Type V synthesis and deposition by chicken embryo corneal fibroblasts in vitro

JACQUELINE SHEA McLAUGHLIN1, THOMAS F. LINSENMAYER2 and DAVID E. BIRK1'*

'Department of Pathology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, USA ^Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA

•Author for correspondence

Summary

Chick embryo corneal fibroblasts were grown in serum. Immunocytochemistry with anti-collagen, culture to study the processes whereby fibroblasts type-specific monoclonal antibodies revealed a regulate the deposition and organization of the homogeneous population of cells synthesizing types collagenous, secondary stroma. The effects of an I and V collagen. The fibrils deposited by cells existing substratum, cell density, grown in a three-dimensional collagen matrix con- and serum concentration on syn- tained a helical epitope on the type V molecule that thesis were investigated. Type V collagen rep- was inaccessible unless the fibrillar structure was resented approximately 20% of the total fibrillar disrupted, mimicking the situation in situ. The collagen synthesized, regardless of whether the production in vitro of heterotypic fibrils, with a cells were subcultured, grown on untreated or constant i/V ratio and molecular packing mimick- collagen-coated plastic, grown under confluent or ing the natural stroma, offers opportunities for subconfluent conditions, or grown in the presence studying in more detail this important process, of low (0.1%) or high (10.0%) serum concen- which is essential for optical transparency. trations. The synthesis of type V collagen remained constant at 20% of the total collagen when cells were grown in 1.0% serum, even though total collagen synthesis increased nearly twofold when Key words: corneal fibroblast, collagen type V, collagen fibril compared to total synthesis in 0.1% or 10.0% formation, cell culture.

Introduction 1988). The content of type V collagen in the corneal stroma is relatively high with respect to type I collagen The corneal stroma is a highly organized tissue composed when compared with other matrices composed of striated of precisely arranged layers of collagen fibrils and fibro- collagen fibrils, i.e. sclera, tendon, dermis and bone blasts. In the chicken, the striated fibrils are of uniform (Broek et al. 1985; Cintron et al. 1981; Davison et al. small diameter, with a relatively constant spacing, 1979; Fessler et al. 1982). One possible consequence of arranged as cholesteric liquid crystal-like orthogonal this increased proportion of type V collagen may be arrays (Hay and Revel, 1969; Trelstad and Coulombre, related to the regularity and size of corneal fibril diam- 1971). All of these attributes may contribute to optical eters, which are uniform and narrow (~25 nm) when transparency. Although type I collagen is the predomi- compared with other tissues containing type I collagen. nant macromolecule in the stroma, types V and VI In vitro self-assembly studies also have demonstrated collagen also are present in significant amounts (Hay et that the type i/V ratio influences fibril diameter with al. 1979; LinsenmayereJa/. 1984, 1986; Blrketal. 1986). higher type V concentrations producing smaller diameter The corneal fibroblasts are neural crest-derived mes- fibrils (Adachi and Hayashi, 1986; Birk et al. unpub- enchymal cells, and are responsible for the deposition and lished data; Linsenmayer et al. in press). organization of this collagenous matrix (Hay and Revel, In the present study, we have used chick embryo 1969; Hay et al. 1979; Johnston et al. 1979). corneal fibroblasts grown in cell culture to investigate Results of immunoelectron microscopy studies indi- whether: (1) the relative synthesis of type V collagen is an cate that collagen types I and V are assembled together inherent property of differentiated corneal fibroblasts or within single fibrils in the corneal stroma to form if it can be modulated; (2) a subpopulation of type V- heterotypic fibrils (Birk et al. 1986, 1988; Fitch et al. secreting cells exists; (3) type V collagen deposited in Journal of Cell Science 94, 371-379 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 371 vitro is assembled with type I collagen as heterotypic (lOCimmol , ICN Radiochemicals) and 10/tCiml ' of 3 1 fibrils, as seen in situ. These studies demonstrate that L-[2,3,4,5- H]proline (lOOCimmol" , ICN Radiochemicals) differentiated corneal fibroblasts in cell culture synthesize for 12 h in complete MEM without FBS. Following the labeling type V collagen, and deposit and organize a collagenous period, both medium and cell layer fractions were collected separately and dialyzed exhaustively against 0.5M-acetic acid. matrix in a manner that closely resembles the situation in Each fraction was treated with pepsin (0.125 J/gml"1; Sigma situ. Chemical Co.) for 36h, lyophilized, then resuspended in non- reducing SDS-sample buffer. Incorporated label was measured Materials and methods in a liquid scintillation counter (Beckman, LS-233) using Hydrofluor (National Diagnostics). SDS-polyacrylamide gel electrophoresis (PAGE) was performed using 7.5% separating Isolation of chick corneal fibroblasts gels with 3 % stacking gels according to the procedure of White Leghorn chicken embryos were incubated at 37.5°C in a Laemmli (1970). Following electrophoresis, gels were fixed, humidified atmosphere. Embryos were removed at 14 days of impregnated with EN3HANCE (DuPont) for fluorographic incubation and staged according to Hamburger and Hamilton detection of radiolabeled bands, dried, and exposed to pre- (1951). Corneas were excised and the central portion isolated flashed X-ray film (X-omat, Kodak; Bonner and Laskey, 1974) using a 2 mm dermal punch and then treated for 15 min with for 3-20 days at —70°C. Chick types I and V were run 0.02% Versene containing 0.025 % trypsin to remove epithelial as standards and stained with Coomassie Blue R-250. Fluoro- and endothelial layers. Tissues were washed in incomplete grams were scanned with an Optronics Raster Photoscanner Minimum Essential Medium (MEM) containing sodium bicar- and the percentage of types I and V collagen were determined bonate buffer, 0.29 me ml" L-glutamine, 2.5/igml~ Fungi- from the band areas and intensities. The sampling error was zone, and 0.05 ^gml~ gentamicin, and then incubated in fresh 5 %, based on three independent measurements. medium containing 200 units ml"1 bacterial collagenase (Sigma Chemical Co.) for 2 hours at 37°C in 5 % CO2 in air and 100% Fractionation of collagens types I and V relative humidity. The tissues were cycled through a 10 ml Confluent primary cultures grown on plastic were labeled with pipette every 30 min to dislodge cells. Fibroblasts were har- 1 radioactive amino acids, and the resulting medium fractions vested by centrifugation at 1000 revs min" for 5 min, washed were pooled and dialyzed against 0.5 M-acetic acid, then treated twice with complete MEM containing 20% fetal bovine serum with pepsin. Chick collagen types I and V, at concentrations of (FBS), tested for cell viability using Trypan Blue exclusion, 12.5 fig ml'1, were added as carriers. Collagen types I and V and counted using a hemocytometer. were fractionated by differential salt precipitation from acetic acid (Silver and Birk, 1984). Culture conditions Approximately 5xlO6 fibroblasts were seeded into a T-25 Itnmunocytochemistry culture flask containing complete MEM with 0.1, 1.0 or 10.0% Monoclonal antibodies against chick collagen type I (I-BA1; FBS and incubated at 37°C in 5% CO2 in air. After 48 h in Linsenmayer et al. 1986) and type V (V-AB12; Linsenmayer culture, ascorbate at 0.05 mgml"1 was added; ascorbate is 4 et al. 1983) were used as primary antibodies for indirect cytotoxic to cells plated at low densities (4xl0 cells cm" ), as immunofluorescence. A monoclonal antibody against chick previously reported by Rowe et al. (1977) and Peterkofsky and collagen type IV (IV-IA8/H8G8; Fitch et al. 1982) served as a Prather (1977). A subconfluent primary culture was typically control when substituted for the primary antibody. All anti- obtained at 2 days of incubation; a confluent primary culture bodies recognize helical epitopes that are collagen type-specific. was typically obtained at 5 days of incubation. A subconfluent Antibodies were affinity purified and used at a concentration of state represented a population of attached cells whose edges did 25^gml~ . Fluorescein isothiocyanate-conjugated goat anti- not contact neighboring cells, while a confluent state rep- mouse IgG (Jackson Immunoresearch Laboratories, Inc.) was resented a population of attached cells in contact with adjacent used as the secondary antibody at a dilution of 1:150 in cells on all sides. Primary cultures were subcultured after phosphate-buffered saline (PBS/0.05% Tween/l% BSA). treatment with 0.25 % trypsin and split 1: 4. Monensin, a monovalent ionophore that has been found to Fibroblasts were grown on or in different substrata. For interfere with the secretory pathways of plasma cells, pancreatic collagen-coated plastic, 100 mm2 Petri dishes were overlaid with 2 exocrine cells, macrophages, myotubes, fibroblasts and chon- 1.3ngcm~ Vitrogen 100 and acid-solubilized bovine dermal drocytes (Tartakoff and Vassalli, 1977, 1978; Smilowitz, 1979, collagen type I (Collagen Corporation), and allowed to dry 1980; Uchida et al. 1979, 1980; Nishimoto et al. 1982), was overnight under sterile conditions. The number of cells plated, used to study the intracellular localization of collagen types I culture conditions and subculturing technique were the same as and V. Corneal fibroblasts were plated onto three-well teflon above. For cells grown within three-dimensional collagen gels, slides (25000cells/well; Cell Line Associates, Inc.) and al- cells from primary cultures were harvested after trypsin treat- lowed to attach in complete MEM with 10 % FBS for 8 h. Cells ment, washed, tested for cell viability, and counted. Collagen were then washed with PBS and incubated with MEM contain- gels were prepared using Vitrogen 100 collagen at a final 5 1 ing 0.5% FBS and 5x 10~ M-monensin (Sigma Chemical Co.) concentration of O.Smgml" type I collagen in incomplete for 4 h at room temperature (modified from Uchida et al. 1980). MEM with a pH of 7.4. Cells were mixed with the unpolym- 5 After exposure to the drug, the cells were washed with PBS and erized gel solution, 2.5 XlO cells per 0.5 ml gel solution, and fixed with 4% paraformaldehyde in PBS, pH7.4, for 15 min at added to the wells of a 24-well tissue culture plate (Fal- 4°C. Following fixation, the cells were again washed with PBS, con 3047). Gelation was initiated by warming the cell/collagen then treated with NaBH4 (50mg/l00ml PBS) for 1 h at room mixture to 37 °C. After gelation the gels were overlaid with 1 ml 1 temperature to quench free aldehydes. A subsequent exposure of MEM with 10% FBS containing ascorbate at 0.05 mgml" . to 100% acetone for 5 min at room temperature was utilized to permeabilize cell membranes. After air drying for 5 min, cells SDS-polyacrylamide gel electrophoresis and were washed in PBS, and blocked in 2 % normal goat serum fluorography (Cappel) for 2h at room temperature. This was followed by Cultures were labeled with 10/iCiml"1 of L-[2-3H]glycine incubation with primary antibody for 2 h at room temperature

372 J. S. McLaughlin et al. under humidified conditions. Cells were then washed exhaus- Results tively with PBS/0.05 % Tween. Following a 2-h incubation period at room temperature with the secondary antibody, cells Type V deposition by chick corneal fibroblasts in vitro were again washed exhaustively and mounted in Gelvetol. In Fibroblasts from the corneal stroma of 14-day chick some cases, cells were exposed to primary antibody for two embryos were isolated and grown on plastic as primary, cycles to amplify the signal (Linsenmayer et al. 1988). 1st passage and 2nd passage cultures in 10% FBS. All Collagen deposition by cells grown in collagen gels for 7 days cultures were grown to a confluent state, then labeled was studied using indirect immunofluorescence with collagen with tritiated amino acids. Culture media and cell layers type-specific, monoclonal antibodies. Gels, with or without were harvested, pepsin treated and analyzed by cells, were incubated in PBS containing 7.0% sucrose for SDS-PAGE. In the medium, type V collagen rep- 30min, then embedded in OCT compound (Tissue-Tek; Miles resented a relatively high and constant proportion of the Scientific), all at room temperature. The embedded tissue was frozen at — 20 °C, and cryostat sections were cut and picked up newly synthesized fibrillar collagen (Fig. 1 A,C). When onto albumin-coated microscope slides. Sections were divided analyzed by densitometry, 18-22% of the total collagen into three groups: (1) acid treatment; (2) cold treatment; and found in primary culture medium is type V collagen (3) no treatment. Sections of gels without cells were cut (Table 1). The type V collagen was present as alpha simultaneously with controls. Cryostat sections were exposed to chains, arl(V)2 and a2(V)i, present in a 2:1 ratio. Type O.lM-acetic acid for 5 min at room temperature, followed by V collagen represented a similar percentage in the media fixation. Alternatively, sections were cold treated at 4°C for 48 h of 1st and 2nd passage cultures, 15-23% and 19-26%, before fixation. No treatment meant that sections were immedi- respectively. ately fixed without acid and/or cold exposure. Sections were fixed and immunocytochemistry was performed as described In the cell layer fractions, type V also represented a above. relatively high and constant proportion of the newly All specimens were observed and photographed using a Zeiss synthesized collagen. In general, the total collagen de- Photomicroscope III equipped for epifluorescence, Kodak Tri- posited was much less than that seen in the medium X film, and Diafine developer. Micrographs were prepared fractions. The cell layers contained between 20 and 30 % under identical exposure and printing conditions to permit of the total collagen (incorporated tritiated amino acids), comparisons. that was found in the medium fractions. The primary, 1st

B

P 1 2 P 1 2

Fig. 1. Collagen type I and V synthesis by chick corneal fibroblasts grown on tissue-culture plastic under confluent conditions in 10% FBS. Fluorograms of SDS-polyacrylamide gels (7.5%) loaded with pepsin-treated primary (P), 1st passage (1), and 2nd passage (2) media (A) and cell layers (B) collected following labeling with tritiated amino acids. Type V collagen represents 15-26% of the total collagen in the medium fractions, and 13-27% of the total collagen in the cell layer fractions (see Table 1). Subculturing has no significant effect on type V collagen synthesis. Deposition occurs primarily into the surrounding tissue culture medium. Fluorograms of gels loaded with pepsin-digested pellets obtained after precipitation of primary culture medium with 0.7M-NaCl-type I collagen and 1.2M-NaCl-type V collagen (C, 1st and 2nd lanes, respectively) were run as standards.

Corneal type V collagen 373 Table 1. Percentage of type V collagen synthesis Passage c Substratum Density (%) Fraction Primary 1st 2nd Plastic Confluent 10.0 M 18-22% 15-23% 19-26% 20±2 (3) 18±4 (3) 23±4(3) Plastic Confluent 10.0 CL 18-26% 13-21 % 22-27% 22±4 (3) 17±4(3) 24±4 (3) Plastic Subconfluent 10.0 M 15-20% 14-17% 18-21 % 18±2 (3) 16±2 (2) 20±2 (2) Plastic Subconfluent 10.0 CL 14-21% 17-22% 15-25% 17±2 (3) 20±4 (2) 20±5 (2) Collagen Confluent 10.0 M 20-28% 18-25% 17-25% 23±4(3) 21±4 (3) 21±4(3) Collagen Confluent 10.0 CL 15-23% 20-22% 18-24% 19±4 (3) 21 ±1 (2) 21 ±4 (2) Plastic Confluent 1.0 M 18-24% 22-28% 20-23% 21±4 (2) 26±5 (2) 22±2 (2) Plastic Confluent 1.0 CL 22-25% 17-22% 25-30% 23±2 (2) 20±4 (2) 28±4 (2) Plastic Confluent 0.1 M 17-24% 18-20% 20-23% 20±4 (3) 19±1 (2) 22±2 (3) Plastic Confluent 0.1 CL 15-25% 14-21 % 15-24% 20±5 (2) 18±5 (2) 20±6 (2)

Type V collagen synthesis as a percentage of total fibrillar collagen (types I and V) deposited into the medium (M) and cell layer (CL) fractions by chick corneal fibroblasts grown under various culture conditions. Type V collagen represents a major percentage of the total fibrillar collagen synthesized. This percentage remains constant when cells are subcultured, grown under confluent or sub-confluent conditions, grown on untreated or collagen-coated plastic, or in the presence of varying serum concentrations. Percentages were obtained by determining the band areas and intensities for types I and V collagen in fluorograms scanned with an Optronics Raster Photoscanner. Data are presented as range, mean±standard deviation, and the number of gels scanned (N). passage and 2nd passage cell layers contained constant The influence of a pre-existing collagen matrix on type collagen i/V ratios as seen in the medium fractions V collagen synthesis (Fig. IB). Type V collagen constitutes 18-26% of the To study the effects of a pre-existing collagen substratum total collagen seen in primary culture cell layers, 13-21 % on type V collagen production, primary, 1st passage and of the total collagen seen in 1st passage cell layers, and 2nd passage cultures were grown on collagen-coated 22-27 % of the total collagen seen in 2nd passage cell plastic in the presence of 10% FBS. As seen in Table 1, layers (Table 1). 20-28 % of the total collagen is type V in primary culture Overall, corneal fibroblasts grown on tissue-culture medium; 18-25% in 1st passage medium; and 17-25% plastic secrete collagen types V and I in an approximate in 2nd passage medium. Again, similar results were 1:4 ratio, both into the medium and cell layer. Subcul- obtained for cell layer fractions. These results indicate turing had no significant effect on the ratio of collagen that corneal fibroblasts continue to synthesize type V types V and I, or the site of deposition, medium versus collagen in a manner similar to cells grown on untreated cell layer. plastic, i.e. in a 1:4 ratio with type I collagen, whether confluent or subconfluent. Thus, a pre-existing collagen matrix does not appear to alter the property of type V The effect of cell density on type V collagen synthesis collagen synthesis. Further evidence for this is illustrated Corneal fibroblasts were plated onto plastic as primary, in Fig. 2, which is a histogram showing total collagen 1 1st passage and 2nd passage cell cultures in 10 % FBS. To synthesis (total incorporated pepsin-resistant ctsmin" ) examine the possible influences of cell density on type V for each medium fraction examined. There is no signifi- collagen synthesis, cells were grown to subconfluent and cant difference in the amount of total collagen in the confluent states and the amounts of collagen secreted medium of cells grown to confluency on plastic in 10 % were compared. The results of these labeling experiments FBS or the medium of cells grown to confluency on can be seen in Table 1, which lists the percentages of type collagen in 10% FBS. V collagen deposited into medium and cell layer frac- tions. Fibroblasts, when grown under subconfluent con- The difference between high and low serum on type V ditions, continue to synthesize type V collagen in relative collagen deposition amounts similar to that of confluent cultures. For To determine the effect of serum-borne factors on type V example, under subconfluent conditions, type V collagen collagen synthesis, corneal fibroblasts were grown in low constitutes 15-20% of the total collagen in primary (0.1% or 1.0% FBS) and high (10.0% FBS) serum culture medium, 14-17% in 1st passage medium, and concentrations. Cells were grown to confluency, then 18-21 % in 2nd passage medium. Similar percentages labeled and analyzed for type V collagen. At all serum were found in the cell layer fractions. concentrations examined, type V collagen continued to

374 J. S. McLaughlin et al. 6- T ^^J primary I | | 1st passage

| | 2nd passage

4--

A B C D Collagen, Plastic, Plastic, Plastic, 10.0% serum 10.0% serum 1.0% serum 0.1% serum

Fig. 2. Total collagen synthesis (total incorporated ctsmin ) in primary, 1st passage, and 2nd passage media collected from cells grown to confluency on: A, collagen-coated plastic in 10% FBS; B, plastic in 10% FBS; C, plastic in 1.0% FBS; D, plastic in 0.1 % FBS. Medium fractions were treated with pepsin, dialyzed and incorporated label was determined by liquid scintillation spectrophotometry. Cells grown in 1 % serum show increased synthesis of collagen when compared with total collagen synthesis by cells grown in either 0.1 % or 10.0% serum. Data represent the means of three experiments±standard deviation. constitute a high proportion of the newly synthesized collagen (Fig. 3 A-C; Table 1). The percentage of type V collagen also remained relatively constant in each case; however, total collagen synthesis was found to increase B substantially in 1.0% serum medium when compared with total collagen synthesis in 0.1 % or 10.0% serum- containing medium (Fig. 2). Intracellular localization of collagen types I and V Chick corneal fibroblasts that had been grown to con- fluency were exposed to the ionophore monensin, a method used to increase the intracellular pool of material. Such cells were analyzed by immunofluorescence for the intracellular localization of collagen types I and V. A large number of intracellular fluorescent vesicles stain m *1 (I) positively for type I collagen (Fig. 4A); a similar number — o2(l) of vesicles also exhibited type V immunoreactivity Fig. 3. Collagen synthesis (types I and V) by chick corneal fibroblasts grown on tissue-culture plastic under confluent conditions in varying serum concentrations. Fluorograms of SDS-polyacrylamide gels (7.5%) loaded with pepsin-treated media collected from cells grown in 0.1 % (A), 1.0% (B), and 10.0% (C) FBS, following labeling with tritiated amino acids. The percentage of type V collagen remains relatively high and constant in each case: 17-24% (0.1 %), 18-28% (1.0%), and 15-26% (10.0%). However, total collagen synthesis was found to increase in 1.0% serum when compared with total collagen synthesis in 0.1 % or 10.0% serum, as seen in Fig. 2. 0.1% 1.0% 10.0%

Corneal type V collagen 375 Fig. 4. Intracellular localization of collagen types I and V. Chick corneal fibroblasts were grown to sub-confluency, treated with 5xlO~:>M-monensin for 4h at 37°C, permeabilized, then prepared for indirect immunofluorescence staining using type I- and V- specific monoclonal antibodies and a fluorescein-conjugated goat anti-mouse IgG. Intracellular vesicles stain strongly with the anti-type I collagen monoclonal antibody (A). Type V collagen staining also shows a positive reaction in intracellular vesicles (B). In both cases, labeled vesicles are found in all cells. An antibody against type IV collagen was used as a control and showed no immunoreactivity (C). A phase-contrast micrograph of cells following monensin treatment shows the number and morphology of the intracellular vesicles in association with cell size and shape (D). All cells stain for collagen types I and V, indicating that a subpopulation of type V secreting cells does not exist.

(Fig. 4B). Both types I and V collagen staining patterns monoclonal antibodies employed are completely species- were observed in all cells. An antibody against type IV specific for chicken collagens, the bovine matrix showed collagen was used as a control and found to be negative no reactivity. When antibody against type I collagen was (Fig. 4C). It is apparent that all cells synthesize type I used, reaction was observed throughout the gel, showing and V collagens. intense staining (Fig. 5A). When antibody for type V was employed on similar specimens without any pretreat- Organization of collagen fibrils in vitro ment, minimal staining was observed in areas around To promote and study fibril formation of newly secreted cells (Fig. 5D). Staining with the control, type IV collagens, cells were grown in a three-dimensional matrix monoclonal antibody, was negative (results not shown). of bovine skin collagen and the deposition of collagen To ascertain whether the absence of reactivity for type fibrils was studied using immunocytochemistry. Fibro- V collagen was due to fibril assembly, as occurs in vivo blasts were grown in gels for 7 days and analyzed by- (the type V epitopes are masked by the co-assembly with indirect immunofluorescence using monoclonal anti- type I collagen into fibrils), cryostat sections of fibro- bodies against types I, V and IV collagens. Since the blast-populated gels were exposed to dilute acid, or

376 Jf. S. McLaughlin et al. Fig. 5. Organization of collagens types I and V deposited by chick corneal fibroblasts grown in three-dimensional collagen gels. Gels were prepared for indirect immunofluorescence using type-specific monoclonal antibodies and a fluorescein-conjugated goat anti-mouse IgG. Pretreatment of gels in 0.1 M-acetic acid (B and E) or in cold (C and F) disrupts fibril structure. In A, B and C, the gels were incubated with a monoclonal antibody against type I collagen; and in D, E and F, the gels were incubated with a monoclonal antibody against type V collagen. Staining reactions are consistent with the concept of heterotypic fibrils consisting of both types I and V collagen molecules assembled so that the epitopes on the type V molecule are unavailable to antibody unless fibrillar structure is disrupted.

Comeal type \' collagen 377 incubated in the cold, before fixation and antibody type V collagen suggests the importance of the 1:4 ratio staining. These steps were done to disrupt fibril structure in the control of the uniform, smaller diameter, corneal partially. Fig. 5B shows that reaction with the type I fibrils. Further evidence for this postulate comes from antibody in the acid-treated gel was still highly positive data that 17-day chick tendon fibroblasts, grown as and indistinguishable from that in the untreated gel primary cultures on plastic in 10 % FBS, deposit between (Fig. 5A). Staining of the cold-treated gel was similarly 3 and 7% (Ar=3) type V collagen into the medium positive (Fig. 5C). The cell-populated collagen gels fraction and between 3 and 5% (N=3) type V collagen treated with either acid or cold also stained positively for into the cell layer fraction (data not given). The mean type V collagen (Fig. 5 E and F). This is in contrast to diameter of a 17-day chick tendon fibril in situ is 46 nm the untreated gels in which a minimum of specific (Birk et al. in press). staining was observed in pericellular locations (Fig. 5D). Previous work has shown that collagen matrix forma- Note the loss of compaction and the dispersion of the tion in the embryonic chick cornea occurs within a series collagen fibers in the gels shown in Fig. 5B, C, E and F. of unique extracellular compartments (Birk and Trelstad, 1984). Accordingly, fibril formation takes place within surface recesses containing fibrils with mature diameters Discussion and constant intrafibrillar spacing. The compartmental- ization of fibril assembly provides an extracellular site Chick embryo corneal fibroblasts synthesize a remarkably where collagen types I and V could be co-assembled. constant proportion of collagen types I and V, with 20 % Alternatively, mixing of procollagen types I and V can type V to 80% type I. When the cells are subcultured, occur during packaging in intracellular vesicles. By grown on different substrata of plastic or collagen, or whatever means, corneal fibroblasts synthesize a constant grown under subconfluent or confluent conditions, this ratio of collagen types I and V and thereby form collagen 1:4 ratio of V: I does not change. fibrils with constant diameters. Since the corneal fibroblast synthesizes types V and I The intracellular localization studies of collagen types I collagen in a 1:4 ratio under all culture conditions, this and V in permeabilized, monensin-treated cells indicate indicates that this phenotype is an intrinsic property of that all corneal fibroblasts contain intracellular vesicles this cell type, is stable and is closely regulated by the that stain for types I and V collagen. These results corneal fibroblast. It also suggests that these two col- demonstrate that a subpopulation of cells that only lagens are constitutive proteins and that the genes associ- produce type V collagen does not exist; instead, all cells ated with them must be continuously active. However, produce both type I and V collagen species. However, it our data show that the net synthesis of collagen can be is still not known if collagen types I and V are packaged in modulated. There is an increase in total collagen syn- the same, or different, vesicles before being deposited thesis by cells grown in 1.0% serum when compared with extracellularly. Also, we do not know what differences are cells grown in either 0.1% or 10.0% serum. This present in the post-depositional enzymatic modifications, observation may be explained by the addition of high such as procollagen processing and covalent cross-link- serum, 10.0%, to fully differentiated cells rapidly in- ing, on these two different collagen species. Future creasing the rate at which non-collagen mRNAs are experiments should unravel these important issues. translated, disrupting the maintenance of high procol- Type V collagen within corneal fibrils in situ is masked lagen production (Valmossoi and Schwarz, 1988). by its supramolecular organization with type I collagen. Alternatively, serum could contain inhibitors of collagen Helical epitopes on type V collagen are buried within synthesis. The lower concentration of growth factors and heterotypic fibrils and therefore inaccessible to antibody growth constituents present in 0.1 % serum may alter the interaction (Linsenmayer et al. 1983; Fitch et al. 1984; cell's metabolism, so that instead of procollagen trans- Birketal. 1986, 1988; Fitchetal. 1988). Our immunoflu- lation, the cell uses its energy for maintenance of vi- orescence data on fibroblasts grown in three-dimensional ability. A serum concentration of 1.0% may be the collagen gels are consistent with the concept of heteroty- balance between these two extremes, i.e. high procol- pic fibrils assembled, so that the epitopes on the type V lagen production and normal metabolism. Nonetheless, molecule are unavailable unless fibrillar structure is the significant point is that the ratio of types I and V disrupted. Brief incubation in dilute acid or extensive continues to remain unchanged when cells are grown cold treatment was necessary to 'unmask' the type V under varying serum concentrations. The strict mainten- molecules. The ability of cold treatment alone, without ance of this ratio of collagens I and V under all conditions prior inhibition of crosslink formation with /3-aminopro- strengthens the concept that the proportion of collagens pionitrile (/SAPN), to disrupt fibril structure, may be produced is of critical importance in fibril formation. explained as follows: analysis of our fluorograms of Small-diameter collagen fibrils are constant through- SDS—polyacrylamide gels from radiolabeled cultures out the entire cornea at all stages of development, revealed that crosslinked collagen dimers and trimers approximately 25 nm. Previous work on collagen types I were negligible. Such forms are readily seen when chicks and V self-assembly has shown that increasing amounts are labeled in ovo and the extracted corneal collagen is of type V collagen result in fibrils of smaller diameter similarly treated. It may be that covalent cross-linking of (Adachi and Hayashi, 1986; Birketal. unpublished data; collagen deposited in aqueous three-dimensional gels is Linsenmayer et al. in press). The fact that corneal less efficient than that seen in the dense corneal stroma. fibroblasts consistently synthesize a high proportion of In summary, type V collagen constitutes approxi-

378 jf. S. McLaughlin et al. mately 20 % of the total fibrillar collagen, i.e. types V and HAY, E. D., LINSENMAYER, T. F., TRELSTAD, R. L. AND VON DER I, synthesized by the chick embryo corneal fibroblast MARK, K. (1979). Origin and distribution of collagens in the developing avian cornea. In Current Topics in Eye Research (ed. J. grown in culture. This percentage is unaffected by A. Zadunaisky & H. Davson), pp. 1-35. New York: Academic subculturing, substratum, cell density or serum con- Press. ditions. All corneal fibroblasts secrete both collagen types JOHNSTON, M. C, NODEN, D. M. AND HAZELTON, R. D. (1979). V and I and when grown in three-dimensional collagen Origins of avian ocular and periocular tissues. Expl Eve Res. 29, 27-43. gels these cells assemble heterotypic fibrils. The forma- LAEMMLI, U. K. (1970). Cleavage of structural proteins during the tion of heterotypic fibrils and the consistent synthesis of a assembly of the head of bacteriophage T4. Nature, Land. "221, high proportion of type V collagen in a 1:4 ratio with type 680-685. I collagen, indicate the importance of type V collagen in LINSENMAYER, T. F., BRUNS, R. R., MENTZER, A. AND MAYNE, R. regulating the formation of uniform, small diameter (1986). Type VI collagen: Immunohistochemical identification as a filamentous component of the of the corneal fibrils. developing avian corneal stroma. Devi Biol. 118, 425-431. LINSENMAYER, T. F., FITCH, J. M. AND BIRK, D. E. (1990). We thank Ed Wong, Emanuel Zycband, and Ann Latrielle for Heterotypic collagen fibrils and stabilizing collagens: controlling their assistance and Kathleen Doane, John Fitch and Robert elements in corneal morphogenesis? Ann. N.Y. Acad. Sci. (in Trelstad for critically reading the manuscript. This work was press). supported by NIH grants EY 05129 and EY 05191 and a LINSENMAYER, T. F., FITCH, J. M. AND MAYNE, R. (1984). Research Career Development award (EY 00254) to DEB. Extracellular matrices in the developing avian eye. Type V collagen in corneal and noncorneal tissues. Invest. Ophthal. Vis. Sci. 25, 41-47. References LINSENMAYER, T. F., FITCH, J. M. AND SCHMID, T. M. (1988). Multiple-reaction cycling: A method for enhancement of the ADACHI, E. AND HAYASHI, T. (1986). In vitro formation of hybrid immunocytochemical signal of monoclonal antibodies, jf. fibrils of type V collagen and type 1 collagen. Limited growth of Histochem. Cytochem. 36, 1075-1078. type I collagen into thick fibrils by type V collagen. Conn. Tis. Res. LINSENMAYER, T. F., FITCH, J. M., SCHMID, T. M., ZAK, N. B., 14, 257-266. GIBNEY, E., SANDERSON, R. D. AND MAYNE, R. (1983). BIRK, D. E., FITCH, J. M., BABIARZ, J. P. AND LINSENMAYER, T. F. Monoclonal antibodies against chicken type V collagen: (1988). Collagen type I and type V are present in the same fibril in production, specificity, and use for immunocytochemical the avian corneal stroma. J7. 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