- Email: [email protected]
Electron Microscopy of Cornea and Conjunctiva in Childhood Cystinosis
ELECTRON MICROSCOPY O F CORNEA AND CONJUNCTIVA I N C H I L D H O O D CYSTINOSIS K E N N E T H R. KENYON, M.D., AND JUDITH A. SENSENBRENNER,
M.D.
Baltimore, Maryland Cystinosis is an autosomal, recessively in herited disorder of amino acid metabolism characterized by the deposition of cystine crystals in the eye, kidney, reticuloendothelial system, and various other tissues.1 Most se vere of the three recognized forms is the childhood or nephropathic form in which progressive renal tubular insufficiency leads to growth retardation, rickets, and early uremic death.1 Goldman and associates2 de scribed a rare juvenile form in which only mild nephropathy develops. Adult cystinosis appears to be a completely benign disorder.3-6 In all three variants, the slit-lamp appear ance of the fine, scintillating, tinsel-like crys tals dispersed throughout the anterior cornea, conjunctiva, and iris may be diagnostic.3'7 Additional distinguishing ocular features of childhood cystinosis are photophobia and mottled pigmentary retinopathy of the pe ripheral fundus.8 Previous electron microscopic studies of ocular tissues affected by cystinosis have focused primarily on the conjunctiva.6'9 The present study is, to our knowledge, the first detailed report of corneal ultrastructure in a cystinotic child.
including marked growth retardation and hypopigmentation of skin and hair (Fig. 1). Ocular find ings included severe photophobia (Fig. 1) and uncorrected visual acuity of 20/20 bilaterally. By slitlamp biomicroscopy, refractile crystalline deposits appeared throughout the anterior corneal stroma (Fig. 2) and bulbar conjunctiva. Indirect ophthalmoscopy revealed typical patchy pigmentary changes in the peripheral f undi. OBSERVATIONS
Biopsy specimens of bulbar conjunctiva and peripheral superficial cornea were im mediately fixed in 2 % osmium tetroxide (in 0.14M Veronal acetate buffer, p H 7.2) for 60 minutes at 0°C, and were embedded in Araldite epoxy resin. Paraphenylenediamine stained 1-jx-thick sections for phase contrast photomicrography. Ultrathin sections were stained with uranyl acetate and lead citrate,
C A S E REPORT
The patient was a 10^-year-old white boy ( J H H 141 03 45) who has been previously reported.10 Al though diabetes insipidus and dwarfism had been noted by age 2 years, cystinosis was initially diag nosed at 4 years of age, when marked photophobia prompted ophthalmic examination, which disclosed corneal crystals. At age 8^4 years (1970), evalua tion at Johns Hopkins Hospital revealed the char acteristic physical features of childhood cystinosis, From the Eye Pathology Laboratory, Wilmer In stitute, and the Moore Clinic of the Johns Hopkins Hospital, Baltimore, Maryland. This study was sup ported in part by the Carrie L. Groedel Fund of Fight for Sight, Inc. (Dr. Kenyon). Reprint requests to Kenneth R. Kenyon, M.D., Eye Pathology Laboratory, Wilmer Institute, 601 N. Broadway, Baltimore, MD 21205.
Fig. 1 (Kenyon and Sensenbrenner). Childhood cystinosis demonstrates severe photophobia and de creased pigmentation of skin and hair.
VOL. 78, NO.l
CORNEA AND CONJUNCTIVA IN CYSTINOSIS
Fig. 2 (Kenyon and Sensenbrenner). Slit-lamp photograph shows strikingly refractile crystals dis persed throughout the anterior corneal stroma.
and were examined with a JEM 100-B elec tron microscope. Cornea—By light microscopy, many cor
69
neal epithelial cells, particularly in the inter mediate layers, contained abnormal perinuclear inclusions, 2 to 5 [x in diameter (Fig. 3). With the electron microscope, these inclusions proved to be double membranelimited vacuoles with homogeneous granular contents of medium electron density (Fig. 4). Although the vacuolar membranes were always closely apposed to the nuclear enve lope, serial sectioning did not reveal con tinuity between the vacuolar membranes and either the nuclear or plasma membranes of the cell. No crystals were visible within the epithelial cytoplasm. Bowman's membrane was of normal thick ness (<—'9 [A) but showed occasional irregu larities where it had been split by large histiocytic cells containing rhomboid-shaped in clusions (Fig. 3). In the superficial stroma, keratocytes exhibited occasional crystalline material in the form of elongated rectangular or fusiform profiles (Figs. 5 and 6). These large, needle-shaped profiles (measuring up to 15 [/, in length) were invariably intracellular, were bounded by single membranes, and were always oriented parallel to the
Fig. 3 (Kenyon and Sensenbrenner). Light photomicrograph of peripheral cornea illus trates perinuclear inclusions (arrows) in corneal epithelial cells and histiocytes (asterisk) that split Bowman's membrane (B) (phase contrast, paraphenylenediamine, X 1,200).
Fisr 4 (Kenyon and Sensenbrenner). By electron microscopy, abnormal inclusions (asterisks) within corned epithelial cells contain homogeneous granular material. N indicates nucleus; BM, < f ^ ^ ment membrane; and B, Bowman's membrane. Inset, At higher magnification, the double membranes (arrows) bounding these vacuoles are visible (X7,300; inset, X42.000).
VOL. 78, NO. 1
71
CORNEA AND CONJUNCTIVA IN CYSTINOSIS
'$£fe
^<^*c-';4- ■!
•31
i*fe
.393
S'V:
Fig. S (Kenyon and Sensenbrenner). Inset, Light microscopy reveals crystal-containing cell (asterisk) in the superficial corneal stroma (S). E indicates corneal epithelium; and B, Bowman's membrane (phase contrast, paraphenylenediamine, X 1,200). By electron microscopy, the needle-shaped profiles of crystals within keratocytes are prominent. No crystalline images are evident in the collagenous stroma (S) ; N in dicates nucleus (X 20,000). plane of the stromal lamellae ( F i g . 5 ) . Smaller crystals (0.5 to 3 [A in length) ap peared to have formed within single mem brane-limited organelles having the typical morphologic features of secondary lysosomes (Fig. 6 ) . N o crystalline accumulations were apparent extracellularly, and derangement of stromal collagen did not indicate the location of extracellular crystals, which might have been dissolved during tissue processing in aqueous solutions. The axonal processes of corneal nerves were ultrastructurally normal. Descemet's membrane and endothelium were not in
cluded in the biopsy specimen. Conjunctiva—By light microscopy, crys talline profiles were occasionally seen in the conjunctival epithelium ( F i g . 7, inset), but perinuclear vacuoles (like those prominent in the corneal epithelium) were absent. Similar crystalline forms were particularly abundant throughout the conjunctival stroma where they appeared to be intracellular (Fig. 8, inset). I n both locations, the crystals were smaller (0.2 to 3 n in diameter) than those within the keratocytes, and were polygonal (mostly rectangular) rather than fusiform in configuration. W i t h the electron microscope,
72
AMERICAN JOURNAL OF OPHTHALMOLOGY
these crystalline forms appeared as sparse granular material within double membranelimited organelles that frequently displayed the marginated electron-dense material char acteristic of secondary lysosomes (Figs. 7 and 8). No extracellular crystals were visual ized by electron microscopy. Vascular endothelial and perithelial cells, lymphatic endothelial cells, Schwann cells, and neuronal processes contained no abnor mal inclusions. DISCUSSION
Histopathologic reports of the eye in child hood cystinosis have revealed crystals, which were abundant in conjunctiva, ciliary body, and choroid, were sparse in cornea, ciliary
JULY, 1974
muscle, iris, and sclera, and were nearly ab sent in retina, optic nerve, lens, and vitre ous.8'11"13 Previous studies of cystinosis retinopathy have demonstrated only focal ir regularities of thickness and pigmentation of pigment epithelial cells in the peripheral ret ina.8'14 More recently, however, in a cystinotic patient with maculopathy, Sanderson and associates12 were able to show crystalline deposits within retinal pigment epithelial cells by both light and electron microscopy. Cogan and Kuwabara 11 consistently noted a distinct distribution of crystalline forms in various ocular tissues, as needle-shaped crys tals were predominantly confined to the cor nea and sclera, whereas rectangular polyg onal forms were evident in the conjunctiva
Fig. 6 (Kenyon and Sensenbrenner). Inset, Light photomicrograph of stromal cell containing a large (—6 n) crystal (phase contrast, paraphenylenediamine, Xl,200). Electron micrograph of keratocyte in which small crystalline forms (asterisks) have seemingly developed within lysosome-like organelles. S in dicates stroma ( x 22,000).
VOL. 78, NO. 1
CORNEA AND CONJUNCTIVA IN CYSTINOSIS
73
I'
3T-*
v:w.
■Sv
-VA, Fig. 7 (Kenyon and Sensenbrenner). Inset, Light photomicrograph occasional crystals (arrow) within conjunctival epithelial cells ( E ) . S indicates stroma; and L, lymphatic (phase contrast, paraphenylenediamine, X 1,200). By electron microscopy, the limiting membrane and sparse content of the intraepithelial crystals are evident. BM indicates epithelial basement membrane; AF, anchoring fibrils; circle, hemidesmosome; and S, stroma (X27,000).
and uvea. Biochemical studies11 have sug gested that both crystalline variants are Lcystine, although definitive characterization by x-ray diffraction has been accomplished only for the conjunctival and uveal de posits.13 Ultrastructural examination of corneal and conjunctival biopsies from our patient with childhood cystinosis has revealed several new findings. First, as earlier histologic studies had observed,11 crystalline profiles within the cornea were exceedingly sporadic and were morphologically distinguished from those of
other tissues by their fusiform shape. In con trast, however, to light microscopic impres sions that most of these crystals were extra cellular,11 we invariably found them located intracellularly within membrane-limited, lysosome-like organdies. Sanderson and asso ciates12 recently confirmed this observation. Second, the presence of large perinuclear vacuoles within the corneal epithelial cells is distinctly abnormal. The granular content of these vacuoles resembles material within the lysosomes of keratocytes (Fig. 6) and may be comparable to ultrastructurally similar
74
AMERICAN JOURNAL OF OPHTHALMOLOGY
accumulations within cystinotic leukocytes and cultured fibroblasts where excessive intracellular cystine is stored in an amorphous noncrystalline form.15'16 Third, our observation of membrane-limited, polygonal crystalline material in both conjunctival epithelial and connective tissue cells extends the previous work of Wong and others,6'9 who described such profiles only in the conjunctival
JULY, 1974
stromal cells. Extracellular cystine deposits, as visualized by light microscopy in unfixed conjunctival specimens,7 were never detected here or in other ultrastructural studies of cystinotic ocular tissues,9'12 and we would, therefore, doubt their existence in vivo. The underlying biochemical defect responsible for the excessive accumulation of cystine remains unidentified. Current morpho-
Fig. 8 (Kenyon and Sensenbrenner). Inset, Light photomicrograph of the conjunctiva shows several crystals (arrows) within connective tissue cells, but none throughout the extracellular stroma (S). E in dicates conjunctival epithelium (phase contrast, paraphenylenediamine, X 1,200). Electron micrograph of conjunctival fibrocyte at high magnification illustrates the double limiting membranes (arrows) and elec tron-dense marginal material (asterisks) of crystal-containing lysosomes. M indicates mitochondrion, and C, stromal collagen fibrils (X61,000).
VOL. 78, NO. 1
CORNEA AND CONJUNCTIVA IN CYSTINOSIS
logic and biochemical evidence, however, indicates that the intracellular cystine de posits are compartmentalized within subcellular organelles having the ultrastructural characteristics, acid phosphatase activity, and phagocytic properties of lysosomes.9'17"20 The inability of cystinotic fibroblasts in vitro to use their intracellular cystine, even when needed for sustaining normal metabolism,17 further implicates a disorder of lysosomal function.21 A defective transport mechanism of the lysosomal limiting membrane, for ex ample, may be responsible for the irreversi ble intralysosomal sequestration of cystine.22 On the basis of static cellular morphologic characteristics, it is difficult to determine whether these intralysosomal crystals arise from autophagy of crystine produced by the cells in which it appears or from endocytosis of extracellular cystine.23 Both mechanisms may be involved. Since cystine is a nonessential amino acid in vivo, many cell types may be capable of its formation from methionine. On the other hand, Schneider and associ ates17 have observed that cystine accumula tion is most prominent in actively endocytotic cells, such as those of the reticuloendothelial system. In the cornea and conjunctiva, we were unable to detect ultrastructural evidence of either synthetic stimulation (e.g., dilated endoplasmic reticulum and hypertrophic Golgi apparatus) or accelerated endo cytosis (e.g., vesicular formation at the cell surface). Thus, in these tissues, the build-up of intracellular cystine by either mechanism would appear only slowly progressive, as is consistent clinically with the gradual increase of corneal crystals in cystinotic infants.8-11-12 For the cornea in particular, the endocytotic capability of the keratocytes,24'26 plus the preferential localization of cystine throughout the entire peripheral stroma and in the subepithelial and subendothelial cen tral stroma8-11-12 suggest that the keratocytic crystals may derive primarily from the limbal blood supply, tears, and aqueous humor. We observed no indication of phagocytic ingestion of preformed crystals by the kerato
75
cytes. On the contrary, the lack of crystalline profiles in the extracellular stroma and the appearance of the smallest intracellular crys tals within lysosome-like organelles suggest that intralysosomal accumulation of noncrystalline cystine precedes the formation of cys tine crystals. Given the de novo formation of intracellular crystals in this manner, local anatomic and physiologic factors may deter mine the shape assumed by the developing crystal. Thus, the compact lamellar organiza tion of the deturgescent corneal stroma could mechanically encourage the fusiform config uration and parallel alignment of crystals within keratocytes, whereas the loose connec tive tissue of the conjunctiva might permit the development of polygonal forms. SUMMARY
Electron microscopic examination of the cornea and conjunctiva of a 10^-year-old boy with childhood cystinosis revealed cor neal stromal cells with occasional fusiform crystalline profiles. The conjunctival epithe lial and connective tissue cells contained nu merous, smaller, polygonal forms. All such crystalline deposits, presumably representing the sites of cystine accumulation, appeared intracellularly within single or double mem brane-limited lysosome-like bodies. Corneal epithelial cells had large, double membranelimited perinuclear vacuoles that may indi cate a noncrystalline form of cystine storage. ACKNOWLEDGMENTS
We thank A. Edward Maumenee, M.D., for cor neal and conjunctival biopsies; Trexler M. Top ping, M.D., and Tiochiro Kuwabara, M.D., for manuscript review; Terry George, R.B.P., for pho tomicrography; and Kenneth A. Linberg for tech nical assistance. REFERENCES
1. Schneider, J. A., and Seegmiller, J. E.: Cys tinosis and the Fanconi syndrome. In Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S.: The Metabolic Basis of Inherited Disease, 3rd ed. New York, McGraw-Hill, 1972, p. 1581. 2. Goldman, H., Scriver, C. R., Aaron, K., Delvin, E., and Canlas, Z.: Adolescent cystinosis. Com parison with infantile and adult forms. Pediatrics 47 -.979, 1971.
76
AMERICAN JOURNAL OF OPHTHALMOLOGY
3. Cogan, D. G., Kuwabara, T., Kinoshita, J., Sheehan, L., and Merola, L.: Cystinosis in an adult. J.A.M.A. 164:394, 19S7. 4. Cogan, D. G., Kuwabara, T., Hurlbut, C. S., and McMurray, V.: Further observations on cys tinosis in the adult. J.A.M.A. 166:1725, 1958. 5. Lietman, P. S., Frazier, P. D., Wong, V. G., Shotton, B., and SeegmiUer, J. E.: Adult cystinosis. A benign disorder. Am. J. Med. 40:511, 1966. 6. Brubaker, R. F., Wong, V. G., Schulman, J. D., SeegmiUer, J. E., and Kuwabara, T.: Benign cystinosis. The clinical, biochemical and morpho logic findings in a family with two affected siblings. Am. J. Med. 49:546, 1970. 7. Cogan, D. G., Kuwabara, T., Kinoshita, J., Sudarsky, D., and Ring, H.: Ocular manifestations of systemic cystinosis. Arch. Ophthalmol. 55:36, 1956. 8. Wong, V. G., Lietman, P. S., and SeegmiUer, J. E.: Alterations of pigment epithelium in cystino sis. Arch. Ophthalmol. 77:361, 1967. 9. Wong, V. G., Kuwabara, T., Brubaker, R., Olson, W., Schulman, J., and SeegmiUer, J. E.: Intralysosomal crystine crystals in cystinosis. Invest. Ophthalmol. 9:83, 1970. 10. Sensenbrenner, J. A., Howell, R. R., Blizzard, R. M., and Kenyon, K. R.: Juvenile cystinosis with hypothyroidism. Birth Defects. In press. 11. Cogan, D. G., and Kuwabara, T.: Ocular pathology of cystinosis, with particular reference to the elusiveness of corneal crystals. Arch. Ophthal mol.. 63:51, 1960. 12. Sanderson, P. O., Kuwabara, T., Stark, W. J., and Wong, V. G.: Cystinosis. A clinical, histopathologic and ultrastructural study. Arch. Ophthalmol. In press. 13. Frazier, P. D., and Wong, V. G.: Cystinosis. Histologic and crystallographic examination of crystals in eye tissues. Arch. Ophthalmol. 80:87, 1968. 14. Frangois, J., Hanssens, M., Coppieters, R., and Evans, L.: Cystinosis. A clinical and histopathologic study. Am. J. Ophthalmol. 73:643, 1972. 15. Schulman, J. D., Wong, V. G., Kuwabara, T.,
JULY, 1974
Bradley, K. H., and SeegmiUer, J. E.: Intracellular cystine content of leukocyte populations in cystino sis. Arch. Intern. Med. 125 :660, 1970. 16. Hummeler, K., Zajac, B. A., Genel, M., Holtzapple, P. G., and Segal, S.: Human cystinosis. In tracellular deposition of cystine. Science 168:859, 1970. 17. Schneider, J. A., Rosenbloom, F. M., Bradley, K. H., and SeegmiUer, J. D.: Increased free-cystine content of fibroblasts cultured from patients with cystinosis. Biochem. Biophys. Res. Commun. 29:527, 1967. 18. Patrick, A. D., and Lake, B. D.: Cystinosis. Electron microscopic evidence of lysosomal storage of cystine in lymph node. J. Clin. Pathol. 21:571, 1968. 19. Schulman, J. D., Bradley, K. H., and Seeg miUer, J. E.: Cystine. Compartmentalization within lysosomes in cystinotic leukocytes. Science 166:1152, 1969. 20. Schulman, J. D., Wong, V. G., Olson, W. H., and SeegmiUer, J. E.: Lysosomal site of crystalline deposits in cystinosis as shown by ferritin uptake. Arch. Pathol. 90:259, 1970. 21. Hers, H. G.: Inborn lysosomal diseases. Gastroenterology 48:625, 1965. 22. Schulman, J. D., and Bradley, K. H.: In vitro studies on cystinosis. In Schulman, J. D. (ed.) : Cystinosis. Washington, DHEW Publication No. (NIH) 72-249, 1972, p. 111. 23. Ericsson, J. L. E.: Mechanism of cellular autophagy. In Dingle, J. T., and Fell, H. B. (eds.) : Lysosomes in Biology and Pathology, vol. 2. Fron tiers in Biology, vol. 14B. Amsterdam, North-Hol land Publishing Co., 1969, p. 345. 24. Kaye, G. I., and Pappas, G. D.: Studies on the cornea. 1. The fine structure of the rabbit cornea and the uptake and transport of colloidal particles by the cornea in vivo. J. Cell Biol. 12:457, 1962. 25. Klintworth, G. K.: Experimental studies on the phagocytic capability of the corneal fibroblast. Am. J. Pathol. 55:283, 1969.
M.D.
Baltimore, Maryland Cystinosis is an autosomal, recessively in herited disorder of amino acid metabolism characterized by the deposition of cystine crystals in the eye, kidney, reticuloendothelial system, and various other tissues.1 Most se vere of the three recognized forms is the childhood or nephropathic form in which progressive renal tubular insufficiency leads to growth retardation, rickets, and early uremic death.1 Goldman and associates2 de scribed a rare juvenile form in which only mild nephropathy develops. Adult cystinosis appears to be a completely benign disorder.3-6 In all three variants, the slit-lamp appear ance of the fine, scintillating, tinsel-like crys tals dispersed throughout the anterior cornea, conjunctiva, and iris may be diagnostic.3'7 Additional distinguishing ocular features of childhood cystinosis are photophobia and mottled pigmentary retinopathy of the pe ripheral fundus.8 Previous electron microscopic studies of ocular tissues affected by cystinosis have focused primarily on the conjunctiva.6'9 The present study is, to our knowledge, the first detailed report of corneal ultrastructure in a cystinotic child.
including marked growth retardation and hypopigmentation of skin and hair (Fig. 1). Ocular find ings included severe photophobia (Fig. 1) and uncorrected visual acuity of 20/20 bilaterally. By slitlamp biomicroscopy, refractile crystalline deposits appeared throughout the anterior corneal stroma (Fig. 2) and bulbar conjunctiva. Indirect ophthalmoscopy revealed typical patchy pigmentary changes in the peripheral f undi. OBSERVATIONS
Biopsy specimens of bulbar conjunctiva and peripheral superficial cornea were im mediately fixed in 2 % osmium tetroxide (in 0.14M Veronal acetate buffer, p H 7.2) for 60 minutes at 0°C, and were embedded in Araldite epoxy resin. Paraphenylenediamine stained 1-jx-thick sections for phase contrast photomicrography. Ultrathin sections were stained with uranyl acetate and lead citrate,
C A S E REPORT
The patient was a 10^-year-old white boy ( J H H 141 03 45) who has been previously reported.10 Al though diabetes insipidus and dwarfism had been noted by age 2 years, cystinosis was initially diag nosed at 4 years of age, when marked photophobia prompted ophthalmic examination, which disclosed corneal crystals. At age 8^4 years (1970), evalua tion at Johns Hopkins Hospital revealed the char acteristic physical features of childhood cystinosis, From the Eye Pathology Laboratory, Wilmer In stitute, and the Moore Clinic of the Johns Hopkins Hospital, Baltimore, Maryland. This study was sup ported in part by the Carrie L. Groedel Fund of Fight for Sight, Inc. (Dr. Kenyon). Reprint requests to Kenneth R. Kenyon, M.D., Eye Pathology Laboratory, Wilmer Institute, 601 N. Broadway, Baltimore, MD 21205.
Fig. 1 (Kenyon and Sensenbrenner). Childhood cystinosis demonstrates severe photophobia and de creased pigmentation of skin and hair.
VOL. 78, NO.l
CORNEA AND CONJUNCTIVA IN CYSTINOSIS
Fig. 2 (Kenyon and Sensenbrenner). Slit-lamp photograph shows strikingly refractile crystals dis persed throughout the anterior corneal stroma.
and were examined with a JEM 100-B elec tron microscope. Cornea—By light microscopy, many cor
69
neal epithelial cells, particularly in the inter mediate layers, contained abnormal perinuclear inclusions, 2 to 5 [x in diameter (Fig. 3). With the electron microscope, these inclusions proved to be double membranelimited vacuoles with homogeneous granular contents of medium electron density (Fig. 4). Although the vacuolar membranes were always closely apposed to the nuclear enve lope, serial sectioning did not reveal con tinuity between the vacuolar membranes and either the nuclear or plasma membranes of the cell. No crystals were visible within the epithelial cytoplasm. Bowman's membrane was of normal thick ness (<—'9 [A) but showed occasional irregu larities where it had been split by large histiocytic cells containing rhomboid-shaped in clusions (Fig. 3). In the superficial stroma, keratocytes exhibited occasional crystalline material in the form of elongated rectangular or fusiform profiles (Figs. 5 and 6). These large, needle-shaped profiles (measuring up to 15 [/, in length) were invariably intracellular, were bounded by single membranes, and were always oriented parallel to the
Fig. 3 (Kenyon and Sensenbrenner). Light photomicrograph of peripheral cornea illus trates perinuclear inclusions (arrows) in corneal epithelial cells and histiocytes (asterisk) that split Bowman's membrane (B) (phase contrast, paraphenylenediamine, X 1,200).
Fisr 4 (Kenyon and Sensenbrenner). By electron microscopy, abnormal inclusions (asterisks) within corned epithelial cells contain homogeneous granular material. N indicates nucleus; BM, < f ^ ^ ment membrane; and B, Bowman's membrane. Inset, At higher magnification, the double membranes (arrows) bounding these vacuoles are visible (X7,300; inset, X42.000).
VOL. 78, NO. 1
71
CORNEA AND CONJUNCTIVA IN CYSTINOSIS
'$£fe
^<^*c-';4- ■!
•31
i*fe
.393
S'V:
Fig. S (Kenyon and Sensenbrenner). Inset, Light microscopy reveals crystal-containing cell (asterisk) in the superficial corneal stroma (S). E indicates corneal epithelium; and B, Bowman's membrane (phase contrast, paraphenylenediamine, X 1,200). By electron microscopy, the needle-shaped profiles of crystals within keratocytes are prominent. No crystalline images are evident in the collagenous stroma (S) ; N in dicates nucleus (X 20,000). plane of the stromal lamellae ( F i g . 5 ) . Smaller crystals (0.5 to 3 [A in length) ap peared to have formed within single mem brane-limited organelles having the typical morphologic features of secondary lysosomes (Fig. 6 ) . N o crystalline accumulations were apparent extracellularly, and derangement of stromal collagen did not indicate the location of extracellular crystals, which might have been dissolved during tissue processing in aqueous solutions. The axonal processes of corneal nerves were ultrastructurally normal. Descemet's membrane and endothelium were not in
cluded in the biopsy specimen. Conjunctiva—By light microscopy, crys talline profiles were occasionally seen in the conjunctival epithelium ( F i g . 7, inset), but perinuclear vacuoles (like those prominent in the corneal epithelium) were absent. Similar crystalline forms were particularly abundant throughout the conjunctival stroma where they appeared to be intracellular (Fig. 8, inset). I n both locations, the crystals were smaller (0.2 to 3 n in diameter) than those within the keratocytes, and were polygonal (mostly rectangular) rather than fusiform in configuration. W i t h the electron microscope,
72
AMERICAN JOURNAL OF OPHTHALMOLOGY
these crystalline forms appeared as sparse granular material within double membranelimited organelles that frequently displayed the marginated electron-dense material char acteristic of secondary lysosomes (Figs. 7 and 8). No extracellular crystals were visual ized by electron microscopy. Vascular endothelial and perithelial cells, lymphatic endothelial cells, Schwann cells, and neuronal processes contained no abnor mal inclusions. DISCUSSION
Histopathologic reports of the eye in child hood cystinosis have revealed crystals, which were abundant in conjunctiva, ciliary body, and choroid, were sparse in cornea, ciliary
JULY, 1974
muscle, iris, and sclera, and were nearly ab sent in retina, optic nerve, lens, and vitre ous.8'11"13 Previous studies of cystinosis retinopathy have demonstrated only focal ir regularities of thickness and pigmentation of pigment epithelial cells in the peripheral ret ina.8'14 More recently, however, in a cystinotic patient with maculopathy, Sanderson and associates12 were able to show crystalline deposits within retinal pigment epithelial cells by both light and electron microscopy. Cogan and Kuwabara 11 consistently noted a distinct distribution of crystalline forms in various ocular tissues, as needle-shaped crys tals were predominantly confined to the cor nea and sclera, whereas rectangular polyg onal forms were evident in the conjunctiva
Fig. 6 (Kenyon and Sensenbrenner). Inset, Light photomicrograph of stromal cell containing a large (—6 n) crystal (phase contrast, paraphenylenediamine, Xl,200). Electron micrograph of keratocyte in which small crystalline forms (asterisks) have seemingly developed within lysosome-like organelles. S in dicates stroma ( x 22,000).
VOL. 78, NO. 1
CORNEA AND CONJUNCTIVA IN CYSTINOSIS
73
I'
3T-*
v:w.
■Sv
-VA, Fig. 7 (Kenyon and Sensenbrenner). Inset, Light photomicrograph occasional crystals (arrow) within conjunctival epithelial cells ( E ) . S indicates stroma; and L, lymphatic (phase contrast, paraphenylenediamine, X 1,200). By electron microscopy, the limiting membrane and sparse content of the intraepithelial crystals are evident. BM indicates epithelial basement membrane; AF, anchoring fibrils; circle, hemidesmosome; and S, stroma (X27,000).
and uvea. Biochemical studies11 have sug gested that both crystalline variants are Lcystine, although definitive characterization by x-ray diffraction has been accomplished only for the conjunctival and uveal de posits.13 Ultrastructural examination of corneal and conjunctival biopsies from our patient with childhood cystinosis has revealed several new findings. First, as earlier histologic studies had observed,11 crystalline profiles within the cornea were exceedingly sporadic and were morphologically distinguished from those of
other tissues by their fusiform shape. In con trast, however, to light microscopic impres sions that most of these crystals were extra cellular,11 we invariably found them located intracellularly within membrane-limited, lysosome-like organdies. Sanderson and asso ciates12 recently confirmed this observation. Second, the presence of large perinuclear vacuoles within the corneal epithelial cells is distinctly abnormal. The granular content of these vacuoles resembles material within the lysosomes of keratocytes (Fig. 6) and may be comparable to ultrastructurally similar
74
AMERICAN JOURNAL OF OPHTHALMOLOGY
accumulations within cystinotic leukocytes and cultured fibroblasts where excessive intracellular cystine is stored in an amorphous noncrystalline form.15'16 Third, our observation of membrane-limited, polygonal crystalline material in both conjunctival epithelial and connective tissue cells extends the previous work of Wong and others,6'9 who described such profiles only in the conjunctival
JULY, 1974
stromal cells. Extracellular cystine deposits, as visualized by light microscopy in unfixed conjunctival specimens,7 were never detected here or in other ultrastructural studies of cystinotic ocular tissues,9'12 and we would, therefore, doubt their existence in vivo. The underlying biochemical defect responsible for the excessive accumulation of cystine remains unidentified. Current morpho-
Fig. 8 (Kenyon and Sensenbrenner). Inset, Light photomicrograph of the conjunctiva shows several crystals (arrows) within connective tissue cells, but none throughout the extracellular stroma (S). E in dicates conjunctival epithelium (phase contrast, paraphenylenediamine, X 1,200). Electron micrograph of conjunctival fibrocyte at high magnification illustrates the double limiting membranes (arrows) and elec tron-dense marginal material (asterisks) of crystal-containing lysosomes. M indicates mitochondrion, and C, stromal collagen fibrils (X61,000).
VOL. 78, NO. 1
CORNEA AND CONJUNCTIVA IN CYSTINOSIS
logic and biochemical evidence, however, indicates that the intracellular cystine de posits are compartmentalized within subcellular organelles having the ultrastructural characteristics, acid phosphatase activity, and phagocytic properties of lysosomes.9'17"20 The inability of cystinotic fibroblasts in vitro to use their intracellular cystine, even when needed for sustaining normal metabolism,17 further implicates a disorder of lysosomal function.21 A defective transport mechanism of the lysosomal limiting membrane, for ex ample, may be responsible for the irreversi ble intralysosomal sequestration of cystine.22 On the basis of static cellular morphologic characteristics, it is difficult to determine whether these intralysosomal crystals arise from autophagy of crystine produced by the cells in which it appears or from endocytosis of extracellular cystine.23 Both mechanisms may be involved. Since cystine is a nonessential amino acid in vivo, many cell types may be capable of its formation from methionine. On the other hand, Schneider and associ ates17 have observed that cystine accumula tion is most prominent in actively endocytotic cells, such as those of the reticuloendothelial system. In the cornea and conjunctiva, we were unable to detect ultrastructural evidence of either synthetic stimulation (e.g., dilated endoplasmic reticulum and hypertrophic Golgi apparatus) or accelerated endo cytosis (e.g., vesicular formation at the cell surface). Thus, in these tissues, the build-up of intracellular cystine by either mechanism would appear only slowly progressive, as is consistent clinically with the gradual increase of corneal crystals in cystinotic infants.8-11-12 For the cornea in particular, the endocytotic capability of the keratocytes,24'26 plus the preferential localization of cystine throughout the entire peripheral stroma and in the subepithelial and subendothelial cen tral stroma8-11-12 suggest that the keratocytic crystals may derive primarily from the limbal blood supply, tears, and aqueous humor. We observed no indication of phagocytic ingestion of preformed crystals by the kerato
75
cytes. On the contrary, the lack of crystalline profiles in the extracellular stroma and the appearance of the smallest intracellular crys tals within lysosome-like organelles suggest that intralysosomal accumulation of noncrystalline cystine precedes the formation of cys tine crystals. Given the de novo formation of intracellular crystals in this manner, local anatomic and physiologic factors may deter mine the shape assumed by the developing crystal. Thus, the compact lamellar organiza tion of the deturgescent corneal stroma could mechanically encourage the fusiform config uration and parallel alignment of crystals within keratocytes, whereas the loose connec tive tissue of the conjunctiva might permit the development of polygonal forms. SUMMARY
Electron microscopic examination of the cornea and conjunctiva of a 10^-year-old boy with childhood cystinosis revealed cor neal stromal cells with occasional fusiform crystalline profiles. The conjunctival epithe lial and connective tissue cells contained nu merous, smaller, polygonal forms. All such crystalline deposits, presumably representing the sites of cystine accumulation, appeared intracellularly within single or double mem brane-limited lysosome-like bodies. Corneal epithelial cells had large, double membranelimited perinuclear vacuoles that may indi cate a noncrystalline form of cystine storage. ACKNOWLEDGMENTS
We thank A. Edward Maumenee, M.D., for cor neal and conjunctival biopsies; Trexler M. Top ping, M.D., and Tiochiro Kuwabara, M.D., for manuscript review; Terry George, R.B.P., for pho tomicrography; and Kenneth A. Linberg for tech nical assistance. REFERENCES
1. Schneider, J. A., and Seegmiller, J. E.: Cys tinosis and the Fanconi syndrome. In Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S.: The Metabolic Basis of Inherited Disease, 3rd ed. New York, McGraw-Hill, 1972, p. 1581. 2. Goldman, H., Scriver, C. R., Aaron, K., Delvin, E., and Canlas, Z.: Adolescent cystinosis. Com parison with infantile and adult forms. Pediatrics 47 -.979, 1971.
76
AMERICAN JOURNAL OF OPHTHALMOLOGY
3. Cogan, D. G., Kuwabara, T., Kinoshita, J., Sheehan, L., and Merola, L.: Cystinosis in an adult. J.A.M.A. 164:394, 19S7. 4. Cogan, D. G., Kuwabara, T., Hurlbut, C. S., and McMurray, V.: Further observations on cys tinosis in the adult. J.A.M.A. 166:1725, 1958. 5. Lietman, P. S., Frazier, P. D., Wong, V. G., Shotton, B., and SeegmiUer, J. E.: Adult cystinosis. A benign disorder. Am. J. Med. 40:511, 1966. 6. Brubaker, R. F., Wong, V. G., Schulman, J. D., SeegmiUer, J. E., and Kuwabara, T.: Benign cystinosis. The clinical, biochemical and morpho logic findings in a family with two affected siblings. Am. J. Med. 49:546, 1970. 7. Cogan, D. G., Kuwabara, T., Kinoshita, J., Sudarsky, D., and Ring, H.: Ocular manifestations of systemic cystinosis. Arch. Ophthalmol. 55:36, 1956. 8. Wong, V. G., Lietman, P. S., and SeegmiUer, J. E.: Alterations of pigment epithelium in cystino sis. Arch. Ophthalmol. 77:361, 1967. 9. Wong, V. G., Kuwabara, T., Brubaker, R., Olson, W., Schulman, J., and SeegmiUer, J. E.: Intralysosomal crystine crystals in cystinosis. Invest. Ophthalmol. 9:83, 1970. 10. Sensenbrenner, J. A., Howell, R. R., Blizzard, R. M., and Kenyon, K. R.: Juvenile cystinosis with hypothyroidism. Birth Defects. In press. 11. Cogan, D. G., and Kuwabara, T.: Ocular pathology of cystinosis, with particular reference to the elusiveness of corneal crystals. Arch. Ophthal mol.. 63:51, 1960. 12. Sanderson, P. O., Kuwabara, T., Stark, W. J., and Wong, V. G.: Cystinosis. A clinical, histopathologic and ultrastructural study. Arch. Ophthalmol. In press. 13. Frazier, P. D., and Wong, V. G.: Cystinosis. Histologic and crystallographic examination of crystals in eye tissues. Arch. Ophthalmol. 80:87, 1968. 14. Frangois, J., Hanssens, M., Coppieters, R., and Evans, L.: Cystinosis. A clinical and histopathologic study. Am. J. Ophthalmol. 73:643, 1972. 15. Schulman, J. D., Wong, V. G., Kuwabara, T.,
JULY, 1974
Bradley, K. H., and SeegmiUer, J. E.: Intracellular cystine content of leukocyte populations in cystino sis. Arch. Intern. Med. 125 :660, 1970. 16. Hummeler, K., Zajac, B. A., Genel, M., Holtzapple, P. G., and Segal, S.: Human cystinosis. In tracellular deposition of cystine. Science 168:859, 1970. 17. Schneider, J. A., Rosenbloom, F. M., Bradley, K. H., and SeegmiUer, J. D.: Increased free-cystine content of fibroblasts cultured from patients with cystinosis. Biochem. Biophys. Res. Commun. 29:527, 1967. 18. Patrick, A. D., and Lake, B. D.: Cystinosis. Electron microscopic evidence of lysosomal storage of cystine in lymph node. J. Clin. Pathol. 21:571, 1968. 19. Schulman, J. D., Bradley, K. H., and Seeg miUer, J. E.: Cystine. Compartmentalization within lysosomes in cystinotic leukocytes. Science 166:1152, 1969. 20. Schulman, J. D., Wong, V. G., Olson, W. H., and SeegmiUer, J. E.: Lysosomal site of crystalline deposits in cystinosis as shown by ferritin uptake. Arch. Pathol. 90:259, 1970. 21. Hers, H. G.: Inborn lysosomal diseases. Gastroenterology 48:625, 1965. 22. Schulman, J. D., and Bradley, K. H.: In vitro studies on cystinosis. In Schulman, J. D. (ed.) : Cystinosis. Washington, DHEW Publication No. (NIH) 72-249, 1972, p. 111. 23. Ericsson, J. L. E.: Mechanism of cellular autophagy. In Dingle, J. T., and Fell, H. B. (eds.) : Lysosomes in Biology and Pathology, vol. 2. Fron tiers in Biology, vol. 14B. Amsterdam, North-Hol land Publishing Co., 1969, p. 345. 24. Kaye, G. I., and Pappas, G. D.: Studies on the cornea. 1. The fine structure of the rabbit cornea and the uptake and transport of colloidal particles by the cornea in vivo. J. Cell Biol. 12:457, 1962. 25. Klintworth, G. K.: Experimental studies on the phagocytic capability of the corneal fibroblast. Am. J. Pathol. 55:283, 1969.