Cells generate energy by reducing oxygen to water during which reactive oxygen species are generated. The generation of free radicals has been reported to lead to tissue injury through induction of inflammation in several disease states including sepsis, ischemic heart and brain disease, atherosclerosis, asthma and AD [8
Increased oxidative stress status, specially elevation of ROS, has been described in patients with AD. Indeed, urine concentrations of 8-hydroxy-2’-deoxyguanosine (8-OHdG), a marker for oxidative DNA damage, and acrolein-lysine adducts, a marker of lipid peroxidation, have been reported to be higher in AD patients compared to controls [8
]. Oxidative stress changes in AD have been reported extensively in Dermatology literature. Niwa et al. confirmed the presence of lipid peroxidation, detected by 4- HNE, in the stratum corneum of patients with AD [24
]. The skin inflammation in AD was histologically characterized by intense infiltration of lymphocytes, monocytes and eosinophils [4
]; all of which may release bioactive substances including inflammatory cytokines and ROS upon immunological and non-immunological stimulation [5
]. There is futher evidence in the literature that circulating polymorphonuclear leucocytes from AD patients are hyperreactive, showing enhanced release of reactive oxygen intermediates [26
], suggesting the possibility that these changes may lead to a vicious cycle further causing immune activation, oxidative damage, and propagation of skin inflammation.
In the field of Ophthalmology, oxidative stress damage has been reported to play a role in several ocular diseases including age-related macular degeneration [28
], cataract [30
], uveitis [31
], retinopathy of prematurity [32
], corneal inflammation [33
], keratitis [35
], and conjunctivochalasis [36
], an aging disease of the conjunctiva characterized by conjunctival laxity.
Although the oxidative stress status in atopic skin disease has been reported to be elevated, there are still no studies related to the status of oxidative stress in atopic ocular surface disease.
In this study, we investigated the lipid oxidative stress related changes in the tear film and brush cytology samples of patients with AD comparing the results with healthy control subjects. We also performed a histopathological assessment of lipid peroxidation staining in papillary resection samples of patients with severe AKC.
The tear stability, quantity, ocular surface epithelial damage and conjunctival injection scores were significantly worse in patients with AKC compared with controls which were consistent with previously published studies. Brush cytology revealed a significantly higher extent of inflammatory infiltrates, mainly of eosinophils in AKC patients. Another piece of evidence for increased ocular surface inflammation was based on significant increases in the concentration of TNF-α, IL-5, and IL-4 in tears of patients with AKC compared with controls. Not only the systemic disease process that favors a Th2 cytokine dominant response in AD but the environmental conjunctival allergen exposure might have led to the recruitment and activation of inflammatory cells in the conjunctiva.
Allergen exposure has been shown to be associated with release of IL-5 and IL-13 from inflammatory cells in allergic airway exposure and associated with oxidative stress in BALB/c mice [37
]. In vitro studies on primary cultures of purified human CD4+
T cells showed evidence that oxidative stress could be induced which in turn modulated T cell polarization toward Th2 cytokine producing cells, including IL-4, IL-5, and IL-13 [38
]. Inflammatory cells are also known to release reactive oxygen products. It is our belief that allergens induce recruitment of inflammatory cells including mast cells, lymphocytes, eosinophils, and neutrophils in AKC, which not only release inflammatory cytokines but free oxygen radicals as well.
Our efforts to investigate the lipid oxidative stress changes interestingly revealed higher percentages of cells positively stained for HEL (early phase lipid peroxidation marker) and 4-HNE (late phase lipid peroxidation marker) in palpebral conjunctival samples suggesting an increased lipid peroxidation status in AKC. The lipid peroxidation marker HEL was also significantly increased in tears of patients with AKC compared with controls.
Although palpebral conjunctival tissue samples from healthy control subjects could not be obtained due to lack of ethic board permission, palpebral conjunctiva covering the papillary formations during papillary resection surgery in patients with severe AKC showed dense mast cells and CD45+ inflammatory cells as well as marked stainings for HEL and 4-HNE antibodies providing further evidence that conjunctival inflammation and lipid peroxidation coexisted in AKC.
There is increasing evidence that atopic diseases are intimately linked to the generation of oxidative stress. While the oxidative stress could be the consequence of oxygen radical production by macrophages, neutrophils, and eosinophils, it is important to understand that oxidative stress could also be an inciting factor in the generation of ocular surface inflammation. Oxidative free radicals directly oxidize various macromolecules including lipids. It has been described in the literature that lipid peroxides and their break-down products, such as HEL and 4-HNE, can directly or indirectly affect many functions integral to cellular and organ homeostasis. As a result, the increased membrane lipid peroxidation may evoke and/or increase the immune and inflammatory response, activate gene expression and cell proliferation, or initiate apoptosis [39
]. Thus, a close relationship between ROS production, peroxidative lipid membrane damage and an inflammatory pathological process may be postulated for the ocular surface disease in AKC.
Our correlation analysis showed a significant positive correlation between tear HEL levels and conjunctival HEL staining whereas another strong positive correlation was observed between eosinophilic inflammatory cell numbers and the extent of early and late lipid peroxidation as evidenced by the higher percentages of positively stained cells for HEL and 4-HNE in specimens harboring higher eosinophil densities. The extent of early and late lipid peroxidation marker stainings also showed a strong positive correlation with corneal epithelial damage.
Peroxidation of membrane lipids might have very well induced perturbation of ocular surface epithelial cellular functions causing breakdown and death of epithelial cells which might have contributed to increased epithelial fluorescein staining scores. TNF-α, which is known to induce inflammation and cell death, was also elevated in tears of AKC patients. The pathways regulating the elevation of lipid oxidation status and linking it to inflammation are still unclear. Whether upregulation of STAT 6 with increased NF-kB activity results in Th2 favored cytokine response and induce oxidative stress in allergic ocular surface disease remains to be investigated in future studies.
It also remains the further goal of future studies to determine short and long-term conjunctival epithelial cytology staining status with oxidative stress markers and their correlation to inflammatory cytokine markers that may open up some new avenues of categorizing, diagnosing, and treating different cryptic elements of AKC and AD. Further studies investigating the simultaneous alterations of the ocular surface anti-oxidant status will also provide invaluable information.
It is also our wish that the observations from the current study will pave the way to newer studies testing the efficacy of anti-oxidant treatment strategies in AKC. Indeed, antioxidant defense impairment in atopic dermatitis has been confirmed in a study reporting that N-acetyl cystein (NAC) was able to down-regulate Th2-secreted-cytokines, such as IL-4, IL-5 with a subsequent over-activation of the Th1 response, suggesting that in Th2 related diseases, such as atopic dermatitis, NAC might serve as a possible therapeutic agent [40
]. Recent studies also suggest squalene monohydroperoxide is a primary oxidized lipid generator which is elevated in atopic dermatitis. Antioxidants, such as catechin, or ascorbic acid which have been shown to markedly decrease the cytotoxicity of squalene monohydroperoxide may find roles in the treatment of AKC in the future [41
In summary, this study provided the first evidence that lipid peroxidation and inflammation coexisted in the conjunctiva of patients with AKC, a finding which might hold the keys to future explanations for the pathogenesis of the ocular surface disease in AKC.