September 2014
Volume 55, Issue 9
Immunology and Microbiology  |   September 2014
Restoring Conjunctival Tolerance by Topical Nuclear Factor–κB Inhibitors Reduces Preservative-Facilitated Allergic Conjunctivitis in Mice
Author Affiliations & Notes
  • Mauricio Guzmán
    Institute of Experimental Medicine, National Academy of Medicine/CONICET, Buenos Aires, Argentina
  • Florencia Sabbione
    Institute of Experimental Medicine, National Academy of Medicine/CONICET, Buenos Aires, Argentina
  • María Laura Gabelloni
    Institute of Experimental Medicine, National Academy of Medicine/CONICET, Buenos Aires, Argentina
  • Silvia Vanzulli
    Institute of Oncologic Research, National Academy of Medicine, Buenos Aires, Argentina
  • Analía Silvina Trevani
    Institute of Experimental Medicine, National Academy of Medicine/CONICET, Buenos Aires, Argentina
  • Mirta Nilda Giordano
    Institute of Experimental Medicine, National Academy of Medicine/CONICET, Buenos Aires, Argentina
  • Jeremías Gastón Galletti
    Institute of Experimental Medicine, National Academy of Medicine/CONICET, Buenos Aires, Argentina
  • Correspondence: Jeremías Gastón Galletti, Pacheco de Melo 3081, Immunology Laboratory, Institute of Experimental Medicine, National Academy of Medicine/CONICET, (1425) Buenos Aires, Argentina;
Investigative Ophthalmology & Visual Science September 2014, Vol.55, 6116-6126. doi:10.1167/iovs.14-14075
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      Mauricio Guzmán, Florencia Sabbione, María Laura Gabelloni, Silvia Vanzulli, Analía Silvina Trevani, Mirta Nilda Giordano, Jeremías Gastón Galletti; Restoring Conjunctival Tolerance by Topical Nuclear Factor–κB Inhibitors Reduces Preservative-Facilitated Allergic Conjunctivitis in Mice. Invest. Ophthalmol. Vis. Sci. 2014;55(9):6116-6126. doi: 10.1167/iovs.14-14075.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose.: To evaluate the role of nuclear factor–κB (NF-κB) activation in eye drop preservative toxicity and the effect of topical NF-κB inhibitors on preservative-facilitated allergic conjunctivitis.

Methods.: Balb/c mice were instilled ovalbumin (OVA) combined with benzalkonium chloride (BAK) and/or NF-κB inhibitors in both eyes. After immunization, T-cell responses and antigen-induced ocular inflammation were evaluated. Nuclear factor–κB activation and associated inflammatory changes also were assessed in murine eyes and in an epithelial cell line after BAK exposure.

Results.: Benzalkonium chloride promoted allergic inflammation and leukocyte infiltration of the conjunctiva. Topical NF-κB inhibitors blocked the disruptive effect of BAK on conjunctival immunological tolerance and ameliorated subsequent ocular allergic reactions. In line with these findings, BAK induced NF-κB activation and the secretion of IL-6 and granulocyte-monocyte colony-stimulating factor in an epithelial cell line and in the conjunctiva of instilled mice. In addition, BAK favored major histocompatibility complex (MHC) II expression in cultured epithelial cells in an NF-κB–dependent fashion after interaction with T cells.

Conclusions.: Benzalkonium chloride triggers conjunctival epithelial NF-κB activation, which seems to mediate some of its immune side effects, such as proinflammatory cytokine release and increased MHC II expression. Breakdown of conjunctival tolerance by BAK favors allergic inflammation, and this effect can be prevented in mice by topical NF-κB inhibitors. These results suggest a new pharmacological target for preservative toxicity and highlight the importance of conjunctival tolerance in ocular surface homeostasis.

Local side effects of medical glaucoma treatment represent a significant challenge in clinical practice because of their negative impact on patients' quality of life and compliance. 1 Allergic conjunctivitis and dry eye can arise or worsen after initiating topical treatment with hypotensive eye drops, 24 and a significant body of evidence suggests that the microbicidal agents in glaucoma formulations are responsible for this phenomenon. 1 The most common among the preservatives approved for human use, benzalkonium chloride (BAK), is found at concentrations ranging from 0.004% (4 × 10−3%) to 0.025% (2.5 × 10−2%). 1 The toxic effects of BAK on the ocular surface epithelium have been extensively described, encompassing apoptosis and/or necrosis induction in vitro 57 and the release of proinflammatory cytokines and increased expression of epithelial activation markers both in vivo and in vitro. 8 In contrast, our understanding of how topical exposure to BAK facilitates allergic conjunctivitis and dry eye is quite limited, and there are few reports on the functional impact of the preservative on ocular surface immunity. We have previously demonstrated that BAK can profoundly disrupt conjunctival immunological tolerance to a harmless antigen in mice, 9 but the clinical implications of these findings and the underlying molecular mechanisms remained unexplored. 
The conjunctiva, as we and others have shown, 911 actively regulates the immune response when an innocuous antigen reaches its confines. Under physiological conditions, conjunctival immunological tolerance sets in and requires the migration of antigen-presenting cells to the draining lymph nodes. The development of a tolerogenic response involves the expansion of antigen-specific regulatory T cells 12 and prevents unwanted inflammation on subsequent encounters with the same antigen. 13 This specific function of the conjunctiva has been proposed as an efficient vaccine delivery route, 11 but mucosal tolerance is not exclusive to the eye. Respiratory immune tolerance can successfully dampen allergic airway inflammation and constitutes one of the bases of specific immunotherapy for asthma. 14 Remarkably, and despite extensive research on bronchial and nasal tolerance, the implications of conjunctival tolerance (and lack thereof) have not been addressed in the clinical approach to immune-mediated ocular surface disorders. 
In any mucosa, the epithelium plays a key role in the immune decision-making process by either exerting a tolerogenic or an immunogenic conditioning on dendritic cells, 15 which in turn drives the expansion of either regulatory or effector T cells. Activation of the nuclear factor–κB (NF-κB) pathway in epithelial cells is paramount to this process, and whereas a low threshold of activity is required for epithelial homeostasis, chronic engagement of this signaling pathway leads to T-cell–mediated mucosal inflammation. 16,17 On the other hand, there are numerous examples of how environmental exposure of the skin or mucosal linings to toxic agents can facilitate an antigen-specific immune response. 1820 Some of these substances are capable of triggering NF-κB in epithelial cells and, more importantly, mucosal inhibition of this signaling pathway can ameliorate disease in animal models. 2123  
Based on preliminary findings that suggested a potential involvement in the immune effects of BAK on cultured epithelial cells, 9 we directly explored the role of the NF-κB pathway in both BAK toxicity and epithelial–T-cell interaction. In addition, we hypothesized that exposure to BAK might facilitate allergic conjunctivitis by the disruptive immune effect already described. 9 To this aim, we designed a murine model of BAK-facilitated allergic conjunctivitis and evaluated the effect of topical NF-κB modulation as a therapeutic strategy. 
Female Balb/c mice (8–12 weeks old), which were bred and maintained in our conventional animal facility, were used for the in vivo experiments. All experiments were approved by the Institute of Experimental Medicine Animal Ethics Committee (approval number 20130610) and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Cell Lines and Cultures
Cell cultures were performed in RPMI 1640 medium supplemented with 10% fetal calf serum, 10 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 5 × 10−5 M 2-mercaptoethanol in a humidified incubator with 5% CO2 at 37°C. The Pam212 cell line, which was originally derived from murine epidermal keratinocytes, 24 was a kind gift from Adriana Casas, PhD (Hospital de Clínicas, University of Buenos Aires, Buenos Aires, Argentina), and was maintained in the same medium. 
Reagents and Antibodies
All reagents were from Sigma-Aldrich (Buenos Aires, Argentina) unless otherwise specified. Fluorochrome-tagged antibodies were from BioLegend (San Diego, CA, USA). Grade V ovalbumin (OVA) was used in all experiments. 
Benzalkonium Chloride Treatment of Epithelial Cells for Supernatant Collection and T-Cell Cocultures
Confluent Pam212 cells were exposed to fresh medium alone or with the specified BAK concentration for 15 minutes at 37°C, washed twice, and finally cultured in fresh medium without serum. Nuclear factor–κB inhibitors were added to some cultures 30 minutes before BAK. After overnight culture, supernatants were collected and the viability of the remaining cells was assessed by crystal violet staining. For coculture experiments, Pam212 cells were grown in 48-well plates and treated likewise before adding 2 × 105 T cells obtained from Balb/c mouse lymph nodes. After 4 days of culture, nonadherent cells were removed by gentle washing and then the epithelial cells were detached by mild trypsin treatment. 
Immunostaining and Flow Cytometry
For surface antigen staining (major histocompatibility complex II [MHC II], CD40, and CD86), Pam212 cells were washed in PBS with 0.5% BSA and then incubated with fluorochrome-conjugated antibodies at previously titrated concentrations for 30 minutes at 4°C. For intracellular antigen staining (IκBα), Pam212 cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature, washed with PBS with 50 mM glycine, permeabilized, and blocked with 0.1% saponin in PBS with 5% goat serum for 30 minutes, and then anti-IκBα antibody (1:100 dilution; Abcam ab32158; Abcam, Cambridge, MA, USA) was added for another 30 minutes. After thorough washing, the cells were labeled with DyLight 488 goat anti-rabbit IgG (1:500 dilution; Abcam ab96899) for 30 minutes. For flow cytometry analysis, cells were thoroughly washed in PBS with 1 mM EDTA before acquisition on a FACScalibur cytometer (Becton Dickinson, Buenos Aires, Argentina). Data were analyzed with Flowing Software (Perttu Terho, Turku Centre for Biotechnology, Turku, Finland; Optimal compensation and gain settings, as well as viable cell gating, were determined as previously described. 25 Overlayed histogram graphs are plotted in normalized form as percentage of maximum for each histogram. 
Western Blotting
After BAK and/or NF-κB inhibitor treatment, Laemmli buffer (with 5% 2-mercaptoethanol) was added to Pam212 monolayers, and the resulting extracts were then heated at 95°C for 5 minutes and subjected to SDS-PAGE in 10% gel. Proteins were later transferred from the gel to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) for 2 hours and then blocked in PBS containing 0.05% Tween 20 and 5% nonfat milk for 1 hour at room temperature. Membranes were incubated overnight with anti-IκBα antibody (1:1000 dilution; Abcam ab32158) and the bound antibodies were visualized with horseradish peroxidase–conjugated anti-rabbit IgG Abs and the ECL Western Blotting System (Amersham Biosciences, Amersham, UK). Signal intensity in digital images was quantified with ImageJ software (; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). 
Enzyme-Linked Immunosorbent Assay
Interleukin-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations in culture and explant supernatants were determined with commercial ELISA kits according to the manufacturer's instructions (Peprotech, Rocky Hill, NJ, USA). 
Confocal Microscopy
Pam212 cells grown on coverslips were treated with BAK and/or NF-κB inhibitors, rinsed with PBS at the specified time points, fixed for 30 minutes in 4% paraformaldehyde, permeabilized with ice-cold methanol for 10 minutes, and washed with 50 mM glycine in PBS. The coverslips were then incubated with 1 μg/mL polyclonal rabbit anti–NF-κB p65 antibody (sc372; Santa Cruz Biotechnology, Dallas, TX, USA) or the corresponding isotype control for 2 hours, blocked with 5% goat serum in PBS for 2 hours and then incubated with DyLight 488 conjugated goat anti-rabbit IgG (1:100 dilution) for 2 hours. Coverslips were finally mounted with 1 μg/mL propidium iodide using antifade medium. Images were acquired with a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan) equipped with a Plapon ×60/1.42 objective and analyzed with Olympus FV10-ASW software. 
Conjunctival Instillation of BAK, OVA, and NF-κB Inhibitors and Parenteral Immunization
Phosphate-buffered saline, 10−2% BAK, 20 μg OVA (2 mg/mL), 0.1 mM pyrrolidine dithiocarbamate (PDTC), and 0.5 mM sulfasalazine (SSZ) or different combinations of these compounds in PBS were instilled daily in a volume of 5 μL to the conjunctiva of both eyes for 5 days. On day 7, mice were injected subcutaneously in the flank with 0.1 mL of 1:1 complete Freund' s adjuvant (CFA) emulsion in PBS containing 100 μg OVA. 
Conjunctival Explants
Mice were instilled PBS or BAK alone or combined with 0.1 mM PDTC and 0.5 mM SSZ in both eyes once daily for 3 days before euthanasia. The tarsal conjunctivas were excised under aseptic conditions with the aid of a dissection microscope, as described elsewhere. 9 The conjunctival explants from each animal (approximately 30 mg tissue) were pooled, washed three times with PBS, and then cultured in 1 mL medium without serum. Supernatants were collected after 24 hours for further analysis. 
Myeloperoxidase Colorimetric Quantification Assay
Conjunctival explants from each mouse were rendered into a suspension with a tissue homogenizer in 0.3 mL PBS and then 0.5% Triton X-100 was added. Suspensions were then centrifuged at 2000g for 5 minutes and the supernatants collected for enzymatic assay. In brief, 50 μL 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Scientific, Waltham, MA, USA) was added to 50 μL of each sample in duplicate in microtiter plates and incubated at room temperature for 15 minutes, after which the reaction was stopped by adding 50 μL 1 N sulfuric acid. Absorbance was measured at 450 nm with the reference filter set at 570 nm. 
Measurement of Delayed-Type Hypersensitivity Responses