Anchoring Fibrils Form O Complex Network in Human and Rabbit Cornea

Home , Anchoring fibrils, Basal lamina, Hemidesmosome, Type IV collagen

Anchoring Fibrils Form o Complex Network in Human and Rabbit Cornea

llene K. Gipson, Sandra J. Spurr-Michaud, and Ann 5. Tisdale

Anchoring fibril distribution, depth of penetration into the stroma, and pattern of histochemical localization of type VII collagen (the anchoring fibril collagen) were studied in normal human and rabbit corneas. Electron micrographs of cross sections and sections taken parallel to the basement membrane demonstrate that anchoring fibrils insert into the basal lamina and then splay out laterally. They are more readily seen in sections taken parallel to the basal lamina, where they are observed to form a complex branching and anastomosing network below the basal lamina. Distal to the basal lamina, anchoring fibrils appear to insert into patches of dense extracellular matrix termed "anchoring plaques." Average depth of penetration of anchoring fibrils into stroma is 0.60 and 0.54 urn for human and rabbit, respectively. Monoclonal antibodies to type VII collagen localize only to the basement membrane-anchoring fibril zone of both human and rabbit corneas. No obvious differences in anchoring fibril structure or distribution were observed between human corneas, which have a Bowman's layer, and rabbit corneas, which do not. Invest Ophthalmol Vis Sci 28:212-220, 1987

Anchoring fibrils are uniquely cross-banded fibrils phica in which epidermal blisters form due to the ab- present on the connective tissue side of the basal lamina sence of the fibrils (for reviews, see references 4 and 5). of all stratified squamous epithelia. They are a com- Other stratified squamous epithelia in which an- ponent of the adhesion complex required for tight ad- choring fibrils have been studied include oral mucosa,6 herence of these epithelia, which are subject to abrasion. ectocervix,7 and cornea.8"10 Jakus in 1962" described The adhesion complex consists of the hemidesmosome, "fine-filaments" that extend from protuberances of which is the adhesion junction joining the basal cell the corneal epithelial basement membrane. Later membrane to the basal lamina. The hemidesmosome reports1213 described accumulations of short fibrous junction is connected in an as yet undefined manner material in pockets of duplicated basement membrane through the basal lamina to another portion of the in Meesmann's corneal dystrophy. Kenyon and complex, the anchoring fibril network. This network Maumenee14 ascribed the term "anchoring fibrils" to splays out into the subjacent connective tissue strata. crossbanded fibers in the central cornea of a case of 15 The structure and distribution of anchoring fibrils congenital corneal dystrophy. Subsequently, Kenyon 9 (formerly termed Special Fibrils) have been most thor- and Alvarado et al described accumulations of an- oughly studied at the epidermis-dermis junction.1"3 choring fibrils in duplicated epithelial basal lamina of Studies of amphibian skin demonstrate that the fibrils diabetic and aged humans, respectively. have a symmetrical transverse band pattern, are ~750 A study using an in-vitro corneal system indicated nm long and attach at both free ends to the undulating that new hemidesmosomes form over specific sites on basement membrane of the epidermis.3 Evidence that basal laminae where anchoring fibrils insert from the these fibrils function in holding the overlying epithe- stromal side.10 This site may be a "nucleation" site for lium and basement membrane to the dermis comes hemidesmosome reformation as basal cells of stratified from studies of forms of epidermolysis bullosa dystro- epithelium divide and constantly change positions in relation to the basal lamina. It is not known whether the anchoring fibril itself passes through the basal lamina to form the nucleation site or whether there are From the Eye Research Institute of Retina Foundation and the Department of Ophthalmology, Harvard Medical School, Boston, linking elements between anchoring fibrils and hemi- Massachusetts. desmosomes. Supported in part by research grant R01 EY03306 from the Na- Bentz et al16 reported isolation of a new type of col- tional Eye Institute, National Institutes of Health, Bethesda, Mary- lagen, designated type VII, purified from human am- land. nion. Immunohistochemical and immunoelectron Submitted for publication: June 11, 1986. Reprint requests: llene K. Gipson, PhD, Eye Research Institute, microscopy studies demonstrated that antibodies to 20 Staniford Street, Boston, MA 02114. type VII collagen localize to epidermal anchoring fi-

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brils.17 Type VII collagen seems to have a tripartite Immunofluorescence globular nonhelical head region at the carboxyl ter- Normal rabbit and human corneas were frozen in minus of the filamentous triple helix portion of the Tissue Tek II O.C.T. Compound (Lab Tek Products; molecule.18 Triple helices of individual molecules as- Naperville, IL). Six-micrometer cryostat sections were sociate to form the cross-banded fibril, and globular placed on gelatin-coated slides and air-dried overnight domains associate in the basal lamina and in patches at 37 C. The sections were rehydrated in phosphate- of electron-dense extracellular matrix (anchoring buffered saline (PBS), pH 7.2, and washed in PBS with plaques) located in the connective tissue region sub- 1% bovine serum albumin (BSA) for 10 min. The pri- jacent to the basal lamina.19 mary antibody (mouse anti-human type VII collagen, Despite the reports regarding the accumulation of clone NP76 or 185) was then applied for 1 hr in a anchoring fibrils in corneal pathologies and in aged moist chamber. The slides were rinsed with PBS fol- humans, little morphological and morphometric data lowed by 10 min in PBS with 1% BSA. The secondary are available on the distribution of anchoring fibrils in antibody (fluorescein isothiocyanate [FITC]-conju- the normal human cornea. We report studies of the gated goat anti-mouse IgG; Cooper Biomedical Inc.; distribution and depth of penetration of anchoring fi- Malvern, PA) was applied for 1 hr in a moist chamber. brils in normal human corneas as well as in rabbit cor- After a PBS wash, coverslips were mounted with a me- neas, which do not have a Bowman's layer. In addition, dium consisting of PBS, glycerol, and paraphenylene- we report the pattern of immunohistochemical local- diamine.21 Negative control tissue sections (primary ization of monoclonal antibodies to type VII collagen antibody omitted) were routinely run with every an- in the epithelial basement membrane zone of both spe- tibody-binding study. The sections were viewed and cies. photographed using a Zeiss photomicroscope III equipped for epi-illumination. The primary antibodies were the kind gift of Dr. Robert E. Burgeson (Shriners Materials and Methods Crippled Children's Hospital, Portland, OR), who has verified their specificity.18 The antibodies recognize the All investigations involving animals reported in this nonhelical globular domain of the type VII collagen study conform to the ARVO Resolution on the Use molecule. of Animals in Research. Normal human donor corneas and corneas from adult (2.5 kg) New Zealand White rabbits were used. The rabbits were killed with an in- Results travenous overdose of sodium pentobarbital. Anchoring fibril structure and distribution is difficult to discern in corneas fixed immediately after dissection Transmission Electron Microscopy in routinely used glutaraldehyde-paraformaldehyde Donor human corneas (N = 9) and excised rabbit fixatives (i.e., half-strength Karnovsky's fixative). We corneas (N = 7) were fixed in either half-strength Kar- noted in previous studies that anchoring fibrils were novsky's fixative (2% paraformaldehyde, 2.5% glutar- more discernible in corneal tissue that had been organ- aldehyde in 0.1 M cacodylate buffer, pH 7.4) or 3.75% cultured for 24 hr prior to fixation in half-strength 10 osmium tetroxide (OsO4) in 0.2 M s-collidine buffer, Karnovsky's fixative. Also, trials of various fixatives pH 7.4. In addition, 3 human stromas and 43 rabbit demonstrated that anchoring fibrils were more dis- corneas were incubated 1-24 hr in a completely defined cernible after fixation with osmium tetroxide in colli- organ culture medium20 prior to fixation in half- dine buffer.22 All electron micrographs in Figures 1 to strength Karnovsky's fixative. 4 are sections of tissues processed after 24 hr culture or after osmium fixation. Morphometric Analysis Anchoring Fibril Ultrastructure and Morphometry A Zeiss Videoplan Image Analysis System (Rainin Instruments; Woburn, MA) was used to measure the In sections of cornea taken perpendicular to the basal depth of penetration into the stroma of the five longest lamina, insertion of anchoring fibrils into the basal anchoring fibrils on each of 8-10 electron micrographs lamina can be seen (Figs. 1, 2). At the insertion site, per cornea (N = 9 for humans, N = 50 for rabbits). the fibril has several filamentous branches (Fig. 1, lower The anchoring fibrils were measured perpendicular to inset). These branches unite distal from the basal lam- the basal lamina from their deepest point in the stroma ina insertion to form the cross-banded fibril with a to their insertion into the lamina densa. The mean and banding repeat characteristic of these fibrils. The cross- standard error of the mean were then computed for banded anchoring fibrils extend into the anterior each group of corneas. stroma, splaying out among the collagen fibrils. Some

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Fig. 1. Electron micrographs of cross-sections of rabbit cornea demonstrating anchoring fibrils (AF) in zone below basal lamina. Large micrograph: Anchoring fibril (large arrows) insertion into basal lamina is particularly evident at site along basal lamina opposite hemidesmosomes (HD). Insertion of anchoring fibrils distalty into electron-dense patches, recently termed anchoring plaques, is indicated by small arrows (X73,9OO). Upper inset: An anchoring fibril that inserts at each end into basal lamina. Such anchoring fibrils are seen less frequently (X73.900). Lower inset: Higher magnification of cross-banded anchoring fibril. The anchoring fibril splays out at insertion into basal lamina at top of micrograph. Anchoring plaque at bottom is site of distal insertion of anchoring fibril (X93,OOO). Tissue cultured 24 hr prior to fixation in half-strength Karnovsky's fixative.

of the fibrils insert distally from the basal lamina in the corneal stroma of human and rabbit were 0.54 patches of amorphous extracellular matrix (anchoring ±0.01 and 0.60 ± 0.01 /im, respectively. Maximum plaques), which have the appearance of isolated bits of penetrations measured were 2.05 fim for human cor- basal lamina (Figs. 1, 2). These patches, as demon- neas and 2.69 nm for rabbit. Anchoring fibrils were strated by antibody localization, represent the accu- most discernible in sections of both human and rabbit mulated globular head regions of the type VII collagen corneas taken tangential to the basal lamina (Figs. 3, molecule plus type IV collagen (Keene et al, unpub- 4). These sections demonstrate an elaborate intercon- lished data). necting network of anchoring fibrils in this plane. The We could not identify anchoring fibrils in cross sec- filamentous regions of anchoring fibrils appear to tion. The width of anchoring fibrils is variable in lon- branch and anastomose, forming the complex inter- I gitudinal section; anchoring fibrils with widths up to woven network. 0.15 /urn have been observed. If the anchoring fibrils The networks appear equally complex in human and of this width were round, they should be visible in cross rabbit corneas. By electron microscopy we could dissection. We could not, however, identify such shapes cern no significant difference of anchoring fibril pen- in cross section. etration and distribution in the human corneas, which Morphometric analyses demonstrate that average have a Bowman's layer, and in rabbit corneas, which i maximum depth of penetration of anchoring fibrils into do not. Basement membranes of human corneas do,

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Fig. 2. Cross sections of human (A) and rabbit (B) corneas through zone of anchoring fibrils. Most anchoring fibrils appear directly adjacent to the basal lamina; an occasional fibril penetrates deeply into stroma (B, large arrow). Anchoring plaques at distal insertion of anchoring fibril are indicated by small arrows. Tissue incubated 24 hr prior to fixation (A, X31,200; B, X25.000).

however, appear to undulate as compared with the flat choring fibril network at the basement membrane zone basal lamina of rabbits (see Fig. 2). of the corneal epithelium (Fig. 5). The localization pattern in human and rabbit appeared similar, but we did Type VII Collagen Localization see variation in intensity of localization in human eyes (Fig. 5 A, B). The pattern of localization on a 9-month- Monoclonal antibodies, which localize to the tri- old human eye is shown in Figure 5B. The region of partite globular nonhelical head region of the type VII and below the basal lamina fluoresces intensely with a collagen molecule, localized to the region of the an- diminution of intensity toward the stromal side. At the

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Fig. 3. Sections of 9-month-old human cornea taken tangential (parallel) to the basal lamina. The basal lamina in this cornea was undulating, not flat. Thus, sections through tips of basal cells are present. A: Branching and anastomosing networks of anchoring fibrils are visible in this plane of section (arrows) (X42.000). B: Higher magnification of region of anchoring fibril network demonstrating insertion of fibrils into basal lamina (arrows) (X93,000). Tissue fixed in osmium collidine fixative.

interface of the stroma with the anchoring fibril net- amorphous extracellular matrix (anchoring plaques) work, discrete small patches of localization are obvi- in which the anchoring fibrils appear to insert ous. These patches may correlate to the patches of (Figs. 1,3).

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Fig. 4. Electron micrographs of sections of rabbit cornea taken tangential (parallel) to basal lamina (BL). A: Low magnification demonstrating the density of anchoring fibril network in this plane (X31,200). B: Higher magnification micrograph of anchoring fibril network. Branching insertion of anchoring fibril into basal lamina is evident at arrow. Branching and anastomosing of fibrils is very obvious in this plane of section (XI 64,400). Tissue was cultured 24 hr prior to fixation.

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Fig. 5. Light micrographs demonstrating immunofluorescent localization of monoclonal antibodies to nonhelical region of the type VII collagen molecule on cryostat sections of human and rabbit corneas. A: Human (age 54) cornea. Binding of antibody appears as a thin band along basement membrane zone only. Although not visible, the epithelium is above the fluorescent band (X600). B: Nine-month-old human cornea. Localization in this cornea was particularly intense and the zone of localization was wide. Localization pattern appears as parallel strands of beads extending into stroma. Electron micrographs in Figure 3 are from this cornea (X600). C: Adult rabbit cornea. Binding of antibody is intense at region near basal lamina. Beaded strands reach into stroma (X600).

Fig. 6. Schematic diagram of anchoring fibril network in & zone below the basal lamina. At top, branching insertions of anchoring fibrils (arrows) into basal lamina below 1 hemides mosomes (HD) are depicted. Branching and anastomosing cross-banded fibrils splay out among type 4 1 collagen fibrils, and some anchoring fibrils insert into dense patches of extracellular matrix which are composed of nonhelical domains of type VII collagen molecules and other basal lamina components. These patches have been termed anchoring plaques (AP).

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Discussion aberrant in these diseases (for review, see references 4 and 5). Whether the anchoring fibril absence is the re- The depth of penetration and the elaborate inter- sult of genetic deletion or enzyme degradation is un- connecting network of the anchoring fibrils in the an- known. The epidermal epithelium (and all other squa- terior cornea have not been previously recognized. mous epithelium) with its basal lamina easily splits Conventional fixation and embedding procedures from underlying connective tissue. probably obscured the network. Glutaraldehyde and A second example indicating the importance of an- paraformaldehyde fixatives plus osmium postfixation choring fibrils in adherence of basal lamina to stroma may fix too well. That is, all soluble components sur- comes from studies of diabetic coraeal epithelium. rounding the network may be cross-linked and fixed Kenyon15 reported that when diabetic corneal epithe- in place, making the network difficult to differentiate. lium that has become opaque during vitrectomy sur- These soluble components may be eliminated by gery is scraped off, the duplicated basal lamina remains preincubation in culture medium, and the procedure with the scraped epithelium. In contrast, when normal for osmium fixation in collidine buffer may not fix all epithelium is removed by scraping, the basal lamina components surrounding the network. One could argue remains on the stroma. Measurements of depth of that preincubation distorts the anchoring fibril network penetration of anchoring fibrils from the deepest layer and that depth of penetration measurements are thus of duplicated basal lamina demonstrate that in diabetics inaccurate. Measurement and distribution patterns do depth of penetration is significantly less than in normal correlate, however, between preincubated tissues and corneas.23 Basement membrane reduplication, as ob- osmium-fixed specimens. served with aging9 or in diabetes,15 may interfere with The observation that the anchoring fibril network the preservation of the relationship between anchoring in cornea is more apparent in tangential section would fibrils and the stroma, setting up the stage for a loose indicate that a majority of fibrils run in a plane parallel adhesion as shown by Kenyon.15 to the basal lamina. However, we did not observe fre- Finally, in-vitro experiments designed to study con- quent examples of anchoring fibrils with both ends in- ditions for hemidesmosome formation by corneal ep- serting into the basal lamina. These looping anchoring ithelium provide evidence that the site on the basement fibrils are more prevalent in the epidermis.3 Perhaps membrane where hemidesmosomes form on the basal such looping anchoring fibrils are more common to cell membrane is where anchoring fibrils insert from squamous epithelium with undulating rather than flat the stromal surface.10 These studies indicate that the basal lamina. We were unable to recognize anchoring anchoring fibril network is linked through the basal fibrils in cross section. It may be that anchoring fibrils lamina (directly to the cell or indirectly through linkage are ribbon-like, with their widest dimension in the cen- molecules) to cell membrane adhesion junctions ter of the fibril. Ribbons in cross section could appear (hemidesmosomes). This association between hemi- as short segments of filaments or fibers. desmosomes and anchoring fibrils, previously noted Anchoring fibrils insert into dense bodies or patches by Susi et al,6 provides continuity of adhesion from of extracellular matrix, distal to their insertion into the the cell through the basal lamina and into the connec- basal lamina. Other anchoring fibrils deeper in the tive tissue. stroma extend between these patches of matrix. Keene In summary, a complex anchoring fibril network is et al19 have recently applied the term "anchoring present in the region of the coraeal stroma just beneath plaque" to these structures. The plaques appear to be the epithelial basement membrane. The fibrils insert the "knots or pivot points" in the three-dimensional into the basal lamina and distally into patches of ex- network of anchoring fibrils. The plaques are appar- tracellular matrix (anchoring plaques). Monoclonal ently accumulations of the globular domain of type antibodies to type VII collagen localize to the region VII molecules in combination with the basal lamina occupied by the network. A diagram depicting our collagen, type IV. Because these plaques have the ap- concept of the distribution of the anchoring fibril net- pearance of bits of basal lamina, other basal lamina work is shown in Figure 6. components may be present as well. The role that anchoring fibrils and the network they Key words: anchoring fibrils, type VII collagen, hemides- form have in adhesion of stratified squamous epithelia mosomes, corneal epithelial adhesion, cell-substrate adhesion, to underlying connective tissue is becoming increas- basement membrane, rabbit cornea, human cornea ingly clear. Three examples give insight into the function of the fibrils; in two, epithelial adhesion is faulty. References Epidermolysis bullosa is a group of diseases char- 1. Brody I: The ultrastructure of the tonofibrils in the keratinization acterized by epithelial blistering. In some instances, process of normal human epidermis. J Ultrastruct Res 4:264, the cornea is involved. Anchoring fibrils are absent or 1960.

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2. Palade GE and Farquhar MG: A special fibril of the dermis. J 14. Kenyon KR and Maumenee AE: The histological and ultra- Cell Biol 27:215, 1965. structural pathology of congenital hereditary corneal dystrophy: 3. Bruns RR: A symmetrical extracellular fibril. J Cell Biol 42:418, a case report. Invest Ophthalmol 7:475, 1968. 1969. 15. Kenyon KR: Recurrent corneal erosion: pathogenesis and ther- 4. Briggaman RA: Is there any specificity to defects of anchoring apy. Int Ophthalmol Clin 19(2): 169, 1979. fibrils in epidermolysis bullosa dystrophica, and what does this 16. Bentz H, Morris NP, Murray LW, Sakai LY, HoIIister DW, and mean in terms of pathogenesis?J Invest Dermatol 84:371, 1985. Burgeson RE: Isolation and parital characterization of a new 5. Tidman MJ and Eady RAJ: Evaluation of anchoring fibrils and human collagen with an extended triple-helical structural domain. other components of the dermal-epidermal junction in dystrophic Proc Natl Acad Sci USA 80:3168, 1983. epidermolysis bullosa by a quantitative ultrastructural technique. 17. Sakai LY, Keene DR, Moms NP, and Burgeson RE: Type VII J Invest Dermatol 84:374, 1985. collagen is a major structural component of anchoring fibrils. J 6. Susi FR, Belt WD, and Kelly JW: Fine structure of fibrillar com- Cell Biol 103:1577, 1986. plexes associated with the basement membrane in human oral 18. Lunstrum GP, Sakai LY, Keene DR, Morris NP, and Burgeson mucosa. J Cell Biol 34:686, 1967. RE: Large complex globular domains of type VII procollagen 7. Younes MS, Steele HD, Robertson EM, and Bencosme SA: Cor- contribute to the structure of anchoring fibrils. J Biol Chem 261: relative light and electron microscope study of the basement 9042, 1986. membrane of the human ectocervix. Am J Obstet Gynecol 92: 19. Keene DR, Sakai LY, Lunstrum GP, Morris NP, and Burgeson 163, 1965. RE: Type VII collagen forms an extended network of anchoring 8. Fogle JA, Kenyon KR, Stark WJ, and Green WR: Defective fibrils. J Cell Biol (in press). epithelial adhesion in anterior corneal dystrophies. Am J Ophthalmol 79:925, 1975. 20. Gipson IK and Anderson RA: Effect of lectins on migration of 9. Alvarado J, Murphy C, and Juster R: Age-related changes in the the corneal epithelium. Invest Ophthalmol Vis Sci 19:341, 1980. basement membrane of the human corneal epithelium. Invest 21. Huff JC, Weston WL, and Wanda KD: Enhancement of specific Ophthalmol Vis Sci 24:1015, 1983. immunofluorescent findings with use of a para-phenylene di- 10. Gipson IK, Grill SM, Spurr SJ, and Brennan SJ: Hemidesmosome amine mounting buffer. J Invest Dermatol 78:449, 1982. formation in vitro. J Cell Biol 97:849, 1983. 22. Kelley DE: Fine structure of desmosomes, hemidesmosomes and 11. Jakus M: Further observations on the fine structure of the cornea. an adepidermal globular layer in developing newt epidermis. J Invest Ophthalmol 1:202, 1962. Cell Biol 28:51, 1966. 12. Kuwabara T and Ciccarelli EC: Meesmann's corneal dystrophy. 23. Gipson IK, Spurr SJ, Kimerer LM, and Tisdale AS: Anchoring Arch Ophthalmol 71:676, 1964. fibrils of the cornea: comparisons between normal and diabetic 13. Burns RP: Meesman's corneal dystrophy. Trans Am Ophthalmol human and rabbit. ARVO Abstracts. Invest Ophthalmol Vis Sci Soc 66:530, 1968. 26(Suppl):93, 1985.

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