Homeostasis of ocular surface epithelium in the rat is regulated by opioid growth factor

Brain Research 759 Ž1997. 92–102

Research report

Homeostasis of ocular surface epithelium in the rat is regulated by opioid growth factor Ian S. Zagon a

a, )

, Joseph W. Sassani

b,c

, Edward R. Kane c , Patricia J. McLaughlin

a

Department of Neuroscience and Anatomy, PennsylÕania State UniÕersity, Milton S. Hershey Medical Center, 500 UniÕersity DriÕe, Hershey, PA 17033, USA b Department of Ophthalmology, PennsylÕania State UniÕersity, Milton S. Hershey Medical Center, 500 UniÕersity DriÕe, Hershey, PA 17033, USA c Department of Pathology, PennsylÕania State UniÕersity, Milton S. Hershey Medical Center, 500 UniÕersity DriÕe, Hershey, PA 17033, USA Accepted 4 February 1997

Abstract Endogenous opioid peptides serve as growth factors in developing, renewing, and neoplastic cells and tissues. This study examined the hypothesis that opioids serve to modulate the homeostatic renewal of ocular surface epithelium in the rat. DNA synthesis in the epithelium of the central ŽCC. and peripheral ŽPC. cornea, limbus ŽLM., and conjunctiva ŽCN. was investigated using adult male rats. Animals received an injection of opioid growth factor ŽOGF., wMet 5 x-enkephalin, OGF and naloxone ŽNAL., NAL alone, naltrexone ŽNTX., or an equivalent volume of sterile water ŽCO. and sacrificed 4 h later Ži.e. 16:00 h.. w 3 Hxthymidine was administered 1 h before sacrifice. With the exception of NTX Ž20 mgrkg., all compounds were given at 10 mgrkg. Examination of 5 time points over an 18-h period revealed no variation in DNA synthesis within a region of ocular surface basal epithelium ŽBE.. OGF depressed DNA synthesis of the BE by 25, 48, and 50% in the PC, LM, and CN, respectively; little labeling was recorded in the BE of the CC. Exposure to OGF-NAL or NAL alone did not alter DNA synthesis of the BE. Complete blockade of OGF-z receptor interaction by administration of the potent opioid antagonist, NTX, increased the number of epithelial cells in the PC, LM, and CN undergoing DNA synthesis by 30 to 72%. The effects of OGF and NTX on DNA synthesis of BE also were observed in an organ culture setting. Utilizing immunocytochemistry, OGF and its receptor z were associated with both the basal and the suprabasal cells of the ocular surface epithelium. These results indicate that an endogenous opioid peptide, OGF, and its receptor are present and govern homeostatic cellular renewal processes in ocular surface epithelium. OGF regulates DNA synthesis in a direct manner, and does so by a tonic, inhibitory, and receptor-mediated mechanism. q 1997 Elsevier Science B.V. Keywords: Corneal epithelium; Limbal epithelium; Conjunctival epithelium; Proliferation; Opioid; Naltrexone; wMet 5 x-enkephalin; Cell renewal; Eye

1. Introduction The ocular surface is covered by a non-keratinizing squamous epithelium that plays an important role in maintaining the structural and functional integrity of the cornea w17x. The corneal epithelium is contiguous with the conjunctival epithelium at the limbus. At this transition, a change in the ocular surface contour can be noted, and the regular arrangement of corneal stromal lamellae changes to the irregular collagen bundles of the sclera. The conjunctiva is marked by goblet cells interspersed among the epithelial cells, and by a vascularized and loosely arranged subepithelial connective tissue. In primates, the corneal

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Corresponding author. Fax: q1 Ž717. 531-5003.

0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 2 3 8 - 2

epithelium covers Bowman’s membrane. The corneal epithelium is thought to be derived from the limbal epithelium, but can be replaced by the conjunctival epithelium when necessary; however, the lineages of the limbal and conjunctival epithelium appear to be different w7,19,22,27,34,41,42x. Little is known about the regulation of homeostasis in ocular surface epithelium. Studies have monitored mitosis w2 ,3 ,5 ,1 3 ,1 8 ,2 8 ,3 0 x a n d D N A s y n th e s is w4,7,9,12,14,15,21,29,40 x of the corneal epithelial cells, and some controversy exists as to whether a daily rhythm with respect to cellular generation can be observed w3,4,13,21,24,28–30x. Depending the region examined, epithelial cells of the ocular surface may have distinct characteristics. These include their ability to replicate, migrate, andror differentiate in tissue culture w6,10,11,20,22,42x,

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behavior when transplanted into nude mice w41x, and biochemical attributes w16,27,43x. Various growth factors have been demonstrated to affect ocular surface epithelium in tissue culture and during wound healing w26,31,32,37,38,44x. The role of these growth factors in the homeostatic renewal and maintenance of epithelial cells has received little attention. In at least two instances, growth factors involved in wound healing do not influence the homeostasis of ocular surface epithelium w26,32x. Endogenous opioids and receptors were first thought to be related to neurotransmission and neuromodulation w1x, but it is now clear that opioid peptides act as growth regulators in cells and tissues during normal development, cellular renewal, malignancy, and wound healing w23,25,33,35,36,39,45–51 x. An opioid growth factor ŽOGF., the naturally occurring pentapeptide wMet 5 x-enkephalin, has been identified in both eukaryotes and prokaryotes as a potent inhibitor of cell replication. In vertebrates, OGF also is thought to be important in cellular maturation, migration, and survival, and has been reported to be produced by autocrine and probably paracrine mechanisms. OGF activity is mediated by the z-opioid receptor w46x. The function Ži.e. growth., distribution Ži.e. neural and non-neural., transient appearance during ontogeny, ligand specificity Ži.e. wMet 5 x-enkephalin., competitive inhibition profile, subcellular location Ži.e. nucleus., and the fact that ligands for other known receptors Že.g. m , d , and k . do not influence growth have provided a unique set of characteristics that distinguish the z receptor from other opioid receptors. Recently, OGF and the z receptor have been reported to be present and function in the corneal epithelium w48,49x. Immunocytochemical studies show that both the peptide and receptor are present in a wide variety of classes of the phylum Chordata, including mammals, birds, reptiles, amphibians, and fish w48x. Both the basal and the suprabasal layers of the human corneal epithelium contain OGF and the z receptor. Investigations with rabbit corneal explants reveal that OGF acts on the z receptor to tonically inhibit cell division w49x. It also appears that cell migration and the architectural organization of corneal epithelial outgrowths from explants are modulated by OGF, suggesting that this opioid peptide is a determinant of cell position and interaction. Given the Ž1. influence of opioids on cellular renewal of non-corneal epithelial structures w50,51x, Ž2. ability of OGF to modulate cellular events in outgrowths of corneal epithelial cells w49x, and Ž3. presence of OGF and the z receptor in cells of the corneal epithelium w48,49x, we hypothesized that the generation of ocular surface epithelial cells during homeostasis is dependent on the regulatory capabilities of opioid peptides. In the present study, we have examined this thesis in the cornea, limbus, and conjunctiva of the adult rat using two experimental

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paradigms: Ž1. blockade of opioid-receptor interaction by a potent and long-acting opioid antagonist Žnaltrexone, NTX.; and Ž2. analysis of the effects of the opioid agonist, OGF. The results of disrupting opioid–receptor interaction by exposure to either an opioid antagonist ŽNTX. or opioid agonist ŽOGF. also were analyzed in an organ culture environment in order to determine whether the action of opioids on the homeostasis of ocular surface epithelium was direct or indirect. Finally, to further assess whether opioids play a role in the biology of epithelial cells of the cornea, limbus, and conjunctiva, immunocytochemical studies were conducted to ascertain the presence and location of the peptide and receptor related to growth in the ocular surface.

2. Materials and methods 2.1. Animals Adult Ž175–200 g. male Sprague–Dawley rats ŽCharles River Labs, Wilmington, MA. were utilized in this study. Animals were housed in an environment of 21 " 0.58C with a relative humidity of 50 " 10%. The room had a complete exchange of air 15–18 = per hour and a 12-h light–dark cycle with no twilight. Water and Purina 5010 Rodent Chow were continuously available. All investigations conformed to the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Research, the regulations of the National Institutes of Health, and the guidelines of the Department of Comparative Medicine of The Pennsylvania State University College of Medicine. 2.2. Drugs and drug treatment Animals were injected i.p. with either 20 mgrkg NTX, 10 mgrkg OGF, 10 mgrkg OGF and 10 mgrkg naloxone hydrochloride ŽNAL., 10 mgrkg NAL, or an equivalent volume of sterile water Žcontrol.; all drugs were administered in separate syringes. In the case of OGF-NAL administration, injection of each drug was made individually and within seconds of each other. For tissue culture studies, NTX Ž10y6 M., OGF Ž10y6 M., or an equivalent volume of sterile water was added to the incubation medium. 2.3. DNA synthesis and histology 1 h prior to sacrifice at least 2 animals from each experimental group were injected i.p. with 2 m Cirg body weight of w 3 Hxthymidine Ž20 Cirmol, Du Pont-New England Nuclear, Boston, MA.. All animals were anesthetized and decapitated, and the eyes proptosed and enucleated. Eyes were stored in 10% neutral buffered formalin for 12 h, embedded in paraffin, and processed for autoradiography. 8-m m sections that included the entire corneal

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surface, limbus, and conjunctiva were collected, coated with Kodak NTB-2 emulsion, stored in light-tight boxes at 48C for 35 days, and developed with Kodak D-19. Tissues were counterstained with hematoxylin and eosin. 2.4. Organ culture Enucleated eyes with as much skirt of conjunctiva as possible, were removed and placed in 6-well tissue culture plates containing RPMI medium with 10% fetal calf serum, penicillin Ž100 Urml., streptomycin Ž10 m grml., neomycin Ž20 m grml., and 1.5% sodium bicarbonate. Cultures were incubated at 378C in a humidified atmosphere of 7% CO 2 and 93% air. Immediately after establishing the organ culture, drugs or sterile water were added. 3 h following initiation of drug exposure, 2 m Cirml of w 3 Hxthymidine were added to the wells for 1 h prior to fixation. The eyes were embedded in paraffin, sectioned, and processed for autoradiography as described earlier. 2.5. Immunocytochemistry To identify whether OGF and the z-opioid receptor were present in ocular surface epithelium, tissues were

stained with polyclonal antibodies to wMet 5 x-enkephalin ŽCO-172. or the z receptor ŽAO-440.. The characteristics of these antibodies have been described earlier Žsee w47,48x.. In brief, the antibody to wMet 5 x-enkephalin was produced in rabbits against an antigen composed of wMet 5 x-enkephalin linked with glutaraldehyde to bovine serum albumin. Using a quantitative immunodot assay, a 1 : 150 dilution of the antisera recognized 25 ng of wMet 5 xenkephalin, but did not detect as much as 500 ng of b-endorphin or 1 m g of wLeu5 x-enkephalin, wMet 5, Arg 6 , Phe7 x-enkephalin, wMet 5, Arg 6 , Phe7, Leu8 x-enkephalin, or dynorphin A1-8. The z-opioid receptor antibody was produced in rabbits against the 17 kDa polypeptide of the receptor w47x. Whole eyes were removed from untreated rats and processed for immunocytochemistry. Tissues were immediately frozen in isopentane chilled on dry ice, and 10 m m sections were collected on poly-L-lysine-coated slides and stored at y208C with Drierite until use. Sections were fixed and permeabilized in ice-cold 95% ethanol and 100% acetone for 10 min each, rinsed with 0.1 M Sorenson’s phosphate buffer ŽSPB., and blocked for 15 min with 3% normal goat serum ŽNGS. in SPB with 0.1% Triton X-100, pH 7.4. Anti-wMet 5 x-enkephalin IgG or anti-z IgG were

Fig. 1. Photomicrographs of hematoxylin- and eosin-stained paraffin-embedded sections of the anterior ocular surface from an adult male Sprague–Dawley rat. A: regions analyzed for DNA synthesis included central cornea Žcc., peripheral cornea Žpc., limbus Žlm., and conjunctiva Žcn.; the limbo-corneal junction is marked by an arrowhead. B: higher magnification of the regions examined for DNA synthesis in the ocular surface epithelium. Bar, 240 m m ŽA. and 35 m m ŽB..

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2.6. Labeling indexes The number of cells with four or more grains Žbackground was F 1 grainrcell. in the basal epithelial layer of the central cornea, peripheral cornea, limbus, and conjunctiva was counted from 4 non-serial sections per eye, with at least 4 corneas assessed. At least 300 cells were examined in each region of the ocular surface. Only cells in the deepest aspect of the basal epithelium were studied. Care was taken that the section evaluated went through the axial portion of the cornea, bisecting the globe so that the superiorrinferior poles were present in the sections. Labeling indexes were computed as the number of labeled basal cells divided by the total number of basal cells with nuclei = 100. As noted above, four areas of the ocular surface epithelium were evaluated ŽFig. 1. at 400 = using an eyepiece reticule consisting of a 0.1 mm2 grid. A region in the middle of the cornea, consisting of a 0.48 mm strip, was

Fig. 2. The labeling indexes Ži.e. percentage of labeled cell nuclei. of basal epithelial cells in the central cornea, peripheral cornea, limbus, and conjunctiva. Specimens were evaluated at 02:00, 08:00, 12:00, 16:00, and 20:00 h, 1 h after an injection of w 3 Hxthymidine. No significant differences in labeling indexes were noted between time points for each region. Data represent mean"S.E.M.

diluted 1 : 300 in SPB with 1% NGS and 0.1% Triton X-100. Tissues were incubated in a humidified chamber at 48C for 16–18 h, rinsed in SPB with 1% NGS, blocked for 15 min in SPB and 3% NGS, and incubated at 48C with rhodamine-conjugated goat anti-rabbit IgG ŽCappel Laboratories. at 1 : 100 for 45 min. Sections were mounted in a solution of 60% glycerolr40% SPB. Immunoreactivity was visualized using an Olympus BH-2 microscope equipped for indirect immunofluorescence. The microscope had a standard Olympus G filter set equipped with a band pass filter ŽBP-545. that excites at 545 nm, along with a dichroic mirror ŽDM-580.. Emission occurred at 625 nm using a long pass emission filter ŽO-590.. A supplementary excitation filter ŽO-515. was used to narrow the excitation band more closely to rhodamine, serving also to reduce background. Controls for specificity included tissues processed with antibodies pre-absorbed with an excess of antigen and specimens stained only with secondary antibody.

Fig. 3. The labeling indexes Ži.e. percentage of labeled cell nuclei. of basal epithelial cells in the central cornea, peripheral cornea, limbus, and conjunctiva from adult rats. Specimens were evaluated at 16:00 h, 4 h after a single i.p. injection of 20 mgrkg naltrexone ŽNTX. or an equivalent volume of sterile water Ž sCO.; w 3 Hxthymidine was given 1 h prior to sacrifice. Data represent mean"S.E.M. Significantly different from controls at P - 0.05 Ž ) . or P - 0.01 Ž ) ) ..

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3. Results 3.1. DNA synthesis in ocular surface epithelium does not Õary with the time of day Prior to examining the role of the endogenous opioid system in DNA synthesis of ocular surface epithelium, analysis of whether the basal epithelial cells undergo a daily rhythm in this regard required evaluation. Examination of 5 time points over an 18-h period indicated no distinct and significant variation in DNA synthesis within any region of ocular surface basal epithelium ŽFig. 2.. It should be noted, however, that some variability occurred within a region necessitating a sufficient number of grids for sampling. Moreover, some differences between time points within a region could be discerned, but statistical measures showed no significant differences. Thus, assess-

Fig. 4. The labeling indexes of cells Ži.e. percentage of labeled cell nuclei. in the basal epithelial layer of the central cornea, peripheral cornea, limbus, and conjunctiva of rats. Specimens were collected at 16:00 h, 4 h after a single i.p. injection of either OGF Ž10 mgrkg wMet 5 x-enkephalin., OGF Ž10 mgrkg. and naloxone ŽNAL, 10 mgrkg., NAL Ž10 mgrkg., or an equivalent volume of sterile water Ž sCO.; w 3 Hxthymidine was given 1 h prior to sacrifice. Data represent mean" S.E.M. Significantly different from controls at P - 0.01 Ž ) ) ..

considered to be the central cornea. The peripheral cornea and limbus each were assessed in 0.48 mm lengths on both sides of the junction between limbus and cornea. The central and peripheral cornea had subjacent stroma, whereas blood vessels and non-stromal material were noted underlying the basal epithelial cells of the limbus. The conjunctiva was analyzed for a 0.48 mm length beginning 1.2 mm from the limbo-corneal junction, with goblet cells distinguishing this region Ži.e. bulbar region.. 2.7. Statistics Data were analyzed using analysis of variance, and subsequent comparisons were made with Newman-Keuls tests.

Fig. 5. The labeling indexes Ži.e. percentage of labeled cell nuclei. of basal epithelial cells in the central cornea, peripheral cornea, limbus, and conjunctiva of rats in vitro. Rats were killed at 12:00 h and tissues placed in culture for 4 h in medium supplemented with 10y6 M NTX or an equivalent volume of sterile water Ž sCO.. 1 h prior to fixation, w 3 Hxthymidine was added to the cultures. Data represent mean"S.E.M. Significantly different from controls at P - 0.01 Ž ) ) ..

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ment at 02:00, 08:00, 12:00, 16:00, and 20:00 h revealed that few cells in the central cornea were labeled Žmean: 0.8 " 0.5%., but a notable number of cells in the peripheral cornea Žmean: 7.6 " 0.4%., limbus Žmean: 6.4 " 0.7%., and conjunctiva Žmean: 10.1 " 0.8%. contained radiolabeled thymidine. The level of DNA synthesis in the central cornea was significantly lower Ž P - 0.01. than all other regions, whereas the labeling index of the conjunctiva was significantly elevated from both the peripheral cornea Ž P - 0.05. and limbus Ž P - 0.01.; the peripheral cornea and limbus were comparable in percentage of radiolabeled cells. Because no statistically reliable differences within a region were recorded during the 18-h period of analysis, all subsequent studies were conducted at 16:00 h. 3.2. Opioid receptor blockade eleÕates DNA synthesis in ocular surface epithelium The effects of blockade of endogenous opioid–opioid receptor interaction on the level of DNA synthesis in the epithelium of the cornea, limbus, and conjunctiva were determined using the potent and long-acting opioid antagonist, NTX ŽFig. 3.. Administration of 20 mgrkg NTX or an equivalent volume of sterile water at 12:00 h, injection of w 3 Hxthymidine at 15:00 h, and examination of the eye at 16:00 h showed that exposure to NTX elevated the labeling indexes in the peripheral cornea, limbus, and conjunctiva by 72, 30, and 35%, respectively, from control levels; these data were markedly different from their control values Ž P - 0.05 for the limbus, P - 0.01 for the peripheral cornea and conjunctiva.. No change in DNA synthesis from control levels was observed in the central corneal epithelium after NTX administration. 3.3. Opioid growth factor inhibits DNA synthesis of ocular surface epithelial cells The influence of the opioid agonist OGF on DNA synthesis of basal epithelial cells in the cornea, limbus, and conjunctiva was ascertained ŽFig. 4.. Treatment of rats with 10 mgrkg OGF depressed DNA synthesis of the peripheral cornea, limbus, and conjunctiva by 25, 48, and 50%, respectively, from control levels. Animals receiving an injection of OGF had a labeling index for basal epithelial cells of the central cornea that was comparable to control values. To determine if OGF interacted with opioid receptors, some animals were given 10 mgrkg OGF and 10 mgrkg NAL, a short-acting opioid antagonist of low potency that, at appropriate dosages and regimen, can sufficiently block the opioid receptor related to growth and eliminate opioid action ŽFig. 4.. NAL blocked the repressive nature of OGF on DNA synthesis in the peripheral cornea, limbus, and conjunctiva, but administration of naloxone alone did not

Fig. 6. The labeling indexes Ži.e. percentage of labeled cell nuclei. of basal epithelial cells in the central cornea, peripheral cornea, limbus, and conjunctiva of rats in vitro. Rats were killed at 12:00 h and tissues placed in culture for 4 h in medium supplemented with 10y6 M OGF or an equivalent volume of sterile water Ž sCO.. 1 h prior to fixation, w 3 Hxthymidine was added to the cultures. Data represent mean"S.E.M. Significantly different from controls at P - 0.05 Ž ) . or P - 0.01 Ž ) ) ..

result in any deviation from control data. Treatment with a combination of OGF-NAL, or NAL alone, had no effect on the labeling index of basal epithelial cells of the central cornea. 3.4. Opioid peptides influence DNA synthesis of basal epithelial cells from the ocular surface in tissue culture To examine whether NTX or OGF exert a direct influence on DNA synthesis in epithelial cells of the ocular surface, the action of these agents in tissue culture was examined ŽFigs. 5 and 6.. The studies showed that both the opioid antagonist Ži.e. NTX. and opioid agonist Ži.e. OGF. altered DNA synthesis in a manner similar to that seen in vivo. As noted in Fig. 5, incubation of the enucleated eyes

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for 4 h with NTX increased the labeling index 106, 24, and 59% for the peripheral cornea, limbus, and conjunctiva, respectively, from control values. The labeling index of the central cornea subjected to NTX was comparable to control levels. Eyes incubated with OGF, and w 3 Hxthymidine added in the last hour, exhibited decreases inIabeling index of 25, 41, and 59% for the peripheral cornea, limbus, and conjunctiva, respectively, from control values. The central cornea of OGF-exposed eyes did not differ from controls with regard to the labeling index.

3.5. [Met 5]-enkephalin and the z opioid receptor are associated with the rat cornea, limbus, and conjunctiÕa Antibodies to wMet 5 x-enkephalin Ži.e. OGF. and to the z opioid receptor were used in immunocytochemical studies to determine the distribution and location of this growthrelated peptide and its receptor in the epithelium of the ocular surface in the rat ŽFigs. 7 and 8.. Both antibodies demonstrated a similar pattern of immunocytochemical labeling in the cornea, limbus, and conjunctiva ŽFig. 7A–D,

Fig. 7. Photomicrographs of frozen sections of the anterior ocular surface of the rat stained with an antibody to the OGF Ži.e. wMet 5 x-enkephalin.. Both the basal and suprabasal layers in the central cornea ŽA., peripheral cornea ŽB., limbus ŽC., and conjunctiva ŽD., cytoplasm Žarrow., but not nuclei, of cells were immunoreactive. Control section of the central cornea stained with antibody pre-absorbed with pure antigen and demonstrating no immunoreactivity in the ocular epithelium ŽE.. Bar, 32 m m.

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Fig. 8. Photomicrographs of frozen sections of the anterior ocular surface of the adult rat stained with an antibody to the z-opioid receptor. Note bright staining of both the basal and suprabasal layers, with immunoreactivity found in the cytoplasm Žarrows. of cells of the central cornea ŽA., peripheral cornea ŽB., limbus ŽC., and conjunctiva ŽD.. Control section of the central cornea stained with antibody pre-absorbed with pure antigen demonstrating no immunoreactivity in the ocular epithelium ŽE.. Bar, 32 m m.

Fig. 8A–D.. The basal and suprabasal layers were intensely immunofluorescent, with staining localized to the cytoplasm. Little immunoreactivity was associated with the nuclei of cells. Control specimens processed with antibodies pre-absorbed with the appropriate antigen Ži.e. wMet 5 xenkephalin or z receptor protein., or samples stained with secondary antibody only, exhibited no immunoreaction ŽFig. 7E, Fig. 8E.. 4. Discussion In this study, evidence was provided that a native opioid peptide, OGF, plays an essential role as a tonic

inhibitory substance in the homeostatic control of DNA synthesis in ocular surface epithelium. OGF influenced DNA synthesis profoundly and rapidly. Furthermore, these effects were not restricted to the epithelium from only one region of the ocular surface, but occurred in the cornea, limbus, and conjunctiva. The short-acting opioid antagonist, naloxone, blocked OGF activity at a dosage that had no independent effect on DNA synthesis, indicating receptor-mediated action of OGF. Finally, OGF influenced DNA synthesis in organ culture preparations of ocular surface epithelium, suggesting that opioid peptides directly regulate cellular events. These data extend earlier reports in a wide variety of organs, tissue types, organisms, and situa-

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tions Že.g. development, homeostasis, and wound healing. that O G F functions as a grow th m odulator w23,25,33,39,45,46,49–51x. Although other opioids require examination as to their growth properties with respect to the ocular surface epithelium, OGF has been shown in numerous instances to be the only growth-related opioid peptide w23,45,46,50x. Thus, these novel observations support the postulate that an endogenous opioid serves as a tonic inhibitor regulating DNA synthesis in ocular surface epithelium and reveal the existence of an important growth regulatory pathway dedicated to the homeostatic maintenance of the epithelium. Previous studies have examined mitosis and DNA synthesis in the ocular surface epithelium w2–5,9,12– 15,18,21,28–30,40x, often focusing on the cornea. Some authors have reported a circadian rhythm in the mitotic index w13,28,30x or in DNA synthesis w4,21,29x of ocular surface epithelium, whereas others w3,24x found no such variation. Within the limited study performed herein, we did not observe a regular daily fluctuation in DNA synthesis of epithelium from any region of the ocular surface examined: peripheral cornea, central cornea, limbus, or conjunctiva. These results support and extend the observations of Bertalanffy and Lau w3x and Messier and Leblond w24x who reported that the mitotic and labeling indexes, respectively, of rat corneal epithelium are not subject to a cyclic variation. A number of reasons may account for these differing conclusions relative to whether a circadian rhythm exists for ocular epithelial cells. First, our study clearly defined the specific regions of the ocular surface epithelium analyzed. With some exceptions Že.g. w7,9,12,21,40x., previous authors often failed to delineate the areas studied. Given that we did see differences in DNA synthesis between ocular surface regions, the inability of some earlier reports to account for regional variation may have resulted from a lack of consideration of the contribution from each region resulting in unintentional obfuscation of the data. Second, the present study focused on the basal layer of cells in order to eliminate errors introduced by the variations in DNA synthesis occurring in different layers of ocular surface epithelium. The basal layer is the proliferative region of this epithelium. Combining the results of this DNA synthesis-rich single layer with the multilayered and largely post-mitotic suprabasal layers would skew the data and have the net effect of altering the labeling index. Unfortunately, few authors Že.g. w21x. specify the layerŽs. of epithelium studied. Third, the present investigation administered labeled thymidine systemically, rather than using such techniques as incubation in tissue culture or injection into the anterior chamber Že.g. w7,9,12,14,21x., thus minimizing artifacts from culturing andror injury. Fourth, our study employed autoradiography. Other studies Že.g. w29x. have used scintillation counting, which does not discriminate between and within ocular surface epithelial regions, and even may involve tissues other than epithelium. Fifth, rigorous statistics were

applied to the analysis of the results in the present investigation. Such stringent evaluation of the data usually was not performed in other reports Že.g. w2,3,5,13 – 15,18,21,40x.. Sixth, some studies Že.g. w5,18x. used a ‘flat preparation’ which distorts structures and excludes the ability to discern the individual epithelial layers. Seventh, interspecies variation andror experimental conditions Že.g. handling. may account for some of the discrepancies dealing with the identification of a circadian rhythm in ocular surface epithelium. Thus, the present observations concerning DNA synthesis were made under extremely controlled parameters and were evaluated with appropriate statistical measures. These data support earlier observations w3,24x that a similar labeling Žand mitotic. index occurs in corneal epithelium throughout a light-dark cycle. Few, if any, basal epithelial cells of the central cornea were found to incorporate radioactive thymidine under in vivo or in vitro conditions, and neither OGF nor NTX influenced DNA synthesis. Thus, these central corneal epithelial cells, which normally exhibit little DNA synthesis, also are not governed by opioids and are presumably post-mitotic. Of course, their response to other stimuli of cell division Že.g. the release of contact inhibition by epithelial wounding. is open for question. Our results are consistent with the observations of Ebato and colleagues w10x, and Lindberg and co-workers w22x who found that explants or disaggregated cells from central corneal epithelium of humans did not thrive in a tissue culture milieu. Kruse and Tseng w20x, however, reported that epithelial cells derived from the central cornea of rabbits grew in a serum-free clonal culture system. The differences in cell replication between the studies of Ebato et al. w10x, Lindberg et al. w22x, and our laboratory, and the investigation of Kruse and Tseng w20x, are unclear. Species and experimental design could be at least partly responsible. Our data indicate how important the peripheral cornea, limbus, andror conjunctiva are as potential sources of cells for maintenance of the central corneal epithelium, and confirm previous studies suggesting that progenitor cells reside ou tsid e th e cen tral corn eal epitheliu m w 9 – 12,19,21,27,34,40x. The results of this study are consonant with those reported earlier in regard to the presence and function of endogenous opioids and the corneal epithelium. Examining the outgrowths of explants of the peripheral corneal epithelium from rabbits, Zagon and colleagues w49x found that OGF depressed DNA synthesis and migration of the epithelial cells in a receptor-mediated manner. Moreover, outgrowths subjected to OGF displayed a disorganization in architectural pattern that suggests opioids participate in governing tissue structure. Both OGF and the z receptor were present in cells composing outgrowths of the rabbit corneal epithelium. In a subsequent report, Zagon and co-workers w48x demonstrated by immunocytochemistry that both OGF and the z receptor are in the corneal epithelium of a wide variety of

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classes of the phylum Chordata, including humans. The present studies extend these observations and show that OGF is a growth inhibitory factor with respect to DNA synthesis of not only corneal epithelial cells, but those of the limbus and conjunctiva. Additionally, we now demonstrate that OGF has an important role in maintaining ocular epithelial homeostasis. Data showing that both OGF and the z receptor are associated with the basal and suprabasal cells of the rat corneal epithelium as well as that of the limbus and conjunctiva are in concert with earlier reports of the presence of peptide and receptor in diverse classes of vertebrates w48x. OGF, wMet 5 x-enkephalin, has been identified in bovine cornea by reversed-phase-high performance liquid chromatography ŽRP-HPLC. followed by radioimmunoassay ŽRIA. w36x. In a subsequent report, Tinsley and co-workers w35x identified wMet 5 x-enkephalin in the bovine cornea by negative ion fast atom bombardment-mass spectrometry after initial purification by RP-HPLC and initial identification by RIA. Cripps and Bennett w8x have detected OGF by RIA in adult rat lacrimal glands. Our finding of OGF immunoreactivity in the cytoplasm of ocular surface epithelial cells is consonant with the results of these earlier studies and, for the first time, documents the location of the peptide in these cells. Despite the presence of a native peptide that plays a vital role in the well-being of the corneal, limbal, and conjunctival epithelium, the sourceŽs. of the opioid peptide remains in question. Northern analysis, reverse transcriptase polymerase chain reaction, and particularly in situ hybridization studies which allow one to focus on the individual cells and layers, are needed to identify and localize the prohormone of OGF-preproenkephalin. It is of interest to note that both OGF and the z receptor, which function to regulate cell replication, are present in both basal and suprabasal cells of the ocular surface epithelium. Yet, the suprabasal layer of the peripheral cornea, limbus, and conjunctiva, and both basal and suprabasal cells of the central cornea, are generally not active in DNA synthesis or mitosis w5,10,14,15,22,40x. The reasonŽs. why OGF and the z receptor are found in these cells isŽare. unclear, but a number of possibilities come to mind. First, it may be that expression of the peptide and receptor is retained, or slowly dissipates, from cells that earlier were actively replicating. Second, these cells may produce OGF as a paracrine growth factor in order to control cellular renewal in proliferating basal epithelial cells. Third, both of these elements may function in suprabasal cells, and basal cells of the central cornea, in order to suppress growth-related events with respect to homeostasis. If this is the case, however, then regulation of growth by OGF-z receptor interfacing must be different from basal epithelial cells actively synthesizing DNA because blockade by NTX has little effect on suprabasal cells Žunpublished observations. or central corneal epithelial cells as observed herein. Fourth, if either suprabasal cells

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of the cornea, limbus, or conjunctiva or basal cells of the central cornea have the potential to undergo replication when injured, then the totipotent nature of these cells would predict the requirement for growth modulatory factors that could influence cell proliferation and migration. A number of other growth factors have been detected in ocular surface epithelium w26,31,32,37,38,44x. However, we have found no report that any of these growth factors influenced cellular maintenance during homeostasis. For example, epidermal growth factor w26x and keratinocyte growth factor w32x have been shown to stimulate proliferation of corneal epithelium in tissue culture andror to accelerate corneal epithelial wound healing, but have no effect on normal corneal epithelium. Thus, OGF is a unique growth factor that is present and functions during homeostasis and in wound healing w25,48x. Alterations in transcription, translation, andror post-translation of either the peptide or the receptor might significantly affect the maintenance andror repair of ocular surface epithelium. Acknowledgements Supported by NIH Grant EY10300 and a grant from the Pennsylvania Lions. Dr. Yan Wu is thanked for performing the immunocytochemistry. References w1x H. Akil, S.J. Watson, E. Young, M.E. Lewis, L.H. Khatchaturian, J.M. Walker, Endogenous opioids: biology and function, Annu. Rev. Neurosci. 7 Ž1984. 223–255. w2x L.B. Arey, W.M. Covode, The method of repair in epithelial wounds of the cornea, Anat. Rec. 86 Ž1943. 75–86. w3x F.D. Bertalanffy, C. Lau, Mitotic rate and renewal time of the corneal epithelium in the rat, Arch. Ophthalmol. 68 Ž1962. 144–148. w4x E.R. Burns, L.E. Scheving, Circadian influence on the wave form of the frequency of labeled mitoses in mouse corneal epithelium, Cell Tissue Kinet. 8 Ž1975. 61–66. w5x W. Buschke, J.S. Friedenwald, W. Fleischmann, Studies on the mitotic activity of the corneal epithelium, Bull. Johns Hopkins Hospital 73 Ž1943. 143–168. w6x J.D. Cameron, R.R. Waterfield, M.W. Stefes, L.T. Furcht, Quantification of corneal organ culture migration, Invest. Ophthalmol. Vis. Sci. 30 Ž1989. 2407–2413. w7x G. Costarelis, S.-Z. Cheng, G. Dong, T.-T. Sun, R.M. Lavker, Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells, Cell 57 Ž1989. 201–209. w8x M.M. Cripps, D.J. Bennett, Proenkephalin A derivatives in lacrimal gland: occurrence and regulation of lacrimal function, Exp. Eye Res. 54 Ž1992. 829–834. w9x S. Danjo, J. Friend, R.A. Thoft, Conjunctival epithelium in healing of corneal epithelial wounds, Invest. Ophthalmol. Vis. Sci. 28 Ž1987. 1445–1449. w10x B. Ebato, J. Friend, R.A. Thoft, Comparison of central and peripheral human corneal epithelium in tissue culture, Invest. Ophthalmol. Vis. Sci. 28 Ž1987. 1450–1456. w11x B. Ebato, J. Friend, R.A. Thoft, Comparison of limbal and peripheral human corneal epithelium in tissue culture, Invest. Ophthalmol. Vis. Sci. 29 Ž1988. 1533–1537.

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w34x R.A. Thoft, J. Friend, The X, Y, Z hypothesis of corneal epithelia maintenance, Invest. Ophthalmol. Vis. Sci. 24 Ž1983. 1442–1443. w35x P.W. Tinsley, C. Dass, J.W. Trimble, D.M. Desiderio, Identification of methionine enkephalin in the bovine cornea by fast atom bombardment-mass spectrometry, Exp. Eye Res. 51 Ž1990. 671–674. w36x P.W. Tinsley, G.H. Fridland, J.T. Killmar, D.M. Desiderio, Purification, characterization, and localization of neuropeptides in the cornea, Peptides 9 Ž1988. 1373–1379. w37x B.J. Tripathi, P.S. Kwait, R.C. Tripathi, Corneal growth factors: a new generation of ophthalmic pharmaceuticals, Cornea 9 Ž1990. 2–9. w38x B.J. Tripathi, R.C. Tripathi, A.M. Livingston, N.S.C. Borisuth, The role of growth factors in the embryogenesis and differentiation of the eye, Am. J. Anat. 192 Ž1991. 442–471. w39x P.M. Villiger, M. Lotz, Expression of prepro-enkephalin in human articular chondrocytes is linked to cell proliferation, EMBO J. 11 Ž1992. 135–143. w40x Z.-G. Wei, G. Cotsarelis, T.-T. Sun, R.M. Lavker, Label-retaining cells are preferentially located in fornical epithelium: implications on conjunctival epithelial homeostasis, Invest. Ophthalmol. Vis. Sci. 36 Ž1995. 236–246. w41x Z.-G. Wei, T.-T. Sun, R.M. Lavker, Rabbit conjunctival and corneal epithelial cells belong to two separate lineages, Invest. Ophthalmol. Vis. Sci. 37 Ž1996. 523–533. w42x Z.-G. Wei, R.-L. Wu, R.M. Lavker, T.-T. Sun, In vitro growth and differentiation of rabbit bulbar, fornix, and palpebral conjunctival epithelia, Invest. Ophthalmol. Vis. Sci. 34 Ž1993. 1814–1828. w43x L. Wiley, N. SundarRaj, T.-T. Sun, R.A. Thoft, Regional heterogeneity in human corneal and limbal epithelia: an immunohistochemical evaluation, Invest. Ophthalmol. Vis. Sci. 32 Ž1991. 594– 602. w44x S.E. Wilson, Y.-G. He, S.A. Lloyd, EGF, EGF receptor, basic FGF, TGF beta-1, and IL-1 alpha mRNA in human corneal epithelial cells and stromal fibroblasts, Invest. Ophthalmol. Vis. Sci. 33 Ž1992. 1756–1765. w45x I.S. Zagon, P.J. McLaughlin, The role of endogenous opioids and opioid receptors in human and animal cancers, in: N.P. Plotnikoff, A.J. Murgo, R.E. Faith, J. Wybran ŽEds.., Stress and Immunity, CRC, Caldwell, NJ, 1991, pp. 343–356. w46x I.S. Zagon, P.J. McLaughlin, Opioid growth factor receptor in the developing nervous system, in: I.S. Zagon and P.J. McLaughlin ŽEds.., Receptors in the Developing Nervous System, vol. 1, Growth Factors and Hormones, Chapman and Hall, London, UK, 1993, pp. 39–62. w47x I.S. Zagon, P.J. McLaughlin, Production and characterization of polyclonal and monoclonal antibodies to the zeta Ž z . opioid receptor, Brain Res. 630 Ž1993. 295–302. w48x I.S. Zagon, J.W. Sassani, G. Allison, P.J. McLaughlin, Conserved expression of the opioid growth factor, wMet 5 xenkephalin, and the zeta Ž z . opioid receptor in vertebrate cornea, Brain Res. 671 Ž1995. 105–111. w49x I.S. Zagon, J.W. Sassani, P.J. McLaughlin, Opioid growth factor modulates corneal epithelial outgrowth in tissue culture, Am. J. Physiol. 268 Ž1995. R942–R950. w50x I.S. Zagon, Y. Wu, P.J. McLaughlin, Opioid growth factor inhibits DNA synthesis in mouse tongue epithelium in a circadian rhythmdependent manner, Am. J. Physiol. 267 Ž1994. R645–R652. w51x I.S. Zagon, Y. Wu, P.J. McLaughlin, The opioid growth factor, wMet 5 x-enkephalin, and the z Žzeta. opioid receptor are present in human and mouse skin and tonically act to inhibit DNA synthesis in the epidermis, J. Invest. Dermatol. 106 Ž1996. 490–497.