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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

rGal-1 modulates L-selectin and β2-integrin. Peripheral leukocytes were labeled with antibodies specific for the surface adhesion molecules CD62L (conjugated with PE) and CD11b (conjugated with fluorescein isothiocyanate [FITC]). A-C: Representative dot plots of cells (each point represents a cell) immunostained for adhesion molecules. A: Control, B: LPS, C: LPS + recombinant galectin-1 (rGal-1), D: percentages of CD62L-positive cells, E: CD11b-positive cells, and F: CD62L/CD11b-positive cells. Data represent the median ± SEM of percentage of cells. *p<0.05, versus control; ##p<0.01 versus LPS. G: Representative immunoblot demonstrates the levels of CD11b and CD62L in the pooled extracts of rat eyes (n = 5 animals/group) from the control, LPS, and LPS + rGal-1 groups at 24 h (data illustrate one representative of two independent experiments). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control.
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f3: rGal-1 modulates L-selectin and β2-integrin. Peripheral leukocytes were labeled with antibodies specific for the surface adhesion molecules CD62L (conjugated with PE) and CD11b (conjugated with fluorescein isothiocyanate [FITC]). A-C: Representative dot plots of cells (each point represents a cell) immunostained for adhesion molecules. A: Control, B: LPS, C: LPS + recombinant galectin-1 (rGal-1), D: percentages of CD62L-positive cells, E: CD11b-positive cells, and F: CD62L/CD11b-positive cells. Data represent the median ± SEM of percentage of cells. *p<0.05, versus control; ##p<0.01 versus LPS. G: Representative immunoblot demonstrates the levels of CD11b and CD62L in the pooled extracts of rat eyes (n = 5 animals/group) from the control, LPS, and LPS + rGal-1 groups at 24 h (data illustrate one representative of two independent experiments). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control.

Mentions: The expression of adhesion molecules on leukocytes in the peripheral blood was investigated under the different experimental conditions via flow cytometry. The analysis of dot plot graphs demonstrated a high incidence of CD62L-positive cells (Figure 3B; p<0.05) compared to the control groups (Figure 3A). However, the LPS + rGal-1 group showed a high incidence of CD62L/CD11b-positive and CD11b-positive cells (Figure 3C; p<0.01) and a low percentage of CD62L-positive cells (p<0.001) compared to the LPS group. Analysis of the percentages of CD62L-, CD11b-, and CD62L/CD11b-positive cells confirmed this observation (Figure 3D-F). Similarly, immunoblot analysis of pooled extracts of rat eyes (n = 5 animals/group) revealed high levels of both adhesion molecules in the LPS + rGal-1 group compared to the untreated LPS group at 24 h (Figure 3G).


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)

rGal-1 modulates L-selectin and β2-integrin. Peripheral leukocytes were labeled with antibodies specific for the surface adhesion molecules CD62L (conjugated with PE) and CD11b (conjugated with fluorescein isothiocyanate [FITC]). A-C: Representative dot plots of cells (each point represents a cell) immunostained for adhesion molecules. A: Control, B: LPS, C: LPS + recombinant galectin-1 (rGal-1), D: percentages of CD62L-positive cells, E: CD11b-positive cells, and F: CD62L/CD11b-positive cells. Data represent the median ± SEM of percentage of cells. *p<0.05, versus control; ##p<0.01 versus LPS. G: Representative immunoblot demonstrates the levels of CD11b and CD62L in the pooled extracts of rat eyes (n = 5 animals/group) from the control, LPS, and LPS + rGal-1 groups at 24 h (data illustrate one representative of two independent experiments). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: rGal-1 modulates L-selectin and β2-integrin. Peripheral leukocytes were labeled with antibodies specific for the surface adhesion molecules CD62L (conjugated with PE) and CD11b (conjugated with fluorescein isothiocyanate [FITC]). A-C: Representative dot plots of cells (each point represents a cell) immunostained for adhesion molecules. A: Control, B: LPS, C: LPS + recombinant galectin-1 (rGal-1), D: percentages of CD62L-positive cells, E: CD11b-positive cells, and F: CD62L/CD11b-positive cells. Data represent the median ± SEM of percentage of cells. *p<0.05, versus control; ##p<0.01 versus LPS. G: Representative immunoblot demonstrates the levels of CD11b and CD62L in the pooled extracts of rat eyes (n = 5 animals/group) from the control, LPS, and LPS + rGal-1 groups at 24 h (data illustrate one representative of two independent experiments). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control.
Mentions: The expression of adhesion molecules on leukocytes in the peripheral blood was investigated under the different experimental conditions via flow cytometry. The analysis of dot plot graphs demonstrated a high incidence of CD62L-positive cells (Figure 3B; p<0.05) compared to the control groups (Figure 3A). However, the LPS + rGal-1 group showed a high incidence of CD62L/CD11b-positive and CD11b-positive cells (Figure 3C; p<0.01) and a low percentage of CD62L-positive cells (p<0.001) compared to the LPS group. Analysis of the percentages of CD62L-, CD11b-, and CD62L/CD11b-positive cells confirmed this observation (Figure 3D-F). Similarly, immunoblot analysis of pooled extracts of rat eyes (n = 5 animals/group) revealed high levels of both adhesion molecules in the LPS + rGal-1 group compared to the untreated LPS group at 24 h (Figure 3G).

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