ABSTRACT
Background Researches showed that thymic stromal lymphopoietin (TSLP) is an interleukin-17-like inflammatory factor and plays important roles in the pathogenesis and development of allergic diseases.However,the study whether TSLP plays roles in allergic conjunctivitis is rare.Objective This study was to investigate the expression change of TSLP and IL-4 in ocular surface tissue and cervical lymph node in the mice with allergic conjunctivitis induced by artemisia annua,a common plant in China,and to explore the role of TSLP and IL-4 in the pathogenesis and development of allergic conjunctivitis.Methods Twenty female BALB/c mice aged 6-8 weeks were randomized into normal control group and model group.Antigen solution was prepared using 400 μl extracting solution of artemisia annua pollen with 400 μl antigen solvent.The mouse models of allergic conjunctivitis were established by injection of 50 μl antigen solution in footpad followed by ocular topical administration of extracting solution once a day from day 10 to 12 after injection,and no any intervention was given in the normal control group.The mice were sacrificed and eyeballs were obtained 13 days after modeling,and corneal epithelium,conjunctiva and cervical lymph nodes were harvested for the detection of TSLP mRNA and IL-4 mRNA by real-time PCR.Immunochemistry was employed to assay the expression of TSLP and IL-4 proteins in corneal,conjunctival and subconjunctival tissues.Results Ocular inflammatory signs appeared 0.5 hours after ocular topical administration of extracting solution,including eyelid edema,conjunctival congestion,tears,scratching eyelids,etc.The symptoms lasted for 6-24 hours,with the model successful rate 80％.Real-time PCR indicated that the expression of IL-4 was absent in corneal epithelium in both model group and normal control group.The relative expression levels of TSLP mRNA in the corneal epithelium,conjunctiva and cervical lymph nodes in the model group were more (1.63±0.20)times,(2.71±0.48) times and (1.48 ±0.05) times than the normal control group,showing significant differences between the two groups (t =4.44,14.16,5.01,all at P＜0.05).Compared with the normal control group,the relative expression levels of IL-4 mRNA in the model group increased (2.94±0.39) times and (1.74±0.09) times,with significant differences between the two groups (t =9.92,14.54,both at P＜0.05).Immunochemistry assay showed that the expression of TSLP protein in the corneal and conjunctival epithelial cells were enhanced in the model group compared with the normal control group.In addition,an intensive expression of IL-4 protein was seen in subconjunctival tissue in the model group,while the expression of IL-4 was absent in the normal control group.Conclusions TSLP is mainly expressed in the cornea,conjunctiva and cervical lymph nodes of the mice with allergic conjunctivitis,suggesting that TSLP promotes the inflammatory process of cornea and conjunctiva;IL-4 is mainly expressed in conjunctiva,showing IL-4 participates in conjunctival inflammatory process.TSLP and IL-4 play synergistic roles in promoting the inflammatory process of ocular surface in the mice with allergic conjunctivitis,which may be new therapeutic targets.
ABSTRACT
Background The pathogenesis of allergic conjunctivitis has not been clearly established.Current researchers indicate that interleukin-4 (IL-4), IL-5 and IL-13 may play an important role in allergic conjunctivitis.But whether the roles of these inflammatory factors are same in different types of allergic conjunctivitis remains unclear.Objective This study was to investigate the expressions of IL-4, IL-5 and IL-13 in ocular surface with different types of allergic conjunctivitis.Methods A prospective cohort study was designed.Eighty individuals were recruited in Shanxi Eye Hospital from April 2013 to September 2014, including 20 patients with vernal keratoconjunctivitis (VKC), 20 patients with seasonal allergic conjunctivitis (SAC), 20 patients with perennial allergic conjunctivitis (PAC) and 20 normal healthy subjects.Surficial tissues were binocularly scraped using disinfected scraper from upper eyelid conjunctiva, and 4 μl of tear fluid was obtained with capillary tube.The expressions of IL-4, IL-5 and IL-13 protein and mRNA in the conjunctival epithelial cells were detected by immunohistochemistry and real-time fluorescence quantitative PCR.The IL-4, IL-5 and IL-13 concentrations in tear fluid were assayed by Luminex method.This study complied with Declaration of Helsinki and the research protocol was approved by the Shanxi Eye Hospital Ethics Committee.Written informed consent was obtained from each subject prior to entering the cohort.Results IL-4, IL-5 and IL-13 were positively expressed in cytoplasm of conjunctival epithelial cells in the VKC group,SAC group and PAC group,but the expressions of IL-4,IL-5 and IL-13 were absent in the normal control group.The relative expression levels of IL-4 mRNA were 4.11±1.24,2.71±0.71 and 2.00±0.80;the relative expression levels of IL-5 mRNA were 4.02±0.43,2.07±0.45 and 1.47±0.50;and the relative expression levels of IL-13 mRNA expression levels were 6.44±0.66,4.35±1.26 and 2.39±0.86 in the VKC group,SAC group and PAC group, showing significant differences among the 4 groups (F =51.32,220.18,162.49, all at P＜0.01).The relative expression levels of IL-4,IL-5 and IL-13 mRNA were significantly higher in the VKC group than those in the SAC group and PAC group;and those in the SAC group were significantly elevated in comparison with the PAC group (all at P＜0.05).No IL-4, IL-5 and IL-13 were detected in the tear fluid in the normal control group;while the concentrations of IL-4,IL-5 and IL-13 in the tear fluid were (14.06±3.50), (10.88±1.82) and (34.28±8.42) pg/ml in the VKC group,and (7.71 ±0.65), (5.10± 1.33), (23.77±6.29) pg/ml in the SAC group as well as (3.30± 1.50) pg/ml, (2.43± 1.28) pg/ml and (17.67 ± 4.28) pg/ml in the PAC group, showing significant differences among the 3 groups (F =200.29,260.49,128.23, all at P＜0.01).IL-4, IL-5 and IL-13 concentrations in the tear fluid were significantly higher in the VKC group than those in the SAC group and PAC group,and those in the SAC group were significantly raised in comparison with the PAC group (all at P＜0.01).Conclusions IL-4,IL-5 and IL-13 participate in the pathogenesis of multiple allergic conjunctivitis,but their expressions in the ocular surficial tissue are discriminatory in different types of allergic conjunctivitis.