Cholinergic regulation of dendritic cell uptake and immune response to topically applied antigens to the conjunctiva

Abstract

Purpose : To evaluate the effects of cholinergic stimulation/blockade on distribution, dendritic cell (DC) uptake and immune response to antigen (Ag) applied to the mouse conjunctiva.

Methods : Experiments were performed in 8-week old female C57/BL6 (B6) mice without and with pre-topical cholinergic stimulation with carbachol (CCh) or blockade with scopolamine (Scop). Distribution and DC uptake of topically applied FITC-labeled 2.3 kDA OVA_323-339 peptide was evaluated by confocal microscopy in whole mount conjunctiva labeled with mucin or DC markers. OVA⁺ DCs were detected in the cervical lymph nodes (CLN) by flow cytometry 24 hr after topical instillation and compared to water controls. Proliferative response to topically applied OVA peptide (daily x 3 days) in the CLNs of OT-II (H-2^b) transgenic mice was evaluated by WST assay.

Results : CCh stimulation prior to topical OVA peptide application increased epithelial mucin binding. In contrast, cholinergic blockade with Scop caused diffuse paracellular antigen migration into the stroma. There were few OVA⁺CD11c⁺ or OVA⁺CD11b⁺ DCs in the epithelium or stroma under homeostatic conditions. After CCh stimulation, weakly OVA⁺ CD11c⁺ and OVA⁺CD11b⁺ cells were noted in the superficial stroma. Following Scop, strongly OVA⁺ CD11c⁺ and CD11b⁺ cells were present in the epithelium and stroma. In the cervical nodes, OVA⁺CD11c⁺ cells were non detectable in the water and OVA peptide groups and detectable in a low percentage of cells in the other groups (CCh⁺OVA = 0.04%, Scop⁺OVA = 0.1%). A greater percentage of OVA⁺CD11b⁺ cells were found in all groups, ranging from 0.11 (OVA) to 0.32 (Scop+OVA), with no statistical difference between groups. The percentage OVA⁺CD11b⁺CD11c⁺ cells was greater in the Scop+OVA group than the OVA and CCh+OVA groups (P ≤ 0.05). The number of proliferating cells in the CLNs following topical Ag challenge was lower in the OVA and CCh+OVA groups than the water control, while there was significantly higher proliferation (P ≤ 0.05) in the Scop+OVA than the other groups.

Conclusions : Cholinergic tone promotes immune tolerance, perhaps through antigen binding to ocular surface mucins that reduces DC antigen uptake. Cholinergic blockade increases the number of antigen positive cells and antigen specific immune response in the draining nodes.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.