Exp. Eye Res. (1979) 29, 479-484

The Chemical Composition of Vitreous Hyalocyte Granules 1. FREE&IAN,*

XELVIN

BERXARD

JACOBSON

AXD

ENDRE

A.

BALAZS

of F&e Structure Reseurch,Boston Biomedical ResearchI&&de, 20 Stnniford Street: Boston, Ma.ss02114, U.S.A. and 3fatrix Biology La,boratory, Depwtment of Ophthalmology, College of Physicians and Suryeons, Columbiu University, 630 West 168th Str.eet,JTew York, fl. Y. 10X2, U.S.A..

Depurtmesat

(Received15 December1978, 5ezv York) Hyalocytes are macrophage-like resident cells of the cortical vitreous gel. Vsing biochemical methods we could demonstrate that the lysosomal granular fraction of these cells, obtained from calves. contain glycosaminoglycans, glycoproteins and nucleic acids. Our previous study demonstrated the presence of characteristic lysosomal hydrolytic enzymes in these granules. Therefore, the presence of these macromolecules may indicate their normal pathway of catabolism. Key zoords: hya!ocytes; glycosaminoglycans; hya!uronic acid; lysosomes; vitreous.

1. Introduction The subcellular organelles of calf vitreous hyalocytes have been separated, according to their sedimentation properties, into “nuclear”, “granular”, and “mitochondrialmicrosomalvl:’ fractions. A previous report (Freeman, Jacobson, Toth and Balazs, 1968) provided proof for the identity of the hyalocyte granules as lysosomes, as evidenced by the presence of characteristic lysomal enzymes, such as acid phosphatase and P-glucuronidase. Despite surrounding collagen and marked polydispersity of fraction was obtained which was characterized by both eo.ranule size, a “granular” the absence of nuclear material under electron microscopic observation and low degree of mitochondrial activity, as determined by the level of XADH oxidation (Freeman et al., 1968). The isolated fractions were assayed for the acid hydrolases, acid phosphatase and P-glucuronidase. Alkaline phosphatase, an enzyme known to be associated with leukocyte granules, was also demonstrated. The ‘igranular’i fraction from calf vitreous hyalocytes was found to exhibit the highest specific activity of acid hpdrolases which, in addition, were found to be in a latent form, suggesting lysosomal activity associated with these granu!es. In addition to these enzymes, the presence of esterase in the cortical tissue layer, containing hyalocytes has been demonstrate (Balazs, 1960). Numerous investigators have described the heterogenous lpsosomal granules in various other tissues including liver, kidney, brain, macrophages, leukocytes, gut and bladder epithelium. The concept of lysosome action, as formulated by DeDuve and Wattiaux (1966), proposes the fusion of a primary lysosome (containing only enzyme) with a phagosome (containing substrate for digestion) to form a secondary lysosome * Present address: The Mason Clinic, 1118 Ninth Avenue, Seattle, Washington Reprint requests to: E. A. Balazs, Department of Ophthalmology, Research Physicians and Surgeons, 630 West 168th Street, New York, N.Y. 10032, U.S.A. 0014-4835/79/110479+06 B

$01.00/O

0 1979 Academic 479

98101, U.S.A. Division, College

Press Inc. (London)

Limited

of

450

M/I. I. FREEMAS,

B. JACOBSON

AND

E. A. BALAZS

wherein digestion takes place. The substrates within the phagosome may be of intracellular or extracellular origin. Detailed histochemical studies on the granules of hyalocytes from bovine and human vitreous indicated that these lysosomal granules may contain glycoproteins and polyanionic macromolecules (Szirmai, Balazs, 1958; Balazs, 1960; Balazs, Toth, Eckl and Mitchell, 1964). Positive periodic acid-Schiff (PAS) reaction was found to be characteristic for the hyalocyte granules, which would indicate the presence of carbohydrate rich proteins (glycoproteins). The hyalocyte granules also stain positively with Feulgen reaction for DNA and show strong basophilia, that is: uptake of cationic dyes (azur A and alcian blue). This latter reaction usually indicates the presence of glycosaminoglycans or nucleic acids. The present studies were undertaken to ascertain with biochemical methods whether or not the hya.locyte granules contain glycoproteins, glycosaminoglycans and nucleic acids. 2. Materials and Methods Isola,tio% mad incubation

of hyalocytes

The eyes of calves (2-4 months old) were collected within minutes after the death of the animals, placed on ice and transported to the laboratory within 1 hr. The combination of enzyme digestion and centrifugation methods described earlier were used to isolate the hyalocytes from the posterior vitreous (Freeman et al., 1968). In a typical experiment hyalocytes of 100 calf eyes were collected and suspended in 2 ml Earles balanced salt solution containing 100 mCi of either uniformly labelled [14C]glucosamine (7.56 mCi/mmol), methyl-[3H]thymidine (6.7 Ci/mmol), or 5-[“Hluridine (8 Ci/mmol). The [3H]thymidine and [3H]uridine were obtained from Schwarz Bioresearch and [14C]glucosamine was purchased from New England Nuclear Corporation. The cells were incubated under constant agitation for 15 hr at 4°C which was immediately followed with 6 hr incubation at 37°C. Some samples were incubated without and others with a mixture of antibiotics (Penicillin, 200 U/ml, Streptomycin, 200 pg/ml). Preparation

of the naacromolecular

fractiola

of the panules

uftev incubation

After incubation, the cells were homogenized and fractionated as previously described (Freeman et al., 1968) to collect the granule fraction which was then sonicated to free the soluble contents. The sonicated granule fractions of 3-4 ml volume Tq-ere dialyzed under constant shaking for 48-72 hr at 4°C against 4 liters of frequently changed distilled water. The retentate, representing the macromolecular fraction of the granules, was then assayed for radioactivity in a Beckman IS-200 liquid scintillation spectrometer. All counts were performed in duplicate for 20 min or to an error of 2 ‘A with appropriate corrections for background. The averages of the duplicate counts were used. Enzyme digestions were carried out by adding 1 ml of enzyme solutions to 0.5 ml of macromolecular fraction, previously dialyzed against distilled water. The digestion was carried out at 37°C for 16-24 hr under constant shaking. The following enzyme solutions were used: Three mg testicular hyaluronidase was dissolved in 1.2 ml of 0.1 Br-Ka acetate buffer (pH 5.0) which contained 0.15 M-NaCl. Ten mg pronase was dissolved in 1 ml 0.1 M-Tris buffer (pH 7.8) which contained 1O-3 M-Call,. One mg Neuraminidase was dissolved in 1 ml 0.1 pi-Na acetate buffer (pH 5.0). Thirty mg ribonuclease /3 was dissolved 30 mg of enzyme was added in 1 ml 0.1 M-Na acetate buffer (pH 5.0), and an additional 24 hr later and the incubation was continued for another 16 hr. Two and a half mg desoxyribonuclease I was dissolved in 1 ml O-08 n;l-potassium phosphate buffer (pH 6.8) which contained 0.1 M-NagI. After 24 hr digestion, an additional 5 mg of enzyme was added and the incubation was continued for another 16 hr. Hyahrronidase, DNAse and

COMPOSITION

OF

HYALOCYTE

GRANULES

4Y1

RNAse were obtained from Worthington Biochemical Corp., pronase from Cabbiochem Inc. and neuraminidase from Sigma Chemical Corp. At the end of the incubation period, the digested samples were dialyzed for 48 hr at 4°C against 2 liters of distilled water which was periodically changed. The dialysis was carried out under constant shaking. The retentate was assayed for radioactivity. The dialyzates in some cases were collected and concentrated on a rotary evaporator and also assayed for radioactivity. i%actionatio?z

extract

of the macromolecular

The undiaiyzable macromolecules of the granule fraction were further fractionated by the cetylpyridinium chloride (C.P.C.) method (Scott, 1965). One percent CPC dissolved in 0.01 3+Na,SO, was used as precipitant. Eight mg hyaluronic acid and 7 mg chondroitin (i-sulfate were used as cold carriers. They were dissolved in 10 ml of the macromolecular fraction of the granules. To one volume of this solution 3 volumes of CPC solution were added. The precipitate formed was washed with the CPC solution several times and then a sequential solubilization of the precipitate with 0.2 If-Na,SO,, 1 n!r-MgCl, and 4 ~-Kc1 was carried out. The radioactivity in the solubilized fractions was assayed. The radioactivity in the insoluble residue was not determined.

3. Results The uptake of the radioactively labelled precursors into the non-dialyzable macromolecular fraction of the granules is shown on Table I. The data represent a typical experiment of several independently prepared granule fractions of hyalocytes. The uptake of [14C]D-glucosamine, [3H]thynGdine and [3H]uridine is significantly higher when the incubation was carried out without antibiotics in the media. The bacterial count in the media after incubation was approximately 10 times greater in the absence TABLE

I

Uptake of radioactive precursors into the macromolecules granular fmction of hyalocytes

of the

Non-dialyzable PiT3CUrSOr

Radioactivity(pCi)

[‘*C]D-Glucosamine [3H]Thymidine [3H]Uridine

The incubation of the hyalocytea ately followed by 6 hr at 37°C.

without

30 75 135

was carried

radioactivity (ctjmin) antibiotics with antibiotics

18 265 6250 44 640

out with

or without

15 525 5300 37 510

antibiotics

for 15 hr at 4°C immedi-

of antibiotics than in their presence. When the incubation of the hyalocytes was. carried out only at 4”C, non-dialyxable radioactivity in the granule fraction was not.. significantly higher than the background. The CPC fractionation of the macromolecules of the granules showed that 25%. of radioactivity wa,s present in the 0.2 M-Na,XO,, 9% in the 1 M-Mgcl, and 28% in the 4 M-KC1 soluble fraction. This means that -38% of the total radioactivity was not recovered and presumably remained in the insoluble residue.

482

M. I. FREEMAX,

B. JACOBSON

AND

E. A. BALAZS

Digestion with DKAse and RP\‘Ase renders dialyzable nearly all of the [3H]thymidine and [3H]uridine labelled macromolecules (Table II). Hyaluronidaae treatment degrades and makes dialyzable only 33% of the [14C]glucosamine-labelled macromolecules. When the retentate of the hyaluronidase treated and dialyzed sample is incubated with neuraminidase 70% of the radioactivity became dialyzable. This indicates that one third of the [14C]glucosamine label is part of macromolecules which are hyaluronidase sensitive glycosaminoglycans (hyaluronic acid and chondroitin sulfates) and approximately 47% is labelled neurominic acid on glycoproteins. Pronase treatment renders dialyzable 71% of all [14C]glucosanline labelling suggesting that approximately two-thirds of this label is associated with glycoproteins (Table II). TABLE

II

Enzyme digestiors of [3H] n?zd[‘*Cl labelled vnacromolecules prepared from the granular fraction of hyalocytes

Hyaluronidase Pronase [14C]-glucosamine

Precursors

7; of radioactivity lost during dialysis after digestion

* This digestion dase.

was carried

33

Digestion by DNAse [3H]-thymidine

71

out on the retentate

93

of samples

which

RNAse [3H]-nridine

Neuroaminidase* [WI-glucosamine

94

were digested

70

first with

hyaluroni-

4. Discussion Although D-ghOS%Iline is not a naturally-occurring physiological precursor, it is readily incorporated, via a seriesof intermediates, into the hexosamine moiety of both glycoproteins and glucosaminoglycans such as hyaluronic acid and chondroitin sulfates. [aH]thymidine was used as a marker for DXA and [3H]uridine asa marker for RNA. One can assumethat the granule fraction of hyalocytes does not contain enzyme systems for the synthesis of glycosaminoglycans, glycoproteins, DNA and RNA. Therefore, considerably greater incorporation of radioactivity from [14C]~glueosamineinto the granule fraction in the absenceof antibiotics can be explained only by two mechanisms.First, impurities can be present in this fraction from other cell organelleswhich are involved in the glycosaminoglycan or glycoprotein synthesis. This explanation is unlikely becausein samplesincubated at 4°C the radioactivity in the macromolecular fraction is not significantly higher than the background. Secondly, the labelled macromolecules may originate from extracellular or intracellular pools of newly synthesized macromolecules. The high radioactivity obtained in the granule fraction after incubation with [3H]thymidine and [3H&ridine strongly suggeststhat the granules of hyalocytes during incubation at 37°C incorporate macromolecules or particles into themselves which are not synthesized by them directly. Since the radioactivity of the macromolecular fraction is 15-2Oo/, lower in the presence of antibiotics, the bacteria propagated in the medium may be one of the sources of the labelled macromolecules, This would suggest that the bacteria are phagocyted or bacterial macromolecules pinocyted into the cells and stored in the granules. The

COMPOSITION

OF

HYALOCYTE

GRANULES

453

low level of radioactivity in the granules at 4°C incubation also suggests that the metabolic (phagocytic) activity of the cells are important in this process. The enzyme digestion studies clearly show that the granular fraction contain RXA and DNA which were synthesized during the incubation period. The source of these nucleic acids must be phagocyted bacteria and cell organelles of disintegrated cells. The possibility of nucleic acid storage in these granules originating from synthetic activities of the same cell can not be ruled out. The presence of glycoproteins in the granules is clearly shown by three findings. First,ly: that pronase digestion degrades 71% of all the [14C]glucosamine labelled macromolecules and that the hyaluronidase insensitive macromolecules when digested with neuraminidase lose a considerable amount (70%) of radioactivity. This radioactivity must represent terminal sialic acid residues on the oligosaccharide chain of glycoproteins. The radioactivity remaining in the macromolecular fraction after hyaluronidase and pronase treatment presumably represents the glucosamine and galactosamine moieties of the oligosaccharide chains of glycoproteins. The CPC fractionation also suggests the presence of glycoproteins. The CPC precipitate solubilized with 4 x-KC1 should represent a large part of glycoproteins. This fraction represented actually 28% of all the [14C] radioactivity in the macromolecular fraction. It is expected that most of the CPC complex which could not be solubilized and contained -38% of the radioactivity also represents denatured. glycoproteins. The presence of the hyaluronic acid in the macromolecular fraction is indicated primarily by the presence of 25% of the [14C] radioactivity in the CPC complex soluble in 0.2 nl-Na,SO,, and supported by the finding that testis hyaluronidase treatment causes degradation of macromolecules representing 33% of this label. Testis hyaluronidase however, degrades chondroitin sulfates as well (Gorham, Olaveson and Dodgson, 1975). Therefore the 9% [14C] present in CPC precipitate solubilized with 1 M-$$l, could very well represent this sulfated glycosaminoglycan. It is of some interest to point out that the glycosaminoglycan present in the extracellular matrix in which the hyalocytes are embedded is predominantly hya,luronic acid in calf vitreous. However, sulfated galactosaminoglycans were described in small quantities (< 5% of the hyaluronic acid content) in bovine (Balazs, Laurent and Laurent, 1959 ; Ba,lazs; 1965) and humanvitreous (Breen, Bizzelland Veinsheim, 1977). The results presented above suggest that macromolecules~, such as hyaluronic acid, sulfated glycosaminoglycans, glycoproteins and nucleic acids, appear in purified granule preparations following incubation of intact hyalocytes with labelled precursors. Enzymes capable of degrading these macromolecules have been shown to be present in lysosomes prepared from several tissues (Weissman, 1965). Therefore, it is possible that hyalocyte granules function in the phagocytic and digestive processes of these cells. It is possible that hyalocytes can internalize the extracellular proteoglycans and hyaluronic acid. The endocytic vesicles then would deliver them to the degradative apparatus of the lysosomes. Pinocytosis of [14C]hyaluronate by cultured rat hepatocytes and human synovial cells were described recently (Truppe, Basner, von Figura and Kresse, 1977). Human synovial cells are mononuclear phagocytes, like hyaloqytes, it was shown that they have endocytic vesicles (Bloom and Balazs, ! 965). Thus the possibility exists that these cells at a certain stage internalize hyaluronic acid from the extracellular environment and at another stage of their life cycle after repair or new synthesis of this polysaccharide they release to the extracellular matrix.

484

M. I. FREEMAN,

B. SACOBSOI\T

AND

E. A. BALAZS

ACKNOWLEDGMEKTS This National Public

project Eye Health

was supported Institute and Service.

by by

Grant Nos EY 01747 and EY a Special Pellowship (lPllNB-03

00810 awarded VSN) from

by the the U.S.

REFERENCES Balazs, E. A. (1960). Physiology of the vitreous. In Importance of the Vitreous Body in Retina Xwrgery with Special Emphasis on Reopen-dons, (Ed, Schepens, C. L.). Pp. 29-48. C. V. Mosby CO., St. Louis, MO. Balazs, E. A. (1965). Amino sugar-containing macromolecules in the t,issues of the eye and the ear. In The Amho Su3ars (Eds Balazs, E. A. and Jeanloz, R. W.). Pp, 401-60. Academic Press, N.Y. Balazs, E. A., Laurent, T. C. and Laurent, U. B. G. (1959). Studies on the structure of the vitreous body. VI. Biochemical changes during development. J. Biol. Chem. 234, 422430. Balazs, E. A., Toth, L. 2. J., Eckl, E. A. and Mitchell, A. P. (1964). Studies on the structure of the vitreous body. XII. Cytological and histochemical studies on the cort,ical tissue layer. Exp. Eye Res. 3, F-71. Bloom, G., and Balazs, E. A. (1965). An electron microscopic study of hyalocytes. Eqx Eye Res. 4,249~56. Breen, >!I., Bizzell, J. W. and Veinshein, H. G. (1977). A galactosamine containing proteoglycan in human vitreous. Exp. Eye Res. 24,409-12. DeDuve, C. and Wattiaux, R. (1966). Functions of lysosomes. Ann. Rev. Physiol. 28,435-492. Freeman, M. I., Jacobson, B., Toth, L. Z. J. and Balazs, E. A. (1968). Lysosomal enzymes associated with vitreous hyalocyte granules. I. Intracellular distribution pattern of enzymes. Ezp. Eye Res. 7, 113-120. Gorham, S. D., Olaveson, A. H. and Dodgson, K. S. (1975). Effect of ionic strength and pH on the properties of purified bovine testicular hyaluronidase. C’o~zn. Tiss. Res. 3,17-25. Scott, J. E. (1965). Fractionation by precipitation with quaternary ammonium salts. In Methods irk Carbohydrate Chemistry. Vol. 5 (Ed. Whistler, R. W.). P. 38. Academic Press, Sew York. Szirmai, J. A. and Balazs, E. A. (1958). Studies on the structure of the vitreous. III. Cells in the cortical layer. Arch. Ophthalmol. 59,344s. Truppe, W., Basner, R., von Figura, K. and Kresse, H. (1977). Uptake of hyaluronate by cultured cells. Biochem. ad Biophys. Res. Comm. 78 (3), 713319. Weissman, G. (1965). Lysosomes. New E@and J. DIed. 273, 1084-1090.