Molecular Vision 2015; 21:1036-1050
Received 01 June 2015 | Accepted 31 August 2015 | Published 02 September 2015
The first two authors contributed equally to this work.
1Departament of Biology, Instituto de Biociências, Letras e Ciências Exatas; São Paulo State University (UNESP), São José do Rio Preto, SP, Brazil; 2Department of Physical and Biological Sciences, Integrated College Padre Albino Foundation (FIPA), Rua dos Estudantes, 225, Catanduva, SP, Brazil; 3Department of Morphology and Genetics, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil
Correspondence to: Sonia Maria Oliani. Departament of Biology, Instituto de Biociências, Letras e Ciências Exatas; São Paulo State University (UNESP), Rua Cristóvão Colombo, 2265, Jardim Nazareth, 15054-000, São José do Rio Preto, SP, Brazil; Phone +55 17 3221-2380; FAX: +55 17 3221-2390; email: firstname.lastname@example.org
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.
Endotoxin-induced uveitis (EIU) is a widely accepted animal model for improving our understanding of ocular inflammation [1-3]. Although EIU is generally considered to be an inflammation of the anterior uvea, changes in the posterior segment involving the vitreous and retina may also occur [3-6]. Lipopolysaccharide (LPS) is an exogenous bacterial toxin used in the induction of EIU because it binds to toll-like receptor 4 (TLR4)  and stimulates the synthesis and the release of proinflammatory chemical mediators, such as nitric oxide (NO) [2,8], platelet-activating factor (PAF), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, monocyte chemotactic protein-1 (MCP-1) , and other cytokines [10,11]. This increased expression of inflammatory mediators exacerbates the development of uveitis by breaking down the blood–ocular barrier, which leads to edema formation and contributes to leukocyte influx [10,12,13].
The pharmacological treatments for uveitis include corticosteroids and chemotherapeutic agents, but the side effects of these drugs, such as increased ocular pressure and cytotoxicity, limit their use and highlight the need for new therapeutic approaches [3,14-16]. Among the available anti-inflammatory mediators, the Galectin-1 (Gal-1) protein acts in particular to limit the development of an acute inflammatory process [17-21].
Galectins are lectin family members defined by their affinity for β-galactoside carbohydrates and their shared consensus amino acid sequences in the carbohydrate recognition domain (CRD). They are widely expressed in various tissues and organs, showing the highest expression in the immune system [22,23]. Gal-1 is a prototypic member of this family, with anti-inflammatory properties described in several models of chronic and autoimmune inflammation, including autoimmune encephalomyelitis , arthritis , uveitis , hepatitis , and diabetes . This protein participates in the interaction between the cell surface and extracellular matrix through binding to glycoconjugated proteins  and inhibits the rolling and extravasation of polymorphonuclear cells (PMNs) into sites of inflammation .
Although the anti-inflammatory activities of Gal-1 have been explored in several in vivo and in vitro investigations [29-33], the exogenous role of this protein in ocular inflammatory processes has been poorly elucidated. Given the common side effects of the current therapies used to treat uveitis [14-16], we evaluated the effects of endogenous and exogenous Gal-1 protein in rodent ocular tissues in EIU and in an in vitro LPS-inflamed RPE human cell system. These analyses shed light on the genesis of the role of Gal-1 in ocular inflammation, especially uveitis, and indicate its potential for use as a therapeutic approach.
Animals-- Male Wistar rats (Rattus norvegicus) weighing 150–200 g were randomly distributed into four groups (n = 10 per group). The animals were housed under a 12h:12h light-dark cycle and were allowed access to food and water ad libitum. All of the experiments were conducted in compliance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The study was approved by the Ethics Committee on Animal Experimentation of São José do Rio Preto Medical School (No. 0467/2009).
Induction of EIU and rGal-1 treatment-- To induce EIU development, the rats were inoculated in the right paw with 1 mg/kg of LPS from Escherichia coli serotype O127:B8 (Sigma-Aldrich, St Louis, MO); Sigma Chemical, Poole, UK) diluted in 0.1 ml of sterile saline . The animals were then maintained under these conditions for 24 h. The therapeutic efficacy of a recombinant Gal-1 (rGal-1) protein was tested in the EIU animals for 24 h. The rats were inoculated with LPS as described above, and after 15 min, they were treated intraperitoneally (i.p.) with rGal-1 (PeproTech EC Ltd., London, UK) at a concentration of 3 µg/100 µl per animal , then euthanized after 24 h. No manipulated animals were used as control. The animals were anesthetized with isoflurane (1%) before all experimental treatments and were euthanized through an overdose of the anesthetic.
Histopathological analysis-- After collecting aqueous humor (AqH), the right eyes of the control and experimental rats (n = 5 per group) were fixed in a 4% paraformaldehyde, 0.5% glutaraldehyde solution (1:1) in sodium cacodylate buffer 0.1 M (pH 7.4) for 24 h at 4 °C and then bisected. A portion of these eye fragments were dehydrated using graded methanol solutions and embedded in LR Gold resin (Sigma-Aldrich)  for histopathology, while the other portion was embedded in paraffin for immunohistochemistry. Quantitative analysis of neutrophils was performed in 1 µm tissue sections from the anterior and posterior eye segments in a blind manner, with counting performed using a high-power objective (40X) on an Axioskop 2-Mot Plus Zeiss microscope (Carl Zeiss, Jena, Germany). The number of neutrophils was quantified in three semiserial sections per animal with a 30 µm distance between each section, and the values are reported as the mean (± standard error of the mean [SEM]) number of cells/mm2.
Quantitative analysis of leukocytes in AqH and blood-- AqH was collected from the right eyes of the rats (n = 5 per group) by puncturing the anterior chamber with a 28-gauge needle, and a 10 µl aliquot of AqH was stained with Turk solution (90 µl). Blood was collected through cardiac puncture (n = 5 per group), and a 10 µl sample of whole blood was diluted in 190 µl of Turk solution. Neutrophils and monocytes/macrophages (mono-Møs) were quantified in a Neubauer chamber (Laboroptik; Friedrichsdorf, Germany). Data are reported as the mean ± SEM of the average number of cells x 105/ml in the AqH samples and the number of cells x 103/ml in the blood samples.
Blood flow cytometry-- Whole blood was collected in EDTA (100 mg/ml) via cardiac puncture (n = 5 per group). Aliquots (10 µl) of animal blood were incubated with 1 μl of a monoclonal antibody against L-selectin conjugated with phycoerythrin (PE; anti-rat CD62L; BD Pharmigen, San Diego, CA) and β2-integrin conjugated with fluorescein isothiocyanate (FITC; anti-rat CD11b; BD PharMingen) diluted 1:10 in PBS (1X; 120 mM NaCl, 20 mM KCl, 10 mM NaPO4, 5 mM KPO4, pH 7.4) for 20 min at 4 °C in the dark. Immediately after incubation, 180 µl of Guava Lysing Solution/Fixative (EMD Millipore, Billerica, MA) was added to lyse and fix the cells for 20 min at 37 °C. The cells were analyzed using a Guava EasyCyte flow cytometer (Millipore Corporation), and leukocytes were gated according to the side and forward scatter parameters. Data were obtained from 10,000 cells (only morphologically viable cells were considered in the analysis) to determine the percentages (%) of CD62L and CD11b-positive cells.
Immunohistochemistry-- To study the specific localization of Gal-1, 5 µm sections of paraffin-embedded eyes were employed. Following an Ag retrieval step using citrate buffer (pH 6), endogenous peroxide activity was blocked, and the sections were incubated overnight at 4 °C with a primary rabbit polyclonal anti-Gal-1 antibody (Ab; 1:200; Zymed Laboratories, Cambridge, UK) diluted in 1% bovine serum albumin (BSA). After washing, the sections were incubated with a biotinylated secondary Ab (KIT Histostain-SP; Invitrogen, Frederick, MD). Positive staining was detected using a peroxidase-conjugated streptavidin complex, and the color was developed using 3,3′-diaminobenzidine (DAB) as a substrate (Invitrogen). The sections were counterstained with hematoxylin. For densitometric analyses, three slides from each animal (n = 5 per group) were used, and 20 points were analyzed in three fields in the cornea, ciliary processes, and retina (plexiform layers, ganglion cells, and PE). In addition, 10 points were marked on the iris and retina to obtain an average related to the intensity of immunoreactivity. The values were obtained as arbitrary units (a.u.) using the AxioVision software (Carl Zeiss).
Western blotting analysis-- To detect total amounts of Gal-1 and adhesion molecules (CD62L and CD11b), the left eyes of rats (n = 5 per group) subjected to different experimental conditions were macerated in liquid nitrogen and lysed on ice for 15 min with 1 ml of a solution containing one complete mini EDTA-free protease inhibitor mixture tablet (Roche Applied Science, Mannheim, Germany), 50 mM Tris-HCl, 150 mM NaCl, and 1% Triton-X (pH 7.4). The samples were then transferred to microtubes and centrifuged at 10,000 ×g for 20 min at 4 °C. The protein concentration in the eye supernatants was measured using the BCA™ Protein Assay Kit (Pierce Biotechnology, Rockford, IL). All protein samples were stored in aliquots at -80 °C until analysis. Equal amounts of rat samples (31.5 μg) and molecular weight markers were separated by electrophoresis in 12% polyacrylamide gel according to the Mini-PROTEAN Tetra Cell (Bio-Rad, Hercules, CA) protocol and transferred to nitrocellulose membranes (Hi-Bond C, Amersham Biosciences, Little Chalfont, UK). Target proteins were detected in rat samples using the rabbit polyclonal anti-Gal-1 AB (3 μg/ml; Zymed Laboratories), anti-CD62L (1:200), anti-CD11b (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-actin (1:500; BioLegend, San Diego, CA) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000; Sigma-Aldrich). The Western Breeze Immunodetection Kit (Invitrogen) was used for the chromogenic detection of Gal-1 and β-actin bands. Adhesion molecules and GAPDH were detected using an enhanced Supersignal West Pico Chemiluminescent Substrate Kit (Thermo Fisher Scientific) and film was developed in dark room. Protein densitometry was performed using the software ImageJ 1.440 (HIH, Bethesda, MD) to determine relative expression (a.u.) of Gal-1/β-actin.
Cell culture and treatment protocols-- ARPE-19 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA; short tandem repeat analysis [STR] was showed in Appendix 1) and grown in a mixture (1:1) of Dulbecco's Modified Eagle's Medium (DMEM):Nutrient F-12 Ham (Sigma-Aldrich)  supplemented with 10% fetal bovine serum (FBS), 200 mM L-glutamine, 0.1 mg/ml streptomycin, and 100 U/ml penicillin (Invitrogen). To determine the optimal length of treatment with rGal-1 (PeproTech EC Ltd.), ARPE-19 cells were initially cultivated in complete medium, as described previously, and incubated with 10 μg/ml LPS (Sigma-Aldrich) for 2, 8, 24, or 48 h. The tested LPS concentration was chosen based on a study that used the same cell line . To evaluate the anti-inflammatory effect of Gal-1, concentrations of 0.04, 0.4, and 4.0 µg/ml of rGal-1 were tested in the LPS-stimulated cells [18,30]. As positive control for the anti-inflammatory effects on LPS-stimulated cells, we used the glucocorticoid Dex (Sigma-Aldrich) at a concentration of 1.0 µM  (five independent experiments per group). Subsequently, the supernatants were collected to measure the levels of the proinflammatory cytokines IL-8 and IL-6. The results revealed that the 4.0 µg/ml concentration of rGal-1 was the most effective in significantly reducing the levels of both cytokines compared with LPS-stimulated cells. After determining the best treatment protocol, we established the experimental design. Cells were subjected to the following experimental conditions: growth in complete medium (control), activation by LPS (10 μg/ml) without treatment or treatment with 4.0 µg/ml of rGal-1 (LPS/rGal-1) or 1.0 µM of Dex (LPS/Dex). The cytotoxic effect of rGal-1 relating to the proliferation and viability of ARPE-19 cells was investigated in all experimental groups at 2, 8, 24, and 48 h. The cells were trypsinized and quantified using a Countess Automated Cell Counter (Invitrogen). The results revealed no significant differences in the rate of cell proliferation or cell viability among the experimental groups compared with their respective controls. Cellular morphology was evaluated using an inverted microscope (CKX41; Olympus). The experimental procedures were conducted in accordance with the rules of the Research Ethics Committee of the Institute of Bioscience, Letters and Exact Science, IBILCE/UNESP, São José do Rio Preto (no 041/11).
Fixation, processing, and embedding for transmission electron microscopy-- ARPE-19 cells were fixed in a 4% paraformaldehyde, 0.5% glutaraldehyde solution (1:1) in sodium cacodylate buffer 0.1 M (pH 7.4) for 24 h at 4 °C. The cells were subsequently dehydrated through a methanol series and embedded in LR Gold resin (Sigma-Aldrich).
Postembedding immunogold labeling-- To detect the localization of the endogenous Gal-1 protein, ultrathin sections (70 nm) of ARPE cells were sequentially incubated with the following reagents at room temperature: i) distilled water; ii) PBS containing 1% egg albumin (1% PBEA) for 10 min; iii) 5% PBEA; iv) rabbit polyclonal anti-Gal-1 Ab (1:100; Zymed Laboratories) for 2 h, with normal rabbit serum as a control (1:100); and v) 1% PBEA containing 0.01% Tween-20 in three washes (5 min each). To detect Gal-1, a goat anti-rabbit IgG Ab (1:50 in 1% PBEA) conjugated with 15 nm colloidal gold (British Biocell, Cardiff, UK) was applied. After 1 h, the sections were washed thoroughly in 1% PBEA and then in distilled water. These sections were stained with uranyl acetate and lead citrate and then examined using a ZEISS Leo 906 electron microscope (Carl Zeiss). Randomly photographed sections of ARPE cells were used for immunocytochemical analysis. The area of the cell compartment was determined with AxioVision software on an Axioskop 2-Mot Plus Zeiss microscope. The density of immunogold particles (number of gold particles per μm2) was calculated and expressed for each cell compartment. Values are reported as the mean ± SEM of 10 electron micrographs analyzed per group.
Immunofluorescence analysis-- To detect cyclooxygenase-2 (COX-2), ARPE-19 cells were grown on coverslips, fixed in 4% paraformaldehyde for 24 h, washed in PBS and Tween-20 (0.4%), blocked with 1% BSA, diluted in 3% normal goat serum, and incubated with a polyclonal rabbit anti–COX-2 Ab (Abcam, Cambridge, UK; 1:200 in normal goat serum 1.5%). After washing, the cells were incubated with goat anti-rabbit Ab conjugated with FITC (Serotec, Oxford, UK; 1:100 in normal goat serum 1.5%) for 1 h. The slides were mounted with a solution containing glycerol and PBS (1:1). Normal goat serum was used in the reaction control. The cells were analyzed using a filter with a wavelength of 546 nm on an Axioskop 2-Mot Plus Zeiss microscope, and the levels of the enzyme were quantified via densitometry.
Quantitative analysis of chemical mediators in the supernatants of ocular tissues and ARPE-19 cells-- The levels of IL-1β, IL-6, and MCP-1, which are chemical mediators generally linked to EIU [9,12], were analyzed in the supernatants of the ocular tissues obtained following maceration (n = 5 per group). However, the cytokines IL-8, IL-6, and MCP-1 were the chemical mediators released in the largest quantities by ARPE-19 cells following LPS activation for 24 h [3,38]; therefore, they were analyzed in the cell supernatants under all experimental conditions. The supernatants of the ocular tissues, obtained following maceration of eyes (n = 5 per group), were analyzed using the MILLIPLEX RAT Cytokine/Chemokine Magnetic Bead panel (catalog number RECYTMAG-65K; Millipore Corporation, Billerica, MA). The kit can detect IL-1β, IL-6, and MCP-1 cytokines simultaneously in a single sample. The samples were brought to room temperature and added in duplicate to 96-well filter-bottom plates. Antibody-coated detection beads were added to the wells and incubated with agitation on plate shaker for 1 hour at room temperature (20-25 °C). Without aspirated after incubation, the beads were further incubated with streptavidin-phycoerythrin for 30 min. Then 125 μl of sheath fluid were added to each well, and the samples were analyzed using Luminex laser-based fluorescent analytical test instrumentation (MAGPIX, Austin, TX). Cytokine concentrations were determined from standard curves prepared on each plate and expressed as picogram per milliliter (pg/ml).
For the in vitro assays, IL-6, IL-8 and MCP-1 levels in the ARPE-19 cell supernatants were detected using a commercially available Human Quantikine ELISA Kits (R&D Systems, Minneapolis, MN) and according to the manufacturer’s instructions. Briefly, 100 μl of each of the standards, controls, and samples were loaded onto a 96-well polystyrene microplates containing 100 μl assay diluent in duplicate. After incubation for 2 h at room temperature, the microplates were washed 4X. Then, 200 μl of specific human IL-6, IL-8 or MCP-1 conjugate solutions were added to each well for 2 h at room temperature, washed 4X and 200 μl of a substrate solution were added. After 20 min of incubation, 50 μl of the stop solution was added, and the optical density was read at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA) with a correction wavelength of 540 nm. The cytokines concentration of each sample was calculated from the standard curve..
Statistical analysis-- Data were presented as mean ± SEM. A statistical analysis was performed with GraphPad Prism (GraphPad Software Inc., version 5.0). The results were previously subjected to descriptive analysis and determination of normality using the Kolmogorov–Smirnov test. For samples with a normal distribution, we applied analysis of variance (ANOVA), followed by a Bonferroni correction for multiple comparisons or t tests for unpaired samples. The Kruskal–Wallis test followed by Dunn’s test was used for samples with a non-normal distribution. A p-value less than 0.05 was considered statistically significant.
In the eyes of control rats, an absence of transmigrated neutrophils was observed in the anterior (Figure 1A-B) and posterior (Figure 1C) segments. In contrast, 24 h after intraocular injection of LPS, an influx of neutrophils was detected in the anterior ocular segment (p<0.001), followed by transmigration to the AqH, as well as in the stroma of the iris, ciliary processes (Figure 1D), and limbus (Figure 1E). In addition, the posterior segment of the eye presented neutrophils that transmigrated to the vitreous and retina (p<0.01), especially in the inner plexiform layer (Figure 1F). Treatment with rGal-1 induced a decrease in the number of transmigrated neutrophils in both the anterior (Figure 1J; p<0.01) and the posterior (Figure 1K, p<0.01) segments compared with the LPS group.
At 24 h after LPS injection, an intense inflammatory response characterized by a significant increase in the number of neutrophils in the AqH (Figure 2A; p<0.01) and blood (Figure 2B; p<0.001) was detected compared to the control group.
The effect of pharmacological treatment with rGal-1 on the recruitment of leukocytes was also evaluated, revealing a significant decrease in the number of neutrophils in the AqH (Figure 2A; p<0.05) and blood (Figure 2B; p<0.01) compared with the untreated LPS group. Quantification of mononuclear phagocytic cells showed an exacerbated number of these cells following the treatment with rGal-1 in the AqH (Figure 2C; p<0.001) and blood (Figure 2D; p<0.001) compared to the LPS and control groups.
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).
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).
Considering that in vivo LPS-induced ocular inflammation enhanced neutrophil infiltration and cytokine secretion, which were reversed by rGal-1 treatment, we investigated the role of Gal-1 in the secretion of chemotactic cytokines and the cellular localization of COX-2 in LPS-stimulated ARPE-19 cells after 24 h. Because LPS induces RPE cells to secrete high levels of IL-8 and IL-6, we checked these mediators in the cell supernatants. We observed a significant increase in the levels of IL-8 (p<0.001) and IL-6 (p<0.01) in the LPS-stimulated cells relative to the control (Figure 4D,E). The rGal-1 (4.0 µg/ml) and Dex treatments markedly reduced the levels of both cytokines (p<0.001 and p<0.01, respectively). For MCP-1, there was no difference in the LPS group relative to the control (Figure 4F). However, treatment with rGal-1 (4.0 µg/ml) increased release the levels of this chemokine compared to the control and to the LPS (Figure 4F; p<0.0.01 and p<0.05).
In addition, LPS-stimulated ARPE-19 cells revealed intense cytoplasmic positivity of COX-2 compared to control cells (Figure 4G; p<0.001), while Dex and rGal-1 treatments efficiently reduced this cytoplasmic immunoreactivity (Figure 4G; p<0.001). Densitometry analysis confirmed these observations (Figure 4H).
Immunohistochemistry analysis demonstrated the presence of Gal-1 expression in the epithelia of the cornea, ciliary processes, and iris (Figure 5A-C). There was an increase in Gal-1 expression in these regions 24 h after LPS injection (Figure 5D-F; p<0.01, p<0.01, and p<0.001, respectively) compared with the control tissues. However, we observed reduced expression of Gal-1 in the cornea and ciliary processes in the animals treated with rGal-1 (Figure 5G,H; p<0.001). There was no immunostaining in the eye used as negative control section (without primary antibody; Figure 5J). The data obtained through western blotting analysis confirmed the modulation of Gal-1 protein 24 h after LPS injection, due to an increase in its endogenous expression in supernatants of ocular tissues (Figure 5L; p<0.05).
The retinal layers showed immunostaining for Gal-1, mainly in the RPE, outer and inner plexiform strata, and ganglion cells of all studied groups (Figure 6A-C). Densitometry revealed a similar immunostaining pattern in these retinal layers among the control and LPS groups, indicating that this protein is a structural component of this region (Figure 6I). No immunostaining was detected in the ocular tissues used as negative control section (Figure 6D).
To correlate the in vivo findings with other quantitative assays, we determined the endogenous expression of Gal-1 in retinal pigment epithelial cells (ARPE-19 cells/RPE) via transmission electron microscopy. Gal-1 immunogold labeling was detected in the RPE in the plasma membrane, cytoplasm, and nucleus (Figure 6E-G). LPS-stimulated cells showed modest Gal-1 immunoreactivity in their subcellular compartments after 2 (p<0.05) and 24 h (Figure 6F; p<0.05) compared to the control group (Figure 6E). Dex treatment increased Gal-1 expression in these cells after 24 h (Figure 6G; p<0.05). These ultrastructural observations were confirmed by the data on the density of gold particles in these cells (Figure 6J). No labeling was detected in the negative control section (Figure 6H).
Despite advances in elucidating the role of Gal-1 in models of autoimmune diseases, its expression and effects on innate immune cells in ocular inflammatory processes have been poorly studied [18,23,39]. In this research, we developed in vivo and in vitro experimental models to evaluate different aspects of the actions of Gal-1 in the ocular tissues under inflammatory conditions.
Treatment with rGal-1 revealed the anti-inflammatory activities of this protein at 24 h through a decrease in the leukocyte influx into ocular tissues, AqH, and blood, as well as a reduction in the production of IL-1β and IL-6 in ocular tissues during EIU. The release of cytokines by activated inflammatory cells has been implicated in the pathogenesis of EIU. Among these cytokines, TNF-α, IL-1β, and IL-6 play an important role in the process of EIU , and they are produced by a wide variety of inflammatory cells, including neutrophils and mononuclear phagocytic cells [41,42], which are the main infiltrating cells in this inflammation . These findings are in line with in vivo tests showing that preadministration of Gal-1 inhibits the extravasation of neutrophils into the peritoneal cavity 4 h after the administration of carrageenan in rats  and IL-1β  and zymosan in mice . In addition, the anti-migratory effect of this protein was shown to be associated with the inhibition of the release of the proinflammatory cytokines TNF-α and IL-1β but not that of IL-6 . Thus, rGal-1 treatment ameliorates EIU manifestation by decreasing leukocyte migration and proinflammatory cytokine release in ocular tissues.
Supporting our in vivo data, rGal-1 also reduced the levels of IL-8 and IL-6 in LPS-stimulated ARPE-19 cells after 24 h, in comparison with untreated LPS-stimulated cells, through the negative regulation of COX-2 expression. It is well known that in several cell types, LPS is the major inducer of redox-sensitive transcription factor nuclear factor-κB (NF-κB), which plays a pivotal role in triggering an array of proinflammatory genes such as TNF-α, IL-1β, IL-6, IL-8, and COX-2 in inflammatory diseases, including endotoxin- and autoimmune-induced uveitis [3,44,45].
Our results obtained from flow cytometry corroborate the quantification of inflammatory cells, demonstrating a significant increase in the percentage of CD62L-positive cells 24 h after LPS injection. This confirmed the occurrence of cell activation caused by LPS. However, at the same experimental time point, treatment with rGal-1 increased the percentage of CD11b- and CD11b/CD62L-positive cells; moreover, in the 48 h LPS group (data not shown), a phase characterized by the diminished influx of leukocytes in this EIU model was obtained . Interestingly, rGal-1 also increased the influx of mononuclear phagocytic cells in the AqH and blood at 24 h, which is consistent with high levels of adhesion molecules detected by immunoblot assays of rat ocular tissues. These results are consistent with in vitro assays demonstrating the role of Gal-1 in monocyte chemotaxis, induced by its carbohydrate-binding function (which was reduced by 65% in the presence of lactose) and the mitogen-activated protein kinase (MAPK) pathways . In addition, the administration of exogenous Gal-1 appears to play a role in the activation of these cells, particularly in dendritic cells, by increasing the secretion of proinflammatory cytokines such as the chemokine MCP-1 . Supporting this, our in vivo and in vitro data exhibited increased MCP-1 levels after treatment with rGal-1.
In a mouse model of zymosan-induced peritonitis, highly significant levels of CD11b expression were detected at 24 h by flow cytometry analysis in blood PMN from rGal-1 pretreated animals. However, this effect was not associated with an increase in cell transmigration on mesentery . In vitro experiments using human neutrophils activated by platelet PAF showed that Gal-1 has no effect on the expression of CD62L and CD11b at low concentrations (~2.75 to 27.5 nM), but is capable of inhibiting the capture and rolling of these cells in the endothelium . Nevertheless, at high concentrations (~275 nM), Gal-1 increases the adherence of neutrophils stimulated by PAF to the endothelium  and stimulates the chemotaxis of these cells . In keeping with this, in our experimental model of uveitis, the inhibitory effect of Gal-1 on neutrophil recruitment may have occurred due to the low concentration of this lectin applied (3 µg/100 µl per animal), causing an imbalance in the expression of adhesion molecules and therefore negatively regulating cell transmigration at 24 h. Researchers have reported that Gal-1 inhibits IL-1β-induced recruitment of PMN leukocytes into the mouse peritoneal cavity, as well as their chemotaxis and transendothelial migration .
Following the detection of the anti-inflammatory action of exogenous Gal-1, western blotting analysis revealed upregulation of endogenous Gal-1 in the eyes 24 h after LPS injection, which was confirmed via immunohistochemistry analysis of the anterior segment. Gal-1 was expressed at particularly high levels in the epithelial cells of the cornea, conjunctiva, ciliary processes, and iris, as well as in endothelial cells. These results support the published finding that Gal-1 is expressed in the anterior segment of the human eye . Modulation of this protein was also observed in a carrageenan-induced rat peritonitis model in which neutrophils showed low levels of endogenous Gal-1 during early acute inflammation (4 h) and high levels after 24 h . In addition, pharmacological treatment with rGal-1 induced a decrease of its endogenous expression in the anterior eye segment, most likely as a negative feedback mechanism. In the posterior eye segment, Gal-1 expression was also observed in the retina in all experimental groups, specifically in the ganglion cells, the inner and outer plexiform layers and the PE, indicating a structural function for this protein in this tissue. Pharmacological treatment with rGal-1 produced no significant change in the endogenous protein levels in relation to the LPS group. Similarly, some studies have demonstrated Gal-1 expression in human, bovine, and rat retinas [52,53] and suggested that this protein functions in the accession of the photoreceptors and the outer plexiform layer through interaction with specific glycoconjugates . Therefore, although Gal-1 can be considered a structural protein of the retina, it may also be secreted into the extracellular medium during the inflammatory process, indicating its anti-inflammatory action.
Based on the relevance of the phenotypic and functional characteristics of human ARPE-19 cells to inflammation  and to confirm the in vivo rodent data, we subsequently assessed Gal-1 expression in ARPE-19 cells activated by LPS and treated with rGal-1 by transmission electron microscopy. Gal-1 immunoreactivity was detected in the plasma membrane, cytosol, and nucleus under different conditions. The activation of cells using LPS decreased the endogenous levels of Gal-1 at 2 and 24 h, indicating the externalization of this protein during the inflammatory process and its participation in the physiology of control RPE cells. This result confirms that the expression of Gal-1 in RPE cells is positively modulated during the process of cell differentiation and proliferation, particularly under the effect of hepatic growth factor (HGF) . In contrast, increased levels of Gal-1 protein expression were detected after Dex treatment (24 h) compared with untreated cells, suggesting a role of the endogenous glucocorticoid in the regulation of this protein in a temporal manner. Previous investigations using nasal polyps [56-58] also demonstrated the upregulation of Gal-1 mRNA and protein following treatment with budesonide and betamethasone glucocorticoids, particularly in epithelial and connective tissues.
Altogether, the in vivo and in vitro data showed that Gal-1 may represent a potential target for the treatment of inflammatory ocular conditions, especially uveitis. Future studies will address the molecular pathways involved in Gal-1 regulation of the inflammatory response.
Appendix 1. STR analysis of ARPE 19 cells.
This study was supported by CNPq (308144/2014-7) and FAPESP (2011/21845–3). CF Zanon and NM Sonehara were supported by FAPESP scholarships (12/02759–1 and 11/05248–5). We thank Eloisa H. Tajara and Giovana M. Polachini from Laboratory of Molecular Biomarkers and Medical Bioinformatics, São José do Rio Preto School of Medicine (FAMERP), for assistance in the western blotting analysis.