Over the past several years a growing body of evidence has suggested that inflammation provides a driving force for the initiation and maintenance of ocular surface disease associated with dry eye. In this study, we examined the hypothesis that a dysregulated immune response (e.g., SS-associated dry eye) or a stress response to chronic deficiencies in the quantity and/or quality of the tear film (e.g., non-SS dry eye or evaporative dry eye) provides the underlying driving force for pathologic keratinization of the ocular surface. With either scenario, inflammation sets off a defense response that leads to increased expression of the cornified envelope proteins present in keratin and loss of mucus-secreting goblet cells. This innate defense is most likely an attempt to “cover up” and protect the mucosal surface from further insult. Unfortunately, it ultimately leads to significant disease that can be sight-threatening when there is corneal involvement (e.g., OCP, SJS).
Given the inherent complexity of deciphering the specific inflammatory events that elicit pathologic expression of keratinizing proteins, we examined the possibility of using SPRR1B as a biomarker for squamous metaplasia in several model systems. SPRR1B provides a convenient endpoint for quantifying pathologic keratinization because it is expressed at negligible levels in nonkeratinized mucosal tissues and quickly becomes overexpressed in the setting of stress-induced and/or immunemediated disease.18,23
Our studies show that SPRR1B is increased across the ocular surface in two different animal models of dry eye disease and in conjunctival samples obtained from the eyes of patients with SS. Adoptive transfer studies using mature lymphocytes from the aire-deficient mouse model confirmed that autoreactive T cells can cause squamous transformation of the ocular surface epithelium in immunodeficient recipients, providing a definitive link between ocular surface inflammation and squamous metaplasia.
To explore inflammation further as an inducer of squamous cell marker expression, we focused on examining the effects of several cytokines reported to be increased in the conjunctival epithelium and/or tear fluid of patients and animals with keratinizing ocular surface disease.5,37,40–43
Some of the mediators are well known to effect differentiation and gene expression in a variety of cell types. For example, in human epidermal keratinocytes, IL1α has been shown to upregulate SPRR1B
Also in keratinocytes, the inflammatory cytokine IFNγ causes irreversible growth arrest and a 7- to 15-fold increase in the expression of both transglutaminase type 1 and SPRR1B
, which precedes expression of the squamous phenotype.45
More recent work has shown a role for IFNγ as an inducer of transglutaminase type 146
in conjunctival epithelial cells, whereas IL1α has no effect.46
Unexpectedly, involucrin gene expression was repressed by IFNγ suggesting that the etiology of squamous metaplasia is complicated and may involve multiple participants.
In our own studies, we found direct induction of SPRR1B
gene expression and protein in response to IL1α, IL1β, IL6, IFNγ, and TNFα, with IL1β proving to be the most potent inducer. We found increased IL1
β and IFN
γ expression in the ocular surface tissues of aire-deficient mice and in bulbar conjunctival cells from patients with SS. Direct induction of SPRR1B
in response to these cytokines suggests that they may function to trigger downstream events that mediate the pathogenesis of dry eye disease, including squamous metaplasia. This possibility is in agreement with a recent study showing a role for IFNγ as an inducer of squamous metaplasia in mice in the desiccating stress model of dry eye.7
In addition to its role as a precursor protein for squamous metaplasia, recent work suggests that the regulation of SPRR genes may not be limited to the induction of epithelial cell differentiation. For example, SPRR1A is involved in axonal outgrowth and is highly expressed at the growth cones of axotomized neurons.47
SPRR1A also confers cardiomyocyte protection in response to IL6 family cytokines after biomechanical –ischemic stress and may play a critical role in the dynamic regulation of actin cytoskeleton assembly.47,48
Of significance to our work is the hypothesis that SPRR genes may play a central role in the induction of an innate defense response after injury or in the setting of primary inflammatory conditions. This notion is supported by studies showing the induction of SPRR2A in mucosal tissue infected by Helicobacter pylori
in models of gastric injury.49
Even the Th2 cytokine, IL13, has been shown to induce SPRR2 in subsets of airway epithelial cells and mononuclear cells associated with inflammation in the asthmatic lung.50
These studies support our hypothesis that SPRR genes are induced in response to stress injury and thereby contribute to cell protection and/or tissue remodeling. They also extend the potential role of SPRR proteins as mediators of genetic or environmental stress where induction may offer protection, such as cardioprotection in the setting of ischemic heart disease, or cause disease, as we find in the setting of keratinizing ocular surface disease.
In summary, we report that SPRR1B is a stress-induced transcript on the ocular surface that is upregulated in both evaporative and immune-mediated, aqueous-deficient dry eye disease. SPRR1B expression is increased in the eyes of patients with mild to moderate KCS and this increase correlates with disease severity. Our results also provide a definitive link between ocular surface inflammation and squamous metaplasia and set the stage for further studies to define the molecular sequence of events that mediates the induction of cornified envelope proteins. Whether a single inflammatory mediator drives the process of squamous metaplasia in autoimmune-mediated keratinizing ocular surface disease is not clear. It is also possible that stimuli such as androgens or tear film hypertonicity could initiate the process of squamous metaplasia. Moreover, studies of SPRR protein and gene expression in the setting of innate immunity may point to additional biological functions for these structural proteins that move beyond their role of forming the cornified envelope. A complete understanding of each role is necessary to preferentially modulate the expression of SPRRs in favor of the host. Further in vitro studies of the gene regulatory mechanisms governing squamous metaplasia in response to cytokines are necessary as well as in vivo studies to identify other key players in the immunopathogenesis of squamous metaplasia. This combination of approaches will guide our studies of therapeutic targets to control this process in sight-threatening ocular surface disease.