Adhesion Complex Formation After Small Keratectomy Wounds in the Cornea

Investigative Ophthalmology & Visual Science, Vol. 33, No. 2, February 1992 Copyright © Association for Research in Vision and Ophthalmology Adhesion Complex Formation After Small Keratectomy Wounds in the Cornea E. Lee Stock,*t Michelle A. Kurpakus4 Barbara Sambol,*t and Jonathan C. R. JonesJ The adhesion complex of the corneal epithelium consists of the hemidesmosome and its associated structures, such as anchoring filaments, lamina densa of the basement membrane, and anchoring fibrils. It contributes to the adhesion of the corneal epithelium to Bowman's layer. To understand the adhesion complex better, an electron microscopic and immunofluorescence analysis was done of the reformation of the adhesion complex in small (1 mm) keratectomy wounds in the guinea pig cornea. In these wounds, the epithelium, hemidesmosomes, basal lamina, anchoring fibrils, and anterior stroma were removed. The wound bed was epithelialized completely by 24 hr after wounding. Immunofluores- cence analyses involved the use of antibodies against plaque components of the hemidesmosome, an antibody against laminin, and an antibody against the collagen VII component of anchoring fibrils. At 18 hr after wounding, there was no morphologic evidence of hemidesmosomes at the epithelial-stromal interface. At 24 hr, hemidesmosomes were observed, with or without subjacent lamina densa. Further- more, plaque components were detected by immunofluorescence in those cells in contact with the wound bed. In contrast, no type VII collagen was detected. On day 7, collagen VII, laminin, and bullous pemphigoid autoantibody markers colocalized along the wound bed as determined by immunofluorescence. However, at the ultrastructural level, even though the lamina densa of the basal lamina was observed primarily where hemidesmosomes were present, it remained incomplete. In this study, the precise temporal sequence in which components are incorporated into the assembling adhesion complex was described during wound healing. Furthermore, the possibility that the hemidesmosomal plaque nucleates the formation of the underlying basal lamina was discussed. Invest Ophthalmol Vis Sci 33:304-313,1992 The corneal epithelium adheres to the underlying to be understood. For example, recent studies have stroma in part by the adhesion complex, consisting of revealed that two high molecular weight polypeptides a hemidesmosome, anchoring filaments that traverse (180 and 230 kD) recognized by autoantibodies in the the lamina lucida region of the basal lamina, and an- serum of patients with the blistering disease bullous choring fibrils that arise at the lamina densa of the pemphigoid (BP) are located in the plaque of the he- basal lamina and splay out into the stroma.12 On its midesmosome.3 Furthermore, both in the skin and cytoplasmic side, the hemidesmosome possesses a tri- cornea, it has been shown that the major component partite plaque.1 This plaque acts as the anchorage site of anchoring fibrils is type VII collagen.24 of bundles of intermediate filaments (IF). The bio- To study the assembly of hemidesmosomes, several chemical nature of the adhesion complex is beginning experimental systems have been used. For example, subepidermal blisters were created and hemidesmosome reformation was monitored as epithelial cells repopulated the denuded basal lamina.5 Others used From the *Cornea and External Eye Disease Laboratory, VA an in vitro system in which epithelial cell sheets were Lakeside Medical Center, and the Departments of fOphthalmology 6 and tCell Molecular and Structural Biology, Northwestern Univer- added back to denuded basal lamina. In this system, sity Medical School, Chicago, Illinois. hemidesmosome reformation is rapid, and it has been Supported in part by the Department of Veterans Affairs Re- proposed that hemidesmosome assembly is nucleated search Service (Washington, DC) grant 176-34-3874-03 (ELS) and by the preexisting anchoring fibrils associated with the National Institutes of Health (Bethesda, Maryland) grant GM- the basal lamina. 38470 to JCRJ. JCRJ is a Junior Faculty Research Awardee of the American Cancer Society (grant JFRA-232). MAK was a National In models where epithelial cells repopulate connec- Eye Institute (Bethesda, Maryland) postdoctoral fellow (grant EY- tive tissue in the absence of basal lamina, there are 06237). conflicting reports concerning the temporal sequence Submitted for publication: May 3, 1991; accepted September 17, of the reformation of the complete adhesion complex. 1991. Some authors found that hemidesmosomes, basal Reprint requests: E. Lee Stock, Cornea and External Eye Disease 7 Laboratory, Northwestern University Medical School, 303 East lamina, and anchoring fibrils appear concomitantly. Chicago Avenue, Chicago, IL 60611. Others report that the hemidesmosome plaque assem- 304 Downloaded from iovs.arvojournals.org on 09/28/2021 No. 2 ADHESION COMPLEX FORMATION IN THE CORNEA / Stock et ol 305 bles before the appearance of the lamina densa region We therefore wished to determine whether this is also of the basal lamina.8 the case in vivo. We analyzed the reformation of In previous studies, it has been shown that, in an in hemidesmosomes, anchoring fibrils, and basal lamina vitro model of wound healing, the plaque compo- in small (1 mm) keratectomy wounds in vivo in the nents of the hemidesmosome appear, in most in- guinea pig cornea by electron microscopy and immu- stances, before the appearance of collagen VII.910 Fur- nofluorescence analysis using BP autoantibodies as thermore, the lamina densa region of the basal lamina markers for the hemidesmosome plaque, a monoclo- appears to form subsequently and immediately subja- nal antibody against collagen VII, and an antibody cent to forming hemidesmosomal plaque structures.9 against laminin. Fig. 1. (A) Light micrograph of keratectomy wound immediately after wounding. (B) Light micrograph of keratectomy wound 24 hr after wounding (XI400). Downloaded from iovs.arvojournals.org on 09/28/2021 306 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / February 1992 Vol. 33 Materials and Methods Medical School (Chicago, IL). The anticollagen VII 9 Keratectomies monoclonal antibody was characterized previously. Antilaminin antibody was purchased from Telios All guinea pigs were anesthetized with xylazine HC1 (San Diego, CA). (8 mg/kg) and ketamine HC1 (120 mg/kg), in accor- dance with the ARVO Resolution on the Use of Ani- Light Microscopic Analysis mals in Research. Topical tetracaine 0,5% (four Cryostat sections were cut at 6 nm and placed on drops) was used to supplement the anesthetic. A 1- polylysine-coated (Sigma, St. Louis, MO) slides. Dou- mm Elliot trephine (Storz, St. Louis, MO) was used ble-label immunofluorescence was done as detailed for the keratotomy, and the keratectomy was com- previously.9 In brief, sections on slides were fixed for 5 pleted with a sharp blade. Chloramphenicol ophthal- min in -20°C acetone and then air dried. A mixture mic ointment (0.5%) was placed on the eye, and the of primary antibodies diluted in phosphate-buffered animals were allowed to recover. The animals were saline (PBS) was overlaid on the sections. The slides killed by an overdose of sodium pentobarbital at 18, were incubated 1 hr at 37°C, washed in PBS, and 24, and 48 hr or 7 days, and the corneas were re- overlaid with a mixture of appropriate secondary fluo- moved. The corneas were either frozen solid in liquid rochrome-conjugated antibodies. After washing in nitrogen, then embedded in O.C.T. compound PBS, cover slips were placed over sections which were (Miles, Inc., Elkhart, IN) forimmunofluorescence mi- viewed on a Leitz Diaplan microscope (Ernst Leitz, croscopic analysis, or fixed in 1.0% glutaraldehyde for Wetzlar GMBH, Germany) with epifluorescence. electron microscopy. Electron Microscopy Antibodies After fixation in 1.0% glutaraldehyde for at least 2 Serum samples from patients with BP were pro- hr, the tissue was washed six times in PBS, placed in vided by Nancy Furey, MD, Northwestern University 1% osmium tetroxide in PBS for 90 min at room tern- Fig. 2. Transmission electron micrograph of keratectomy wound at 18 hr. Note that there are no hemidesmosomes along regions of epithelial-stromal interaction or an obvious basal lamina (arrows). Intermediate filaments (IF) are present but are not associated with the basal surface of the cell (original magnification X60.000). Downloaded from iovs.arvojournals.org on 09/28/2021 No. 2 ADHESION COMPLEX FORMATION IN THE CORNEA / Srock er ol 307 perature, and rinsed in distilled water three times for 1 Results minute each. The tissue was dehydrated in graded Keratectomy wounds of 1 mm resulting in the re- ethanols, and two 15-min changes of propylene oxide moval of both epithelium and basal lamina were were completed. It then was placed in 1:1 propylene made in the cornea. At various times after wounding, oxide-Epon/Araldite resin mix (Electron Microscopy \-nm sections of Epon-embedded corneas were pro- Sciences, Fort Washington, PA) for 2 days at room cessed for light microscopic observation. Immediately temperature for infiltration. On day 2, the cap was after wounding, the normal corneal epithelium ends loosened to allow propylene oxide to evaporate from abruptly on either side of the wound site (Fig. 1A). the mixture. Final embedding was accomplished in However, within 24 hr, epithelial cells have repopu- 100% Epon/Araldite resin, and the tissue was placed lated the wound bed completely, and the cells already in a 60°C dry oven for 48 hr. are

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