Reports. Cytochemical studies on endogenous peroxidase

Transcription

1 Reports Cytochemical studies on endogenous peroxidase in conjunctival and corneal epithelial cells. TAKESHI IWATA. Endogenous peroxidase activity in conjunctival and corneal epithelial cells was studied cytochemically by the diaminobenzidine method. Many peroxidase-positive cells were found in the fornical conjunctiva. They were also seen, but less frequently, in the tarsal conjunctiva, limbus, and cornea. Three types of peroxidase-positive (cytochemically reactive) cells were differentiated besides goblet cells: (1) the most superficial cells with several reactive secretory granules, (2) motheaten cells near the surface with abundant reactive microgranules, and (3) deeper cells with reactive microgranules and a few densely reactive granules. Some goblet cells shared the same characteristics with other types of cells. The relationship among these cells, functional differences between granules, and the function of peroxidase are discussed. Peroxidase has been thought to act as an antiinfectious agent in tears. 1 ' 4 It is secreted from the lacrimal gland, 1 " 3 the Harderian gland, 1 and goblet cells in the conjunctival epithelium. 4 In the present experiment, peroxidase activity in conjunctival and corneal epithelial cells has been further investigated cytochemically. Various peroxidase-positive cell types are differentiated. Materials and methods. Normal adult albino rats of both sexes (Wistar strain) weighing 200 to 300 grams were used. After luxation of the neck, eyes were enucleated together with conjunctiva. One millimeter thick sagittal sections of the upper anterior quadrant of the eye were obtained from the central part. Lids of the other side were dissected with conjunctiva, and treated in the same way as in the previous studies. 4 These two kinds of specimens were put into 2.5 per cent glutaraldehyde fixative buffered with 0.1 M sodium cacodylate (ph 7.4) for 25 minutes. For cytochemical studies 20 nm and 50 /«n thick frozen sections were cut. They were incubated for 25 minutes at 37 C. in a reaction medium of peroxidase of 0.05 M Tris HC1 buffer (ph 8.5) containing 0.1 per cent 3,3'-diaminobenzidine tetrahydrochloride and 0.02 per cent H 2 O 2, slightly modified after Graham and Karnovsky's medium 5 and Novikoff's medium. 6 After being rinsed with the buffer, 20 nm thick sections were mounted on glycerin jelly for light microscopical study. Sections 50 /tm thick were fixed with 1 per cent osmium tetroxide for one hour. They were dehydrated and embedded in Epon 812. Ultrathin 297 sections were cut with an LKB Ultrotome and examined with a JEM-100B electron microscope without electron staining or with light lead staining. Semithin sections were also examined with a light microscope. Observations were made on 20 rats. Control study. Other rats were used. (1) Sections prepared as described above were incubated in the reaction medium not containing H;O 2. To ensure elimination of endogenous H2O2, some sections were also incubated in the reaction medium containing no FLO:, but 0.1 per cent catalase. (2) Sections were incubated in the reaction medium with 10" 2 to 10" 1 M potassium cyanide added or 10" 2 M sodium azide, or 10-2 M 3-amino-l:2:4-triazole. (3) Sections were incubated in the reaction medium after 10 minutes' boiling at 100 C. Results. Light-microscopical observations. In the fornical conjunctiva, many goblet cells displayed browncolored cytochemical reaction product of peroxidase (Fig. 1, A). In addition to these, many cells were found in various cell layers which demonstrated scattered reactive granules and diffuse reaction products. Especially superficial cells had a remarkable number of such granules. In the tarsal conjunctiva, reactive cells became few except superficial cells (Fig. 1, B). In the limbus and cornea, only some superficial cells showed scattered reactive granules (Figs. 1, C and D). Electron-microscopical observations. Among the cytochemically reactive epithelial cells, three types of cells were differentiated besides goblet cells: (1) cells with a motheaten appearance (with holes in them) near their cell membrane closest to the surface were seen and were usually found in the second or third cell layer counting from the surface (Figs. 2 and 3). The cells demonstrated electron-dense reaction product. The reaction appeared in the rough-surfaced endoplasmic reticulum including the perinuclear cisterna, in microgranules (or microvesicles), and along the internal wall of the motheaten spots, but rarely in Golgi saccules. The microgranules were about the same size in diameter (0.1 to 0.2 (im) as the rough-surfaced endoplasmic reticulum. They were abundant in and between the motheaten spots. They were not vertically cut rough-surfaced endoplasmic reticulum, because in the motheaten area the rough-surfaced endoplasmic reticulum was only poorly developed. (2) The most superficial cells revealed the reaction in several scattered granules of various sizes (up to about 2 jun in diameter) as well as in the rough-surfaced endoplasmic reticulum and microgranules (Figs.

2 298 lc Investigative Ophthalmology April 1976 Reports C^ Id Fig. 1. A, fomical conjunctiva. Many goblet cells (G) have cytochemical reaction product of peroxidase. Many superficial cells also have scattered reactive granules. In addition, there are cells which have reactive granules and diffuse reaction products, in various cell layers (arrows). E: erythrocytes with pseudoreaetion in the subconjunelival vessels. (*200.) B, tarsal conjunctiva. Superficial cells have reactive granules. There are also cell*) in various cell layers which have reactive granules and diffuse reaction products (arrows). R: lidrand. F: fornix. (x 00.) C, limbus and cornea. There are reactive granules in some superficial cells (arrows). B; border between the limbus and the cornea. (The cornea was differentiated from the limbus by the regularly arranged stromal collagen fibers because there is no Bowman's membrane in the cornea of rats.) C: ciliary body. (xloo.) D, cornea. Some superficial cells have reactive granules (arrows). (*260.) 3 and 4). These reactive granules assumed various intermediate appearances between fine granular secretory granules and dense and rather uniform granules found in goblet cells.1 In many granules there were some clear spaces between the contents and the surrounding membrane. They were found to be the gathering of smaller ones or mierogranules (Fig. 3). Some of them were seen to be secreting their contents (Fig. 4). (3) Other cytochemically reactive epithelial cells were seen in deeper cell layers (Fig. 2). They revealed the reaction in the rough-surfaced endoplasmic ret ionium including the pcrinuclear cisterna, in mierogranules, and in a lew granules of various sizes (up to about 1 ^m), but rarely in Colgi saccules. The granules had a dense and rather uniform appearance. As conceivable from the light microscopy, the cells of (1) and (3) types were found in the fomiail and tarsal conjunctiva, and group 2 were found everywhere. Occasionally, goblet colls were found which shared the same characteristics with and resembled other types of cells ( Fig. 2, A). Control study. Sections incubated in the same reaction medium not containing I-I;Os showed positive but weak cytochemical reaction. Sections incubated in the reaction medium containing catalase, the reaction was never found. A 10-- M solution of KCN did not inhibit the reaction, but a K)-1 M solution of KCN strongly inhibited it. Sodium azide (10"- M) completely inhibited the reaction. Aminotriazole (10-- M) considerably reduced the reaction. After boiling, the reaction was never present. DISCUSSION. The control study which I con-

3 Volume 15 Number 4 Reports 299 Fig. 2. Conjiinctival epithelium of the fornix. There are cells (M) with a motheaton appearance near the surface. The second cells from the surface have cytochemical reaction in the roughsurfaced endoplasmic reticulum (rer) including the perinuclear cisterna and in microgranules (m). The microgranules are abundant in and between the motheaton spots. In deeper cell layers, many colls have a few reactive granules ( C J of various sizes which take on a dense and rather uniform appearance, in addition to the reaction in the same places as the motheaten cells. Goblet ceils show the reaction in the fine-granular secretory granules (sc) and dense granules (Ci). (x7,300.) The inset (A) shows an atypical goblet cell. It has a somewhat motheaten appearance. There are many reactive microgranules besides secretory granules. (x6,600.)

4 300 Reports Fig. 3. Conjunctival epithelium of the fornix. The most superficial cell has reactive granules of various sizes. Smaller granules conglomerate into large ones (arrows). Most of the granules are rather dense and have clear.spaces between the contents and the surrounding membrane. The second cell from the surface has reactive microgranules (m) and deposits in the motheaten area, (x6,500.) ducted provided strong evidence that the cytochemical reaction product in comeal and conjunctival epithelial cells demonstrates peroxidase activity.--4- > 10 With regard to three types of peroxidase-positive cells found in this experiment, they probably transform into another type of cell as they approach the surface. On the other hand, some goblet cells shared the same characteristics with other types of cells, supporting the thought7-8 that goblet cells develop from common epithelial cells. In these three types of cells, the presence of peroxidase-positive microgranules was noteworthy. Their origin was unknown, but they acted like condensing vacuoles. In the cells near the surface with a motheaten appearance, such microgranules were well developed. In the most superficial cells, they conglomerated into secretory granules which stained more densely than fine granular ones in goblet cells. The cause of difference in appear- Investigative Ophthalmology April 1976 Fig. 4. Conjunctival epithelium of the fornix. One of the reactive granules in the mast superficial cell is secreting its contents. These granules have a somewhat fine-granular appearance, but smaller granules are discernible within them. (xl0,900.) ance between the two kinds of secretory granules is unknown, but it may be because they differ in function. Many cells in the conjunctival epithelium, including goblet cells, showed peroxidase activity in granules which assumed a dense and rather uniform appearance. Such dense granules have been found in the cells of many other peroxidaseproducing organs and are suspected to be lysosomes.2- " 10 The dense staining of some of them was not due to peroxidase.n- 10 However, in the conjunctival epithelium, it is caused by peroxidase as control studies show. To confirm that they are lysosomes, further investigations are called for. In addition to peroxidase in the conjunctival and comeal epithelium, peroxidase (probably lactoperoxidase) has also been found in the lacrimul gland'-3 and the Harderian gland.1 As suggested in the previous report,' these peroxitlases are supposed to function as an anti-infectious agent in combination with H:Oa and thiocyanate or with H;O-j and halides. However, I conducted the same experiment at different ph values, that is, at ph 7.6, 8.0, 8.5, and 9.0. At ph

5 Volume 15 Number 4 Reports , the reaction in epithelial cells was almost absent, but at ph 8.0 to 9.0, the cells demonstrated strong reaction. On the other hand, the optimal ph value of the peroxidase in the exorbital lacrimal gland is 6.5. These peroxidases may differ in their function. I thank Mrs. H. Ueno for her help in preparing this paper. From the Departments of Bacteriology and Cytochemistry, Chest Disease Research Institute, and Department of Ophthalmology, Faculty of Medicine, Kyoto University, Kyoto, 606, Japan. Submitted for publication Nov. 7, Reprint requests: Dr. Takeshi Iwata. Key words: peroxidase, epithelium, conjunctiva, cornea, goblet cell, cytochemistry. REFERENCES 1. Morrison, M., and Allen, P. Z.: Lactoperoxidase: Identification and isolation from Harderian and lacrimal glands, Science 152: 1626, Essner, E.: Localization of endogenous peroxidase in rat exorbital lacrimal gland, J. Histochem. Cytochem. 19: 216, Herzog, V., and Miller, F.: The localization of endogenous peroxidase in the lacrimal gland of the rat during postnatal development, J. Cell Biol. 53: 662, Iwata, T., Ohkawa, K., and Uyama, M.: The fine structural localization of peroxidase activity in goblet cells of the conjunctival epithelium of rats, INVEST. OPHTHALMOL. 15: 40, Graham, R. C, and Karnovsky, M. J.: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem. 14: 291, Novikoff, A. B.: Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine, J. Histochem Cytochem. 17: 675, Virchow, H.: Mikroskopische Anatomie der au/?eren Augenhaut und des Lidapparates, in: Graefe-Saemisch Handbuch der Gesamten Augenheilkunde, Ed. 2. Leipzig, 1910, Wilhelm Engelman, Vol. I, Part I, Chap. 2, pp Bindner, R.: Beitrag zur Kenntnis der Schleimzellen in der Conjunctiva bulbi bei Macacus rhesus, v. Graefes Archiv Ophthalmol. 153: 477, Fahimi, H. D.: The fine structural localization of endogenous and exogenous peroxidase activity in Kupffer cells of rat liver, J. Cell Biol. 47: 247, Novikoff, A. B., Beard, M. E., Albala, A., et al.: Localization of endogenous peroxidases in animal tissues, J. Microscopie 12: 381, Distribution of hexos amines in bovine cornea. FREDERICK A. BETTELHEIM AND DENNIS GOETZ. Excised bovine cornea were sectioned from epithelium to endothelium into 6 to 8 fractions. The glucosamine/galactosamine ratio steadily increases from epithelium to endothelium in all bovine corneas investigated. The glucosamine/ galactosamine ratio reflects the keratan sulfate/ chondroitin-4-sulfate ratio in the cornea. The significance of this topographic distribution is discussed in terms of the different hydration properties of proteoglycans containing predominantly keratan sulfate or chondroitin-4-sulfate chains. About 60 per cent of the corneal glycosaminoglycans is keratan sulfate and the remaining 40 per cent is mainly chondroitin-4-sulfate. 1 Small amounts of dermatan sulfate and chondroitin-6- sulfate 2 have also been reported. It has been alleged 3 that although the glycosaminoglycans are largely responsible for the hydration of the cornea, the keratan sulfate plays a different role than the chondroitin-4-sulfate in the hydration process. For one, the corneal wound healing shows reduced keratan sulfate and increased chondroitin- 4-sulfate synthesis and injury to the Descemet membrane and endothelium transforms keratccytes to dermatan sulfate-producing cells. 4 Furthermore, non-swelling shark cornea contains little keratan sulfate and considerable amount of chondroitin-4-sulfate. 5 The water vapor absorption properties of these two glycosaminoglycans are also quite different. While keratan sulfate absorbs large amounts of water, it retains very little in the dehydration process 6 ; the reverse is true for chondroitin-4-sulfate. 7 However, the glycosaminoglycans in the cornea are in the form of proteoglycans. These are heterogenous macromolecules carrying keratan sulfate and chondroitin-4-sulfate in different proportion. In our laboratory we isolated from cornea one proteoglycan with predominantly keratan sulfate and one with predominantly chondroitin-4- sulfate side chains. 8 In characterizing the proteoglycans we also found that the water absorption and retention power of these proteoglycans were different, reflecting the behavior of the side chains. If the different proteoglycans differ so much in their hydration properties, it is possible that there is some specific distribution in the cornea reflecting these roles. Anseth 1 reported that there was no difference in the distribution of glycosaminoglycans in the central or peripheral parts of the cornea. The glucosamine/galactosamine ratio "seemed to be higher in the anterior than in the posterior part. This difference, however, was not