Mast cells initiate early recruitment of neutrophils following corneal injury

Abstract

Purpose : Injury-induced innate immune responses, primarily the recruitment of activated neutrophils to the eye, lead to deleterious inflammatory tissue damage. This study investigated the contribution of resident mast cells to the initiation of neutrophil recruitment following corneal injury.

Methods : Corneal injury was created by removal of the epithelium and anterior stroma using a hand-held motor brush. Corneas and conjunctivae were harvested to investigate frequencies of CD45⁺ inflammatory cells, CD11b⁺Ly6G⁺ neutrophils and c-Kit⁺FcεR1⁺ mast cells by flow cytometry. Mast cell-activating factors IL1β and IL33 were evaluated using real-time PCR and ELISA. In vitro, mast cells were stimulated with mechanically damaged corneas in the presence or absence of IL1β or IL33 neutralizing antibodies and assessed for the expression of β-hexosaminidase, CXCL1 and CXCL2 using real-time PCR and ELISA. Cromolyn (2% in PBS) or IL1β neutralizing antibodies (1 mg/ml) were administered locally for mast cell inhibition in vivo.

Results : Flow cytometry analysis demonstrated early neutrophil infiltration to the cornea within 1 hour of injury (3±0.6%, p<0.05), and this infiltration increased progressively over the first 12 hours following injury (12±1%, p<0.001) compared to normal cornea (1±0.2%). Frequencies of activated mast cells at the ocular surface were significantly (3-fold) increased until 6 hours post-injury (0.9±0.3%, p<0.05) and then declined to baseline levels by 12 hours (0.3±0.2%). Mast cells constitutively expressed CXCL2. Mast cells cultured with injured corneas secreted 2.5-fold higher levels of CXCL2 compared to mast cells cultured alone (p<0.01). Corneal injury resulted in significantly increased levels of IL1β and IL33 at the ocular surface compared to normal corneas (p<0.05). IL1β neutralization in injured cornea-mast cell cocultures significantly inhibited mast cell activation and CXCL2 secretion compared to isotype-treated control cocultures (p<0.05). Moreover, in vivo topical blockade of IL1β significantly inhibited mast cell activation, expression of CXCL2 (p<0.05) and limited the early infiltration of neutrophils to the ocular surface compared to control isotype-treated injured corneas (1.9±0.3% vs. 9.8±2%, p<0.01).

Conclusions : Our data suggest that corneal injury activates mast cells via IL1β and amplifies mast cell secretion of CXCL2, which is associated with early neutrophil recruitment to the ocular surface.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.