| Sterile keratitis and ulcers are a series of corneal diseases, which are characterized as poor curative effect to antibiotic and repeating disease-process. Now, it has become one of the basic causes that lead to blindness. Recent researches indicated that the pathogeny of Sterile keratitis may have much correlation with corneal nonspecific inflammation induced by physical and chemical stimulation. For rare clinical specimens and lack of valid animal models, there are few studies on the pathogeny of sterile keratitis. As other antigen-presenting cells (APC), the maturation and costimulatory molecules expression of corneal Langerhans cells (LCs) are greatly influencing their function in corneal immunoreactions. New evidence shows that Th1 and Th2 chemokines are a group of potent cytokines which perform a significant function in Th1/Th2 priming and polarizing. Interferon γinducible protein-10 (Ip-10/CXCL10) and thymus and activation-regulated chemokine (TARC/CCL17) are representative Th1/Th2 chemokines which are paid close attention to recently. Up to now, there are few reports on the etiopathogenesis of sterile keratitis, especially with respect to LCs and chemokines. Here, we constructed a new nonspecific keratitis animal model and preliminarily discussed the related immunological pathogeny from the two important aspects: APC and chemokines. In the course of constructing mice nonspecific keratitis models, Suture and Lipopolysaccharide (LPS) were used as stimulative factors and the pathologic inspections was used to certify the validity of models. For further analyzing, CD11c was stained as LCs specific marker and CD86 was stained as LCs mature marker. Central, paracentral and peripheral areas for each cornea were assessed with confocal microscope separately. Meanwhile, we observed the LCs morphologic characteristics in different mature degree by transmission electron microscopy (TEM). LCs were evaluated on distribution, density and maturation under normal or stimulative conditions. And the functions of pyrrolidine dithiocarbamate(PDTC), an inhibitor of nuclear factor-κB (NF-κB) were assessed from above indexes. In nonspecific inflammation, the mRNA level of corneal IP-10 and TARC were examined with semi-quantity reverse transcription and polymerase chain reaction (RT-PCR). Further, the mediating function of Th1 and Th2 chemokines in nonspecific keratitis were discussed. We constructed mice nonspecific keratitis models successfully. CD11c+LCs were mainly found in the peripheral and paracentral areas, and scattered in the center area of the normal cornea. Some CD11c+CD86+ and CD11c-CD86+ cells were also detected in the peripheral area of the normal cornea. The LCs density of three corneal areas increased significantly after induced by suture and combined with LPS subconjunctival injection. And the LCs quantity increased more promptly within 3~7d postoperative. At the same time point, the accumulation of LCs were not uniform, with a more rapid rate of LCs density increase in peripheral and paracentral cornea than that in central cornea. PDTC impaired the LCs corneal migration effect induced by suture and LPS 1~7d postoperatively, whereas the corneal LCs quantity in PDTC treated groups turned to an upwards trend 14d postoperatively. CD11c+CD86+ cells were found in the central cornea of treated groups 3d postoperatively, and its quantity had a time-dependent increase within the observing period. TEM results showed that the LCs number and dendritic prominence of LCs surface increased obviously in treated groups 3d~14d postoperatively. In suture and combined with LPS groups 3d~14d postoperatively, the sheer increased number of CD11c+CD86+ cells is higher than that of CD11c+ cells in central cornea, whichindicated that most of mature LCs in central cornea derived from CD11c+ cells existed inherently. The corneal LCs in PDTC treated groups expressed fewer CD86 markers than that in suture and combined with LPS groups when comparing at the same time point. IP-10 mRNA expression was detected in normal BALB/C mouse cornea slightly and went to a higher level by the induction of corneal suture or combined with LPS. IP-10 mRNA level reached its Peak value 1d postoperatively, then tended to decline with time-dependence. Comparing with IP-10 mRNA expression of S groups, that of S+L groups is much higher, especially within 1~7d postoperatively. Moreover, the corneal inflammatory corpuscles increased persistently when the IP-10 mRNA expression showed a steep descend, which indicates that IP-10 mRNA expression may have much relation with the aggravation more than the number of inflammatory corpuscles in cornea. Within 7d postoperatively, IP-10 mRNA expression in PDTC treated groups was higher than that in control group, but much lower than that in S and S+L groups. TARC mRNA expression was also detected in normal mouse cornea slightly. No relationship was found between TARC mRNA expression and the increased number of inflammatory corpuscles in cornea, when analyzing the results of semi-quantity RT-PCR and pathologic inspections accordingly. To understand the possible balance of Th1 and Th2 chemokines, the ratios of TARC and IP-10 mRNA expression in nonspecific keratitis were analyzed at different time points. The ratio of Th2 and Th1 chemokines in PDTC treated groups had an obvious rise and were much higher than that in S and S+L groups 3~7d postoperatively. That indicated the predominant status of Th2 chemokine in PDTC treated groups. The corresponding HE inspections showed a slight inflammatory corpuscles rise in PDTC treated groups, which is lower than that in S and S+L groups 3~7d postoperatively. 7~14d postoperatively, the ratio of Th2 and Th1 chemokines in PDTC treated groups got a sharp descent and that in S and S+L groups began to rise. Accordingly, the HE inspections showed a sudden rise of corneal inflammatory... |
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