Bowman's Layer Structure and Function

Bowman’s Layer Structure and Function Critical or Dispensable to Corneal Function? A Hypothesis

Steven E. Wilson, M.D., and Jong-Wook Hong, M.D.

Purpose. The purpose of this article is to review available infor- detail and delineate a hypothesis regarding the formation and mation regarding development, structure, and function of Bow- maintenance of this structure. man’s layer in the cornea. Disease-related abnormalities of Bow- man’s layer are described. A hypothesis is advanced to explain the development and maintenance of Bowman’s layer. Methods. Lit- DEVELOPMENT AND STRUCTURE OF erature review and hypothesis formulation based on previous studies. Results. Information is presented that supports the hypothesis BOWMAN’S LAYER that Bowman’s layer forms as a result of cytokine-mediated interactions occurring between corneal epithelial cells and keratocytes Bowman’s layer is classically described as an acellular conden- 1–5 that include chemotactic and apoptotic effects on the keratocytes. sation of the anterior stroma of the cornea. It is positioned This hypothesis suggests that Bowman’s layer results from such between the epithelial basement membrane and the anterior stroma interactions beginning in early development and continuing into populated with keratocytes. Bowman’s layer is first detectable adulthood in humans and other animals, such as chickens. Con- between 13 and 19 weeks of gestation in the human.2,4 Impor- clusions. Bowman’s layer may be a visible indicator of ongoing tantly, at least two investigators have noted that keratocytes are stromal-epithelial interactions in the human and have no critical present in Bowman’s layer early in development of the cornea.6,7 function in corneal physiology. Bowman’s layer is commonly de- Apple et al.8 also noted that keratocytes were retained in a cornea stroyed in diseases such as advanced bullous keratopathy where with congenital corneal opacification secondary to Bowman’s stromal-epithelial interactions may be interrupted. Bowman’s-like layers often form in response to epithelium, for example when layer dysgenesis associated with a markedly thickened Bowman’s epithelial plugs extend into the stroma in corneas with radial ker- layer. atotomy incisions. Other species, such as the chicken, also have Bowman’s Key Words: Cornea—Bowman’s layer—Keratocytes— layer.1–5 Even a species, such as the rabbit (which does not have Apoptosis—Cytokines—Chemotaxis. a Bowman’s layer in the adult), may have a Bowman’s-like acellular layer in the anterior cornea early in development.9 Some investigators have suggested that the epithelium may con- tribute to the formation of Bowman’s layer. Tisdale et al.2 have suggested that Bowman’s layer develops from a palisade of fila- What is Bowman’s layer and what is its function? These are ments that extend perpendicularly from the basal lamina into the questions that have vexed scientists and clinicians who are inter- anterior stroma beginning at approximately 13 weeks gestation.2 ested in the cornea for more than a century. The questions have These studies imply that the developing corneal epithelium may gained increasing importance with the advent of excimer laser make a major contribution to the formation of Bowman’s layer. photorefractive keratectomy. This has led to another related ques- This has also been suggested by Gordon et al.10 These investiga- tion; what effect, if any, will be noted in the estimated one mil- tors provided data indicating that smaller collagen fibrils in Bow- lion+ eyes that have had photorefractive keratectomy and are, man’s layer, when compared to the underlying stroma, were at- therefore, missing Bowman’s layer over a 5–6 mm diameter area tributable to greater collagen type V content and proposed that of central cornea? This paper will examine these questions in more production of collagen type V by the epithelium contributed to the formation of Bowman’s layer. Bowman’s layer in the human is laden with collagen fibrils that are randomly interwoven to form a dense, felt-like sheet. This contrasts with the collagen fibrils of the underlying stroma that run Submitted September 10, 1999. Revision received November 11, 1999. in alignment across the diameter of the cornea to form character- Accepted November 23, 1999. istic lamellae.5,10,11 Studies have found that the fibrils of Bow- From the Department of Ophthalmology, University of Washington 1 2 School of Medicine, Seattle, Washington, U.S.A. man’s layer are only ⁄2 to ⁄3 as thick as the fibrils that comprise 5,11 Supported in part by National Eye Institute grant EY10056. the underlying stroma. The authors have no commercial or proprietary interests in any of the The fibrils of Bowman’s layer are composed primarily of col- products discussed in this manuscript. lagen types I, III, and V.11–14 Collagen VII, associated with an- Address correspondence and reprint requests to Dr. Steven E. choring fibrils of the overlying epithelium, is also present within Wilson, Department of Ophthalmology, University of Washington School 2 of Medicine, Box 356485, Rm. RR801, HSB, Seattle, WA 98195-6485, Bowman’s layer. Little is known regarding differences between U.S.A. Bowman’s layer and the stroma in other matrix components. How-

417 418 S.E. WILSON AND J.-W. HONG ever, a recent study demonstrated that Bowman’s layer has the by unknown regulatory systems that are compromised by the onset highest concentration of beta Ig-h3-coded protein keratoepithelin of epithelial dysfunction. The factors that are derived from this when compared with other regions of the cornea.15 The function of system could modulate keratocyte localization, phenotype, and/or this matrix alteration remains uncertain. death.

DEVELOPMENTAL AND PATHOLOGIC CHEMOTACTIC BOWMAN’S ALTERATIONS TO BOWMAN’S LAYER LAYER HYPOTHESIS

Developmental anomalies of Bowman’s layer have been noted What are the regulatory systems that may function to maintain in several studies. Congenital absence of Bowman’s layer has been the characteristic morphology of the normal anterior cornea and reported in association with Peter’s anomaly,16,17 sclerocornea,18 the relationship between the epithelium and the underlying and osteogenesis imperfecta type II.19 Kasner et al.20 reported stroma? That remains to be conclusively determined. Our working bilateral congenital absence of Bowman’s layer in three patients hypothesis is that there are ongoing interactions between the epi- with normal vision and otherwise normal, clear corneas with no thelial cells and keratocytes mediated by soluble cytokines that other ocular or systemic abnormalities. maintain the normal corneal organization. We postulate that these Congenital absence of Bowman’s layer associated with hyper- cellular interactions are likely similar to those that occur during cellularity of the corneal stroma has also been noted in infants with development of the cornea when the movement and differentiation trisomy 18.21 Conversely, bilateral congenital corneal opacifica- of neural crest-derived cells are likely regulated by surrounding tion associated with dysgenesis of Bowman’s layer in an otherwise cells that include the overlying ectodermal tissue.32 normal infant has also been reported.8 In this case, both the pri- It remains to be determined which specific cytokines may per- mary corneas and a corneal graft that failed within a few months form critical functions in maintaining Bowman’s layer in the adult showed thickening of Bowman’s layer to three to four times the in vivo. However, the localization of expression of candidate cy- thickness found in normal corneas. In addition, there were kera- tokines and receptors, as well as the in vitro and in vivo effects of tocytes within Bowman’s layer of each of these corneas and as- these cytokines on keratocytes, suggest mechanisms through sociated irregular bundles of collagen produced by these ectopic which regulation could occur. cells. Fetal alcohol syndrome has also been reported to result in The first indication that there could be cytokines derived from varying anomalies of Bowman’s layer. These alterations extended the corneal epithelium that modulated keratocyte location was ob- from complete absence to thickening associated with corneal stro- tained from in vivo experiments evaluating the effect of interleukin mal edema.22 (IL)-1 on keratocyte apoptosis.29 In these studies, mouse IL-1 was Acquired abnormalities of Bowman’s layer can be associated injected into the central corneal stroma of the mouse cornea using with disease. The most commonly acquired abnormality is break- a microinjection system. Keratocytes at the site of injection un- age or disruption of Bowman’s layer associated with the ectasia of derwent apoptosis. However, keratocytes that were farther re- keratoconus.23–26 It is unclear whether these changes are associ- moved from the injection site appeared to be affected differently ated with the underlying pathophysiology of the disease or merely by the exogenous IL-1. These keratocytes were redistributed with secondary changes. Breakage or disruption of Bowman’s within the stroma such that the cells in the stroma anterior to the layer has also been associated with Salzmann’s nodular degenera- injection site appeared to form a line parallel to, but separated tion.27 Stromal haze at the level of Bowman’s layer has been noted from, the overlying epithelium. It was as though the keratocytes in Ehlers–Danlos syndrome.28 had redistributed to a point of equilibrium between the injected An interesting and potentially revealing morphologic change in IL-1 and the overlying corneal epithelium.32 These studies sug- Bowman’s layer is associated with advanced bullous keratopa- gested that IL-1 had a negative or repulsive chemotactic effect on thy29,30 and Fuchs”s corneal dystrophy.31 Stromal edema is typical the keratocyte cells. The negative chemotactic effect of IL-1 on of the early stages of either disease. Edema occurs as corneal keratocytes was subsequently confirmed in vitro.33 Previous stud- endothelial compromise becomes manifest in either disease. Bow- ies demonstrated that IL-1 alpha34 and IL-1 beta35 are expressed at man’s layer remains normal in the early stages of either disorder. high levels in the epithelium, but not the keratocytes, in the un- As the disease advances, however, the epithelium becomes in- wounded cornea. creasingly edematous and dysfunctional. As the later stages of These findings suggested that epithelial IL-1 had a negative these diseases progress, changes are frequently noted in Bowman’s (repulsive) chemotactic effect on keratocytes in vivo. We also layer. The earliest finding is that keratocytes no longer respect showed that platelet-derived growth factor (PDGF) was expressed their normal boundaries and enter into Bowman’s layer. Kerato- at high levels in the corneal epithelium and deposited in the base- cytes with the appearance of wound-associated fibroblasts even- ment membrane of the epithelium and confirmed that PDGF has a tually advance to the immediate subepithelial region. Subepithelial positive (attractive) chemotactic effect on keratocytes.33 The im- fibrous pannus is a very late finding in either bullous keratopa- portance of the PDGF system in organization of the cornea during thy29,30 or Fuchs’ corneal dystrophy.31 This pannus is populated development was confirmed by the finding of corneal dysgenesis by fibroblast-like cells that are probably derived from keratocytes. in Patch mice that have a deletion of the gene encoding the plate- Subepithelial fibrous pannus is also commonly vascularized. In let-derived growth factor receptor alpha subunit.36 some cases, the keratocyte-derived cells enter into the epithelium Thus, keratocytes in the anterior stroma are likely influenced by and form partitions within the epithelial layer. Eventually, Bow- different cytokines derived from the epithelium that have attractive man’s layer is completely destroyed.29,30 These changes suggest or repulsive influences on these cells. If this is the case, then the that Bowman’s layer may be actively maintained during adult life furthest anterior position they assume within the stroma relative to

Cornea, Vol. 19, No. 4, 2000 BOWMAN’S LAYER STRUCTURE AND FUNCTION 419 the overlying epithelium may be determined by the balance be- infect the corneal epithelium. Although not a physical barrier to tween the opposing chemotactic effects of these cytokine systems. virus penetration into the posterior cornea, this acellular layer If a keratocyte moves too close to the overlying epithelium despite could serve as a biological barrier to extension since these viruses negative chemotactic effects, then high concentrations of cytokines require cells for propagation and spread. A recent report suggested such as IL-1 and Fas ligand could eliminate them by triggering that epidemic keratoconjunctivitis or response to toxic injury may apoptosis.29,37 be worse in eyes that have had photorefractive keratectomy.39,40 There is evidence that the epithelium has a critical role in for- However, there is no convincing data in large numbers of patients mation and maintenance of the acellularity of Bowman’s layer. demonstrating this is the case. Each of these anecdotal reports Radial keratotomy is performed by making nearly full-thickness could represent unusually severe responses that may also have radial incisions into the corneal epithelium. Epithelial tissue plugs occurred in eyes with Bowman’s layer. may be retained indefinitely within the stromal incisions following It is clear that any hypothesis that suggests a critical function for healing of the radial incisions. Melles et al.38 demonstrated that a Bowman’s layer will need to account for the apparent lack of Bowman’s-like layer frequently develops around this ectopic ep- significant complications in hundreds of thousands of eyes that are ithelial tissue 5–10 years after radial keratotomy. It will be of devoid of Bowman’s layer over the central cornea following pho- considerable importance to determine whether a similar Bowman’s torefractive keratectomy. Almost 12 years has elapsed since the like layer develops years following photorefractive keratectomy. first photorefractive keratectomy procedures were performed in The Bowman’s-like layer that develops in this situation may only humans. As further time passes, it becomes less and less likely that be grossly similar to the native Bowman’s layer that formed in Bowman’s layer serves some critical role in corneal structure or utero, because the cytokine-mediated effects in the adult may be function. different than those in the embryo. 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Editor search: The Castroviejo Cornea Society is seeking applications for the position of Editor- in-Chief of the journal Cornea to succeed Dr. Mannis whose term ends at the end of 2001. The editor must have clinical and research expertise in the area of cornea and external ocular disease. Submit applications to Joel Sugar, M.D., Search Committee Chair, 1855 W. Taylor, Chicago, IL 60612, U.S.A. Applications with CV should be received before October 1, 2000.

Cornea, Vol. 19, No. 4, 2000