Ординатура / Офтальмология / Английские материалы / Dry Eye and Ocular Surface Disorders_Pflugfelder, Beuerman, Elliot Stern_2004

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II.CORNEAL EPITHELIAL DISEASE

Ocular surface epithelial disease is the most clinically recognizable manifestation of LKC. The ocular surface epithelium is a normally well lubricated smooth surface that attracts tear constituents and stabilizes the tear film. In LKC, it changes to a poorly lubricated and irregular surface that does not attract tear components and destabilizes the tear film. Because of risks associated with corneal biopsy of eyes with LKC, our understanding of the corneal manifestations of this condition is based on clinical observations and animal models. The unstable precorneal tear film in LKC is detected clinically as rapid fluorescein tear breakup, and in more severe cases as visible discontinuities or pits in the tear film [3–5]. Corneal surface irregularities that accompany this unstable tear film may be visualized by biomicroscopy in eyes with severe aqueous tear deficiency, such as in Sjögren’s syndrome, or may be readily detected in most cases by computerized videokeratoscopy surface regularity indices [6,7]. The unstable tear film and corneal epithelial irregularity are responsible for the blurred and fluctuating vision symptoms frequently reported by patients with LKC, as well as their reduced contrast sensitivity [5,8,9].

Another well-recognized manifestation of corneal epithelial disease in LKC is disruption of corneal epithelial barrier function. Because of its barrier function, which is important in maintaining corneal smoothness and clarity, the normal corneal epithelium is much less permeable than the conjunctival epithelium. Disruption of corneal epithelial barrier function is identified clinically by fluorescein dye staining, and fluorometrically by increased permeability to sodium fluorescein dye. Corneal epithelial permeability in patients with untreated dry eye is 2.7–3 times greater than in eyes with normal tear function [10,11]. Rabbit studies showed that the full-thickness cornea was permeable to mannitol (MW 182) but not to larger molecules such as insulin (MW 3000) and dextran (MW 20,000), whereas the conjunctiva was permeable to all three molecules [12]. Removal of the corneal epithelium increased corneal permeability 40-fold, while removal of endothelial cells had no effect. Following corneal epithelial wounding, epithelial defects that healed with nonvascularized corneal or limbal epithelium showed an initial increase in permeability that returned to normal after 3 days. By contrast, the permeability of wounded corneas that reepithelialized with vascularized conjunctival epithelium correlated with the degree of surface vascularization. Avascularized or minimally vascularized conjunctival epithelium that transdifferentiated into a cornea-like morphology showed long-term permeability similar to that of normal corneal epithelium, whereas vascularized conjunctival epithelium that retained a conjunctival phenotype showed increased permeability similar to that of conjunctival epithelium [13].

The cell membrane-associated mucins that coat the superficial corneal epithelium, and the tight junctional complexes that connect adjacent cells, are

important factors in maintaining the corneal epithelial barrier [14]. Derangement of this barrier in LKC is due to death, loss, or dysfunction of well-differentiated apical corneal epithelial cells. Loss of these cells in LKC exposes poorly differentiated subapical cells that lack a mature glycocalyx and tight junctions. Also, disruption of tight junctions in the apical corneal epithelium has been reported to occur in response to activation of stress-related transcription factors, such as NF- κB and AP-1. Exposure of cultured bovine corneal epithelium to surfactants, such as sodium dodecyl sulfate or benzalkonium chloride, induced disruption of tight junctions and increased paracellular permeability in a time-and concentrationdependent manner. Increased DNA binding of the transcription factors NF-κB and AP-1 was also observed following treatment with these surfactants at low concentrations that typically cause mild ocular irritation [15].

Exposure of cultured human corneal epithelial cells to a pro-inflammatory stimulus, such as lipopolysaccharide, also resulted in tight junction disruption, which appeared to be mediated by altered expression or proteolytic degradation of tight junction complex proteins, such as ZO-1, ZO-2, and occludin [16]. One protease that may play a role in this process is matrix metalloproteinase 9 (MMP-9). MMP-9 knockout mice showed significantly less alteration of corneal epithelial barrier function than wild-type mice. This protective effect was abrogated by topical application of MMP-9 to the ocular surface [17]. A significant increase in the concentration and activity of MMP-9 in the tear film has been reported in human patients with LKC [2,18,19]. Hyperosmolar stress and inflammatory cytokines that are elevated in LKC (i.e., IL-1β, TNF-α, and TGF-β1) increase expression of MMP-9 by the corneal epithelium [20]. Both hyperosmolar stress and inflammatory cytokines activate NF-κB and AP-1, transcription factors that regulate development of corneal epithelial tight junctions [21]. These findings explain how the pro-inflammatory ocular surface environment in LKC may disrupt corneal epithelial barrier function.

III.CONJUNCTIVAL EPITHELIAL DISEASE

A well-recognized pathological change in conjunctival epithelial phenotype, termed squamous metaplasia, occurs in LKC (Fig. 1). Squamous metaplasia is a condition of hyperproliferation and abnormal differentiation of the conjunctival epithelium. It is associated with altered histological appearance, reduced expression of cell membrane glycoproteins (e.g., mucins), a decreased number of periodic acid-Schiff (PAS)-stained goblet cells, and altered gene expression (Fig. 2).

Squamous metaplasia of the conjunctiva is found in a variety of ocular surface inflammatory and tear film disorders. Hyperproliferation of the conjunctival epithelium is associated with conjunctival squamous metaplasia, regardless of its cause. For example, increased epithelial cell mitotic rate and decreased goblet

Figure 1 In lacrimal keratoconjunctivitis (LKC), squamous metaplasia with increased stratification and loss of goblet cells on the bulbar conjunctiva is accompanied by increased lymphocytic infiltration (predominantly CD4+ cells) of the conjunctival epithelium and stroma as well as the accessory lacrimal glands. Exfoliation of the metaplastic epithelium exposes the sensory nerves to noxious environmental stimuli.

cell density was observed in the conjunctiva of children with systemic vitamin A (retinol) deficiency. This pathological feature was observed in patients with clinical retinol deficiency (as defined by the presence of fine punctuate keratopathy), whether or not the serum retinol level was below normal (serum concentration ≤70 M) [22]. An increased conjunctival epithelial cell mitotic rate and decreased goblet cell frequency has also been observed in ocular cicatricial pemphigoid [23]. In Stevens-Johnson syndrome and in drug-induced pseudopemphigoid, DNA synthesis (measured by tritiated thymidine uptake) in the conjunctival epithelium was greater than in normal controls [24]. In patients with Sjögren’s syndrome, significantly increased epithelial stratification and increased uptake of the nucleoside analog bromo-deoxyuridine (another measure of DNA synthesis, and cell proliferation) were noted in the bulbar conjunctival epithelium [25]. Kunert and colleagues also noted an increased number of bulbar epithelial cells stained for the cell cycle-associated protein KI-67 in conjunctival

Figure 2 Impression cytology from the bulbar conjunctiva of a patient with Sjögren’s syndrome. There is complete loss of goblet cells, with abnormal mucus strands spanning the metaplastic epithelial cells.

biopsies obtained from non-Sjögren’s KCS patients, compared with normal eyes [26].

A second feature of conjunctival squamous metaplasia is a significant reduction in the number of PAS-stained conjunctival goblet cells observed in the conjunctival biopsies and impression cytology specimens taken from patients with Sjögren’s or non-Sjögren’s LKC [27–30]. The number of RNA transcripts for the goblet cell-specific mucin MUC5AC in the conjunctival epithelium of patients with Sjögren’s syndrome was also found to be significantly less than in normal individuals [31]. Furthermore, protein levels of MUC5AC were significantly reduced in the tear fluid of patients with Sjögren’s and non-Sjögren’s aqueous tear deficiency [31,32].

Alterations in mucin production and processing by the stratified conjunctival epithelium have been reported for LKC patients. A significant difference in binding patterns of membrane mucin antibody H185 to conjunctival cells was found in normal eyes compared with those of patients with dry eye symptoms. In normal eyes, this antibody bound apical epithelial cells in a mosaic pattern,

exhibiting either light, medium, or intense binding [33]. In patients with dry eye symptoms the mosaic pattern was replaced by a “starry sky” pattern in which there was a lack of apical cell binding (hence, dark sky) but increased binding to goblet cells (hence, stars in the sky). The starry sky pattern correlated with the severity of conjunctival rose bengal staining. This study concluded that an alteration in either mucin distribution or mucin glycosylation on the surfaces of apical conjunctival cells is associated with dry eye, and that glycosylation of goblet cell mucins changes with the disease. In a separate study, reduced expression and abnormal aggregates of a cell membrane mucin (termed MEM) were found to a greater extent in conjunctival cytology specimens obtained from Sjögren’s syndrome patients than from patients with other forms of dry eye [34].

Increased expression of certain genes, including transglutaminase 1, involucrin, filagrin, and the cytokeratin pair 1/10, has been detected in eyes with severe squamous metaplasia associated with Stevens-Johnson syndrome and ocular cicatricial pemphigoid [35,36].

Accelerated apoptosis of conjunctival epithelial cells has been observed in eyes of patients with KCS. Dogs who develop spontaneous KCS also exhibit increased apoptosis of conjunctival epithelial cells and decreased apoptosis of conjunctival stromal lymphocytes [37]. Pro-apoptotic markers (Fas, FasL, p53) were highly expressed in the conjunctiva of dry eye dogs, whereas levels of the anti-apoptotic marker bcl-2 were low. These phenomena reversed after treatment with the immunomodulatory agent cyclosporin A. Flow cytometry of conjunctival epithelial cells from dry eye patients showed increased levels of pro-apoptot- ic and pro-inflammatory markers compared to normal eyes, and these markers normalized following cyclosporin A therapy [38]. Experimental induction of dry eye in mice by systemic administration of anticholinergic agents and a dessicating environmental significantly increased apoptosis in ocular surface epithelial cells of the cornea and the bulbar and tarsal conjunctiva [39]. The greatest apoptosis was noted in goblet cell-rich areas of the bulbar conjunctiva. Induction of apoptosis was inhibited with topically applied cyclosporine in this experimental model [40].

IV. INFLAMMATION

The results of numerous immunopathological studies and the therapeutic response of LKC to anti-inflammatory therapies underscore the importance of inflammation in the pathogenesis of this condition. Ocular surface inflammation in LKC involves both cellular and soluble mediators. Inflammatory mediators exacerbate LKC in a number of ways: (1) by increasing the expression of adhesion molecules on conjunctival blood vessels and epithelial cells; (2) by stimulating chemotaxis of inflammatory cells, including T cells, polymorphonuclear,

and macrophages, onto the ocular surface; (3) by activating these cells once they arrive; (4) by altering epithelial proliferation and differentiation; (5) by stimulating the production and activation of proteases that disrupt cell–cell and cell–matrix adhesions; (6) by promoting apoptosis; and (7) by sensitizing ocular surface pain receptors.

An increased number of T lymphocytes and a change in their distribution in the conjunctiva have been detected in eyes with aqueous tear deficiency (Fig. 1). In 1990, Pflugfelder and colleagues observed CD3-positive T cells infiltrating the tarsal conjunctival epithelium of patients with Sjögren’s syndrome-associated KCS, but not in controls [30]. Lymphocytic infiltration of the substantia propria of the conjunctiva was also observed. In a more comprehensive study that evaluated conjunctival biopsies, a significantly increased number of T cells was found in the epithelium and substantia propia of conjunctival biopsies from both Sjögren’s syndrome (Fig. 3) and non-Sjögren’s syndrome patients with KCS, suggesting that this is a common feature of KCS regardless of cause [41]. In addition to the elevated T cell population, the proportion of T cells in the conjunctival epithelium shifted from predominantly CD8 cells (cytotoxic TKiller cells) to CD4 cells (TH cells). Increased expression of CD11a and CD23 indicated an activated phenotype of the CD4-positive T cells [42]. Treatment of these patients with topical cyclosporine decreased the number of T cells, which corresponded to an improvement in ocular surface disease and irritation symptoms.

Figure 3 CD3+ T cells in the conjunctival epithelium (arrows) and stroma in a patient with Sjögren’s syndrome LKC.

There is also evidence of immune activation in the conjunctival epithelium in KCS. Increased production of a number of pro-inflammatory cytokines in the conjunctival epithelium has been detected, including IL-1α and β, IL-6, IL-8, TGF-β1, and TNF-α [1,2,43]. Increased epithelial production of these cytokines was accompanied by increased levels of these cytokines (IL-1α and β, IL-6) in the tear fluid of Sjögren’s syndrome patients with KCS [2,44]. A significant increase in the amount of activated IL-1β and a decreased ratio of IL-1α to its physiological antagonist, IL-1 receptor antagonist (IL-1RA), was also observed [2]. Increased expression of a number of immune activation molecules, including CD54 (ICAM-1) (Fig. 4), HLA-DR, CD40, and CD40 ligand, has been found in the conjunctival epithelium of both Sjögren’s syndrome and non-Sjögren’s syndrome patients with aqueous tear deficiency [38,43,45]. These findings indicate that the ocular surface epithelial cells are direct participants in the ocular surface inflammation of LKC.

The exact mechanisms responsible for the ocular surface inflammation in LKC have not been firmly established. Desiccating environmental stress appears to be an important trigger for ocular surface inflammation. This pro-inflammato- ry stimulus may be exacerbated in certain patients with systemic autoimmune diseases (e.g., Sjögren’s syndrome) by dysfunction of their intrinsic immunoregulatory pathways and in others by age-related androgen hormone deficiency.

Figure 4 ICAM-1 mRNA expression (brown cells) in the conjunctival epithelium of a patient with non-Sjögren’s LKC.

Exposure of human corneal epithelial cells to a hyperosmolar environment both in vitro and in vivo activates intrinsic stress-related signaling pathways in a dose-dependent fashion, which then stimulate production of the same proinflammatory molecules that have been detected on the ocular surface of human patients with LKC [20,21]. Our group has found that exposure of primary human corneal epithelial cultures to increasing concentrations of sodium chloride, elevating the osmolarity of the culture media from 300 to 500 mOsm, results in activation (phosphorylation) of stress-associated protein kinases, such as p38, c-jun n-terminal kinase (JNK), and ERK 1 and 2 [21]. The activated kinases in turn activate transcriptional regulators (such as AP-1) that increase the production of inflammatory cytokines (e.g., IL-1 and TNF-α) and matrix-metallopro- teinases (MMPs). The pro-inflammatory effects of hyperosmolar stress can be inhibited by treating cells with pharmacological inhibitors of these kinases. Inflammatory mediators released from activated ocular surface epithelial cells in response to hyperosmolar stress could certainly initiate an inflammatory cascade on the ocular surface that leads to dysfunction of tear-secreting glands/cells and ocular surface disease.

Cytokines released from activated epithelial cells can trigger production of adhesion molecules by vascular endothelial and epithelial cells in the conjunctiva in a paracrine fashion. Expression of adhesion molecules together with epithelially produced chemokines (e.g., IL-8), could lead to diapedis and retention of inflammatory cells in the conjunctiva [1].

Cytokines produced by activated ocular surface epithelia in LKC may also alter epithelial proliferation, differentiation, or apoptosis, directly or indirectly. The resulting paracrine stimulation of stromal fibroblasts causes secretion of growth factors such as keratinocyte growth factor (KGF), a potent epithelial mitogen [47]. Finally, inflammatory cytokines are potent stimulators of matrixmetalloproteinase production by the ocular surface epithelial cells and infiltrating leukocytes. The pro-inflammatory cytokines IL-1α, TNF-α, and TGF-β1 all significantly increased the production of three classes of MMPs (gelatinases, collagenases, and stromelysins) by cultured human corneal epithelial cells [48–50]. The stimulatory effects of these cytokines on MMP production can be blocked with their physiological antagonists (e.g., IL-1RA in the case of IL-1β) or by neutralizing antibodies. Once activated, these MMPs can activate latent pro-inflammatory cytokines, such as IL-1β, TNF-α, and TGF-β, and neural peptides such as substance P. MMPs also degrade tight junctions in the superficial corneal epithelium and the proteins that anchor the corneal and conjunctival epithelium to their basement membrane [51–55]. It appears that interaction between pro-inflammatory cytokines and the MMPs, including inflammatory stimulation of MMP synthesis and activity, creates a vicious cycle of escalating inflammation on the ocular surface in LKC.