeutic approaches.

No MeSH data available.


Related in: MedlinePlus