Detection of specific collagen types in normal and keratoconus corneas - IOVS
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Detection of specific collagen types in
normal and keratoconus corneas
David A. Newsome, Jean-Michel Foidart, John R. Hassell, Jay H. Krachmer,
Merlyn M. Rodrigues, and Stephen I. Katz
Keratoconus is a corneal disease of unknown cause that involves a progressive thinning and.
scarring of the corneal connective tissue. We examined normal human and keratoconus cor-
neas, including one healed penetrating keratoplasty specimen. Organ cell cultures of normal
and keratoconus corneal specimens were labeled with radioactive proline and analyzed, by
CM-cellulose chromatography and slab gel electrophoresis to determine collagen biosynthesis.
Collagen types I and HI were synthesized in similar amounts by normal and keratoconus
stromacytes in culture. Specifically purified antibodies were used to determine the distribution
of collagen types in tissue sections by immunofluorescence. The distribution of collagen types I,
III, and IV in keratoconus was also similar to that in normal corneas, except that scarred
regions in keratoconus and at the host-graft: juncture were largely type HI. Immunofluorescent
reaction of the anti—type IV collagen antibodies with Bowman's layer, in particular, and
Descemet's membrane in keratoconus specimens indicated extensive destruction. Basement
membrane destruction may play an important role in this disease.
Key words: keratoconus, corneal collagens, collagen types, immunofluorescence,
normal and abnormal corneal extracellular matrix
Ke.;ratoconus is a slowly progressive, seri- tosomal recessive mode of inheritance has
ous corneal disorder of uncertain heritability been postulated,1 most cases appear to be
and unknown cause. The clinical hallmark of sporadic. In other clinical reports, kera-
the disease is the irregular, cone-shaped pro- toconus has been linked to a heritable disor-
trusion of the central cornea which results der of connective tissue, Ehlers-Danlos syn-
from weakening and thinning of the stromal drome.2"4
elements. Although the disease has been re- Extensive biomicroscopic and pathological
ported to exhibit familial patterns and an au- studies of keratoconus have so far failed to
demonstrate conclusively whether the pri-
mary site of the disease is the corneal stroma
From the Section on Retinal and Ocular Connective Tis- or the epithelium and its basement mem-
sue Diseases (D. N., J. H.) and Ocular Pathology brane.5 Ectasia of the stroma likely involves
(M. R.), Clinical Branch, National Eye Institute, the either primary or secondary alterations in its
Laboratory of Developmental Biology and Anomalies, major structural macromolecules or in their
National Institute of Dental Research (J-M. F.), Der-
matology Branch, National Cancer Institute (S. K.),
interactions with other matrix elements. Col-
National Institutes of Health, Department of Health, lagen, the most abundant stromal element
Education and Welfare, Bethesda, Md., and the De- (about 71% of dry weight) has naturally
partment of Ophthalmology, University of Iowa received much attention. Histopathological
(J. K.). J-M. F. is a Fellow of the FNRS in Belgium. studies have documented stromal scarring
Submitted for publication July 5, 1979.
Reprint requests: Dr. David Newsome, Building 10,
and degradative changes in the stromal lamel-
Room 10 D-17, National Institutes of Health, Be- lae but have been of little help in elucidating
thesda, Md. 20205. pathogenetic mechanisms.
738
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Number 6 Collagen types in normal and keratoconus corneas 739
Fig. 1. Fluorescence micrograph of a frozen section of normal cornea reacted with anti-type
IV collagen antibodies demonstrates a wide, discrete area of fluorescence in the epithelial
basement membrane area. (x250.)
In the present study we have used spe- obtained at autopsy from individuals aged 7, 22, 28,
cifically purified antibodies to characterize 35, and 56 years. Corneoscleral specimens were
the distribution of collagen types in normal embedded in OCT embedding medium (Ames) and
human corneal and anterior scleral tissue as then frozen in liquid nitrogen. Frozen sections (4
well as in keratoplasty specimens from pa- jum thick) were transferred to albumin-coated glass
slides to serve as substrate for a modified direct
tients with well-documented keratoconus,
immunofluorescent staining reaction. Keratoconus
including one postmortem specimen from a specimens from patients in the age range 28 to 49
patient who had successful perforating kera- years, all with typical clinical criteria for the dis-
toplasty for keratoconus. We also charac- ease as documented by one of us (J. K.), were
terized biochemically the collagens synthe- placed into cold M-K corneal storage medium at
sized by normal and keratoconus stromal the time of surgery or autopsy and embedded,
cells and normal scleral cells. Our data dem- frozen, and sectioned within 20 hr by the methods
onstrate the cornea-specific distribution of described above. The keratoconus specimens had
collagen types as well as some alterations in variable amounts of obvious scarring; all had irregu-
collagenous components in keratoconus speci- lar thinning and Fleisher pigmented rings.
mens. Corneal stromal cell cultures were established as
previously described6 from explants of pure stroma
Materials and methods
prepared under a dissecting stereomicroscope.
Corneal specimens. Five normal and five kera- Cell cultures were similarly started from cleanly
toconus corneas were studied. Normal tissue was dissected explants of sclera and conjunctival
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740 Newsome et al. June 1981
Fig. 2. Fluorescence micrograph of a frozen section of keratoconus cornea reacted with anti-
type IV collagen antibodies demonstrates a marked reduction in and discontinuity of the
amount ol fluorescence in the epithelial basement membrane area as compared with that seen
in the normal specimen in Fig. 1. (X500.)
stroma. Primary outgrowths of cells as well as cells umn (1.6 by 5 cm) as described elsewhere, which
in the third serial passage forming confluent layers permitted an average (n = 4) recovery of 74% of
were used. Cells were maintained in polystyrene applied counts. 7 " 9 Peak fractions in the effluent
culture dishes (Falcon) in Eagle's minimal essen- were pooled, lyophilized, and electrophoresed in
tial medium with 5% fetal calf serum, 100 u/m] the presence of 0.05M urea on sodium dodecyl
penicillin, and 50 /ng/ml streptomycin, and ex- sulfate (SDS)-polyacrylamide slab gels.10 Purified
posed to 37°, 100% humidity, and a 95% air-5% types I and III collagen standards were included
CO2 atmosphere. Confluent cultures were ra- with each electrophoresis. Reduced samples were
diolabeled by 24 hr incubation in serum-free obtained by dividing the original 250 /xl samples
Dulbecco-Vogt medium (NIH) with 100 ju.Ci/ml into two and adding 5 ju.1 of/3-mercaptoethanol to
3
H-proline (NET-323; New England Nuclear, Bos- one set of the samples. After electrophoresis the
ton), 100 jti.g/ml vitamin C, and 50 yu-g/ml gels were stained with Coomassie blue and were
/3-aininoproprionitrile fumarate as a lathyrogen. impregnated with 2,5-diphenyloxazole (PPO) in
Medium and cells together with carrier collagen dimethylsulfoxide. Autofluororadiograms on Ko-
from skins of lathyritic rats were extracted in 0.5M dak X-Omat film were prepared by exposing the
acetic acid containing 1.0 mg/ml pepsin. The colla- film at —74° for periods up to 7 days.
gen was purified by NaCl precipitations (25%; two Preparation of antigens. Types I and 111 colla-
precipitations) in the presence of carrier collagen gens were isolated from fetal calf skin,8 type II
from skins of lathyritic rats and eluted from a car- from a rat chondrosarcoma," and type IV from a
boxymethyl (CM)-cellulose chromatography col- murine sarcoma.12 Type I procollagen was ex-
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Number 6 Collagen types in normal and keratoconus corneas 741
tracted from the skin of dermatosporactic calves Table I. Distribution of ocular connective
and was a gift from Dr. C. M. Lapiere. Type III tissue proteins in normal and
procollagen was purified from fetal calfskin. 8 The keratoconus specimens as detected
identity and purity of the collagens was deter-
by immunofluorescence
mined by amino acid analysis after hydrolysis in
6N HC1 at 110° for 18 hr ; by CM-cellulose and Site Proteins detected
diethylaminoethyl-cellulose elution profiles, and
Cornea] epithelium No reaction for
by SDS-polyacrylamide slab gel electrophoresis
collagens/fibronectin
with and without /3-mercaptoethanol reduction Cornea! epithelial basement Type IV collagen
and with and without cyanogen bromide diges- membrane
tion. Human fibronectin was purified from fresh Cornea! stroma* Type 1 collagen;
human plasma by affinity chromatography on a fibronectin
Descemet's membrane Type IV collagen;
gelatin column. The material eluted with 1M po- fibronectin
tassium bromide was electrophoresed after reduc- Corneal scars Type III collagen;
tion of the disulfide bonds with 20 mM dithio- some type I
treitol. The 220,000 dalton chain of fibronectin Conjunctival blood vessels Type IV collagen
was eluted from slices of the acrylamide gels and Sclera Type III collagen;
some type I
used to immunize rabbits.
Preparation of antibodies to collagens. Rabbits *Type III collagen has been extracted from corneal stroma and
characterized biochemically but was not reliably detected by
or guinea pigs were immunized by repeated sub- immunofluorescence in our studies.
cutaneous or foot pad injections of 0.5 to 5.0 mg of
the antigen in Freund's complete adjuvant. significant inhibition was observed when the anti-
Antibodies were isolated and purified by cross- body was preincubated with the heterologous col-
immunoabsorption and checked for cross-reac- lagen. Indirect immunofluorescence studies were
tivity to collagen and proteoglycans by indirect performed on fresh-frozen sections of human skin,
immunofluorescence, micropassive hemaggluti- liver, tendon, and spleen. They established that
nation assay,13 and radioimmunoassay. Antibodies the purified antibodies reacted only with those
to collagens were purified from the antisera by structures known to contain these molecules. Fi-
affinity chromatography and cross-immunoab- nally, indirect immunofluorescence blocking and
sorption. H Serum from immunized animals was competition studies confirmed the specificity of
passed sequentially over columns of types II and the anti-type I and anticollagen antibodies, since
III collagen and types IV and V collagens which a preincubation with the specific antigen blocked
had been covalently bound to Sepharose 4B. The the binding of antibodies to known high-affinity
unbound material was then passed over a type I substrates. For example, a preincubation with
collagen column which was extensively rinsed type I collagen blocked the binding of anti-type I
with phosphate-buffered saline (PBS). Antibodies collagen antibodies to human skin but did not in-
were eluted from the column with 0.5N acetic terfere with the binding of anti-type III collagen
acid-0.5M NaCl. This eluate was neutralized with antibody.
3M Tris, dialyzed against PBS, and concentrated Preparation of antibodies to fibronectin.
by filtration on a UM20 Amicon filter. Antibodies Specific antibody to fibronectin was purified by
to the other antigens were purified according to a affinity chromatography. H Serum from im-
similar protocol. munized rabbits was passed over a column of
Testing of antibodies to collagens. The spe- fibronectin which had been covalently bound to
cificity of the antibodies was determined by radio- Sepharose 4B. The unbound material was then
immunoassay (RIA), by immunofluorescence la- washed away with PBS. Antibody to fibronectin
beling of target tissues, and by enzyme-linked was then eluted from the column with 0.5M acetic
immunoabsorbent assay (ELISA) as reported pre- acid, pH 2.9, neutralized with 3M Tris, dialyzed
viously.15 For RIA, types I and III collagens were against PBS, and concentrated on a UM20 Amicon
labeled with iodine-125 by the chloramine-T filter.
method16 (sp. act. 1 to 2 fxCi/ixg). The RIA was Testing of antibody to fibronectin. The spec-
performed according to the method of Rohde et ificity of the antibody was determined by RIA, by
al. 17 In quantitative inhibition tests preincubation ELISA, 15 by radial immunodiflusion according to
of the purified antibody with increasing amounts Ouchterlony, and by immunoelectrophoresis. For
of the autologous collagen inhibited the binding of RIA, labeled fibronectin was purified from the cul-
the antibody with the labeled antigen, whereas no ture medium of human fibroblasts grown with
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742 Newsome et al. June 1981
Fig, 3. Inset of the superficial area of a scarred keratoconus cornea shows distruption (asterisk)
of Bowman's layer (arrows) and superficial stromal fibrosis. E, epithelium; S, stroma. (To-
luidine blue; X350.) The transmission electron micrograph demonstrates irregular subepithe-
lial connective tissue excrescences in the basal epithelium (E). Bowman's layer is replaced by
irregular connective tissue. F, fibroblast. (x27,000.)
[U- 3 H]leucine. l8 Fibronectin was also labeled nodiffusion and immunoelectrophoresis a single
with l2oI by the chloraniine-T method. 16 Preincu- line of precipitation was obtained with an an-
bation of the antibody with other antigens, includ- tifibronectin antibody and purified fibronectin or
ing types I and III collagens, laminin, and type IV normal human serum.
collagen, did not inhibit the binding of the anti- Immunofluorescence reaction. Modified direct
bodv to labeled fibronectin. Bv radial immu- immunofluorescent staining was performed on
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Number 6 Collagen types in normal and keratoconus corneas 743
Fig. 4. These sections of a healed perforating keratoplasty specimen from a patient with
keratoconus were reacted with anti-type I (A) and anti-type III (B) antibodies. Note the
strongfluorescenceof the scar with anti-type III and the similarity of staining of the graft and
host stromas with both antibodies. (X300.)
sections of cornea and corneoscleral tissue with area was strikingly decreased and irregular
antibodies and control sera in two dilutions: 10 and (Fig- 2).
20 /Ag/ml PBS. After a 30 min incubation the sec- Bowman's layer. Bowman's layer reacted
tions were washed in PBS and then exposed to similarly with antibodies to types I and III
fluorescein-conjugated goat-anti-rabbit immuno- collagen as did the anterior stroma. How-
globulin for 30 min, washed again in PBS, and ever, the positivity for anti-type I was
mounted with glycerine. Specimens were exam-
noticeably reduced as compared with more
ined with a Leitz ultraviolet microscope equipped
with a camera attachment and a BG 12 filter. Ex- posterior layers of the stroma. Bowman's
posures were made with an automatic exposure layer reacted more strongly with antibody to
meter on Kodak Ektachrome 200 film. fibronectin than did the stroma. Except for
the presence of type Ill-containing scars that
Results included part of the Bowman's area in some
With the use of the techniques described keratoconus specimens, there was no differ-
herein it was not generally possible to assess ence between the types of detectable colla-
immunofluorescent reactivity quantitatively. gens in the normal and keratoconus material.
We were able to localize the heterogeneous The histological appearance of a typical scar
population of human corneal collagens to can be seen in Fig. 3.
specific sites, This distribution is summarized Corneal stroma. In both normal and kera-
in Table I. toconus tissue the stroma was strongly posi-
Corneal epithelium. The epithelial layer it- tive for type I. Procollagen type I reaction
self did not react with any antibodies. This was consistently weaker in all specimens of
finding was similar in normal and keratoco- cornea. In some of the heavily scarred
nus corneas. The zone just deep to the epi- keratoconus specimens there was a patchy
thelium showed a strong reaction with an- increase in detectable procollagen type I in
tibodies to type IV collagen {Fig. 1). In the the subepithelial anterior stroma.
keratoconus specimens with more advanced Type III collagen was not clearly detected
stromal thinning, the,reaction with anti-type in the normal corneal stroma by fluorescence
IV in the epithelial basement membrane methods. Consistent reactivity in normal tis-
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744 Newsome et al. June 1981
Fig. 5. Fluorescence micrographs (A, X450; B, X300) of a section of normal cornea reacted
with antifibronectin antibody exhibits fluorescent anterior and posterior portions of Des-
cemet's membrane identical to that pattern seen with anti-type IV antibodies. Right panel,
Transmission electron micrograph of normal tissue. The fluorescence pattern was seen in both
normal and undisrupted keratoconus Descemet's membrane and may correspond to the nor-
mal 100 nm wide-spaced collagen arrow zone (immiinoHuorescent dark interleaf) and anterior
and posterior granular basement membrane-like zones of material. S, stroma; DM, posterior
Descemet's membrane; E, endothelial cell. (x30,000,) Fibronectin is also detectable in stroma
itself in the fluorescence micrographs.
sues was detected only on the edges of specimen the healed junction between donor
stromal lamellae. No differences were visible keratoconus recipient reacted strongly with
between the reaction for type III and procol- antibodies to type III and procollagen type
lagen type III in normal and keratoconus un- III. The zone of scarring extended into both
scarred stroma. the host and donor tissues (Fig. 4). Type III
In the specimen of a successful graft of collagen could be clearly detected also in the
normal donor into a keratoconus corneal bed, more thinned and scarred keratoconus spec-
both areas showed the same reaction with imens as patches corresponding to stromal
antibodies to types I and III. In the grafted scars.
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Number 6 Collagen types in normal and keratoconus corneas 745
Fig. 6. Fluorescence micrograph of a normal sclerocorneal section reacted with anti—type III
antibodies reveals strong fluorescence in Tenon's and the sclera at the liinbal transition zone.
Note the relative lack of reaction in the clear cornea (C). This strong reaction was evident in all
areas of sclera studied in normals and keratoconus. (X450.)
Both normal and keratoconus corneal stro- Descemet's membrane was often irregular,
mas exhibited high levels of detectable and the laminar pattern less defined. This
fibronectin. There was a particularly concen- finding appeared to be associated with the
trated positive reaction at the level of the frequent folds in Descemet's membrane near
stroma adjacent to Descemet's membrane and the thinned stroma.
in the epithelial and Descemet's basement Normal Descemet's membrane had de-
membrane areas themselves (Fig. 5). finitely detectable fibronectin by the immu-
Descemet's membrane. Descemet's mem- nofluorescent stain. The distinctive laminar
brane reacted strongly with antibodies to pattern was present, with more intense stain-
type IV collagen, exhibiting a striking lami- ing on the stromal as compared with the endo-
nar pattern with bright fluorescence on the thelial surface. The interruption of the pattern
stromal and endothelial faces and a dark, ap- of fibronectin reaction in most of the
parently unstained interleaf (Fig. 5). When keratoconus specimens was in contrast to the
measured on a fluorescence micrograph, the picture in the normal tissues (Fig. 5).
bright leaves each were about 1 /am wide at Sclera. Normal human sclera had a pre-
4x, and the dark interleaf 2 /xm. These rela- ponderance of detectable type III collagen
tive widths were comparable to the three over type I collagen. This pattern, opposite
anatomical zones discernible ultrastructural- to that seen in the corneal stroma, was most
ly: the anterior zone of granular basement apparent at the scleral spur region, where the
membrane-like material at the stromal inter- two tissues are juxtaposed (Fig. 6). Procolla-
faces, the middle zone of wide-spaced colla- gen type I reaction was consistently weaker in
gen, and the posterior zone of basement all scleral specimens than that of type I itself.
membrane-like material (Fig. 5). In kerato- Conjunctiva. Normal human conjunctival
conus specimens, the staining pattern of stroma reacted strongly with antibodies to
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746 Newsome et at. June 1981
type I collagen. Walls of blood vessels in the
conjunctiva had clearly detectable type IV
collagen and fibronectin. The conjunctival
40,000
epithelium itself did not react with any an-
tibodies.
30,000 Identification of collagen synthesized in
vitro. Normal and keratoconus corneal
20,000 stromal cells synthesized both types I and III
collagen in vitro. The presence of these two
10,000 -
collagens was confirmed by the elution posi-
tion from CM-cellulose columns (Fig. 7) and
SDS slab gel electrophoresis patterns. SDS
gel profiles of the migration patterns of
pooled fractions from the three collagenous
peaks seen on CM-cellulose chromatography
B showed co-migration of peak 1 collagen
50,000 r
chains with purified alpha 1 (I) collagen and
peak 3 with authentic alpha 2 collagen chains
40,000
(Fig. 8). Peak 2 materials migrated largely
with the standard type III and exhibited
30,000 •
disappearance of the trimeric form when
electrophoresed under reducing conditions
20,000 •
(Fig. 8).
10,000 • The ratio of type I to type III collagen as
determined from the CM-cellulose profiles
was 10:1 for normal corneal tissue and 9:1
for keratoconus tissues (average offivespec-
imens). The preponderance of type III colla-
gen in the sclera was demonstrated by a ratio
12,500 of 2.1:1, type I to type III (average of five
specimens). An example of this large amount
10.000 of type III collagen synthesized by scleral
fibroblasts in culture can be seen in Fig. 7.
7500 The material from peak 2 of the chromato-
gram of the scleral collagens again showed
reduction with beta-mercaptoethanol when
subjected to SDS gel electrophoresis.
2500 -
Discussion
20 30 40
Corneal transparency depends upon the
TUBE NUMBER
orderly deposition of collagen and glycocon-
jugates in the stroma.19' 20 Maintenance of
Fig. 7. CM-cellulose chromatograms demonstrate transparency must depend, at least in part,
the elution profiles of the radioactively labeled col- on the controlled turnover of this extracel-
lagens synthesized by cultured corneal stroma- lular matrix. Collagens, the most plentiful
cytes from normal (A) and keratoconus (B) corneas
and sclera (C). Arrows indicate the elution posi-
group of structural macromolecules in the
tions of the carrier rat skin type I collagen peaks. corneal stroma, are present largely as fibrillar
Note the large peak containing the type III colla- proteins that interact with the stroma-specific
gen in C and the similarities of the profiles in A proteoglycans to produce a normal optically
and B. useful stroma. Thus modifications of the
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Number 6 Collagen types in normal and keratoconus corneas
Type Id
ai(l)
a2
1 1R 2R 3R
Fig. 8. Fluororadioautograph of an SDS-polyacrylamide slab gel of the collagen chains eluted
in the three peaks of the CM-cellulose chromatogram demonstrates the presence of the types I
and III collagen synthesized by cultured keratoconus stromaeytes. 1, Pooled fractions from
the a-1 peak region; 2, pooled from the interpeak; 3, pooled from the a-2 peak. Note the
disappearance of the type III trimer after reduction with b- mercaptoethanol (2R). The posi-
tion of the radiolabeled type III collagen was identical to that of coelectrophoresed authentic
type III, as was that of the labeled type I. These patterns were similar between normal and
keratoconus cell synthetic products; the scleral samples had more prominent type III bands on
the gels.
types of collagens synthesized or defects in matrix, as in Ehlers-Danlos type IV, where
posttranslational processing of the molecule the synthesis of type III collagen is low to
could lead to corneal disease. absent. 27 Maumenee 28 reported that two
Recent advances in collagen characteriza- keratoconus corneal specimens contained in-
tion have revealed that there are at least four creased type III collagen as detected by im-
genetically distinct types which are now re- munofluoesence. In our specimens type III
ferred to as types I, II, III, and IV.21 The collagen was not reliably detected in unin-
tissue-specific distribution of collagen types volved corneal stroma. We did demonstrate
appears to play an important role in mor- collections of type III collagen in keratoconus
phogenesis, particularly in the cornea,22'23< 27 corneal scars and at the site of host-graft
and in association with the sensory retina. 24 ' 25 wound healing in a perforating keratoplasty
Corneal stromal matrix stability may de- specimen. Type III collagen has previously
pend in part upon the proportion of the types been reported in scars in nonocular tissues,
of collagen present. We have shown that even those such as tendon which do not nor-
there are two major collagen types synthe- mally have type III. 29
sized in vitro by normal and keratoconus cor- The presence of type III collagen in normal
nea cells, with a similar proportion of types I cornea, including human, has been contro-
and III in both. This mixture of types I and versial,30' 3l and type V collagen preliminarily
III is similar to the collagens that accumulate identified.32 Type III had been identified in
in vivo and have been extracted and iden- bovine material,33 and type V in lapine. 32
tified.26 It is possible that an alteration in the Newsome et al. 26 have recently submitted
proportion of these major collagen compo- evidence from collagenase sensitivity, cyano-
nents could destabilize the connective tissue gen bromide peptides, trypsin sensitivity,
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748 Newsome et al. June 1981
and slab gel electrophoresis with and without epithelial areas of either the normal or
reduction identifying types III and V in nor- keratoconus specimens.
mal human cornea. These data support the The laminar staining pattern of Descemet's
observations of others noted above. Differ- membrane may indicate a regional distribu-
ences with other reports30 may be due at least tion of type IV collagen and of the noncol-
partially to technique, since type III is heav- lagenous fibronectin within the membrane.
ily crosslinked and difficult to extract from Such a specific distribution could explain, at
native tissues. It is more difficult to explain least in part, the distinct ultrastructure of this
the conflict of the present data from cul- membrane. Jakus38 and later Hay and Revel39
tures,31 since extraction is not a problem in described the approximately 100 nm period-
vitro. We used different media with a short icity of the anterior third of Descemet's
labeling period and early passage cells, in membrane, presumably reflecting the pack-
contrast to Stoesser et al.34 In the present ing of membrane subunits in this area. Such
study preliminary results with anti-type V orderly packing could be in response to the
antibodies revealed a positive reaction pat- presence here of type IVfibrilsor aggragates.
tern identical to that for type IV in both nor- The interface between the stroma and
mal and keratoconus corneas. We have not Descemet's membrane is also distinguished
emphasized these results, since our anti- ultrastructurally. Patchy, basement mem-
bodies were not, at the time of this work, brane-like material has been reported here
rigorously proved to be completely specific. by transmission electron microscopy. The
Subsequent purification has, however, con- strong reaction in this region with an-
firmed the type V specificity (J-M. F., un- tifibronectin suggests that this material con-
published data). sists, at least in part, of fibronectin. The pre-
Our results emphasize some similarities cise localization of fibronectin within the
and indicate certain differences between the basement membrane-like material at the
developing chick cornea and human tissue. stromal-Descemet's interface must await ul-
Type I collagen was the major detectable col- trastructural studies.
lagen in our study of the human corneal Type IV collagen was also prominently de-
stroma, a finding corresponding to what has tected at the interface of the endothelium with
been reported for chick.35 Type III collagen Descemet's membrane. This observation
is a small component of the human cornea offers further evidence that this cell layer, so
but is reportedly absent in the chick. The important to the maintenance of proper cor-
synthesis of both types I and III by a uniform neal hydration, synthesizes basement mem-
cell population has not been previously re- brane.40' 41 The presence offibronectinhere in
ported for corneal stromacytes but is known posterior Descemet's membrane is consistent
to occur with smooth muscle cells36 and with its detection in a wide variety of human
fibroblasts.29 We could not reliably detect basement membranes.42 This protein is also
type III collagen in all normal stromas, commonly associated with and synthesized by
perhaps due to the limits of the immu- fibroblasts (for review, see ref. 43). It is possi-
nofluorescent reaction. Factors such as mask- ble that the stromacytes synthesize the
ing of type III by other matrix components fibronectin that accumulates in Descemet's
may also have influenced its detectability. membrane. However, the corneal endothe-
Type II collagen is in the chick a promi- lium is embryologically derived from the
nent component of Bowman's layer, the sub- neural crest, as are the stromacytes proper,44
epithelial stroma, and Descemet's mem- and thus may share common attachment pro-
brane.35 The presence of type II-bearing teins. This observation contrasts with the ap-
primary corneal stroma, thought to be critical pearance of the epithelial basement mem-
to the morphogenesis of the adult stroma in brane area which had no detectable fibronec-
the chick, has not been confirmed in human tin. And, of course, the epithelium is not a
tissue (for review, see ref. 37). We could not neural crest derivative.
demonstrate type II in the Bowman's or sub- The striking amount of type III collagen in
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Number 6 Collagen types in normal and keratoconus corneas 749
the sclera may in part explain its anatomical syndrome. A new aspect of keratoconus. Med J Aust
differences from the type I-rich corneal 1:571, 1975.
4. Kuming BS and Joffe L: Ehlers-Danlos syndrome
stroma. The proportion of collagen types syn-
associated with keratoconus. A case report. S Afr
thesized may affect fibril aggregation and uni- Med J 52:403, 1977.
formity. In the sclera the matrix fibrils are 5. Pollack FM: Contributions of electronmicroscopy to
randomly arrayed in a feltwork with no signs the study of corneal pathology. Surv Ophthalmol
of the orderly lamellae so characteristic of the 20:375, 1976.
corneal stroma. Scleral fibril diameters are 6. Newsome DA, Takasugi M, Kenyon KR, Stark WF,
and Opelz G: Human corneal cells in vitro: mor-
much more heterogenous, and the fibrils phology and histocompatibility (HL-A) antigens of
much larger than those of the cornea. Lapier pure cell populations. INVEST OPHTHALMOL 13:23,
et al. 45 have shown that type III collagen 1974.
influences the size and type of bundles 7. Chung E and Miller EJ: Collagen polymorphism:
formed by type I collagen in vitro. Such characterization of molecules with the composition
[ a l (III)]3 human tissues. Science 183:1200, 1974.
an interaction may condition the collagen 8. Epstein EH Jr: [ a l (III)]3 human skin collagen: re-
bundle organization in various connective lease by pepsin digestion and preponderance in fetal
tissues. life. J Biol Chem 249:3225, 1974.
It is significant that the collagen profiles 9. Trelstad RL: Human aorta collagens: evidence for
three distinct species. Biochem Biophys Res Com-
obtained from cultured corneal, scleral, mun 57:717, 1974.
and conjunctival stromacytes differed among 10. Lammli UK: Cleavage of structural protein during
themselves, and that these differences corre- the assembly of the head of bacteriophage T4. Na-
sponded to differences in immunofluorescent ture 227:680, 1970.
reactivity. For example, the sclera reacted 11. Smith BD, Martin EJ, Miller EJ, Dorfman A, and
Swarm R: Nature of the collagen synthesized by a
strongly with anti-type III and synthesized
transplanted chondrosarcoma. Arch Biochem Bio-
the largest proportion of type III collagen in phys 166:181, 1975.
vitro. The persistence of these differences in 12. Orkin RW, Gehron P, McGoodwin EB, Martin GR,
patterns of synthesis of collagens, each type Valentine T, and Swarm R: A murine tumor produc-
of which is a distinct gene product, is in con- ing a matrix of basement membrane. J Exp Med
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trast to the rapid decay of keratan sulfate pro-
13. Andreopoulos NA, Mestecky J, Wright GP, and Mil-
teoglycan synthesis by in vitro corneal ler EJ: Characterization of antibodies to the native
stroma, even in organ culture. 46 human collagens and to their component a chains in
The present work has extended our knowl- the sera and joint fluids of patients with rheumatoid
edge of the site-specific distribution of the arthritis. Immunochemistry 13:709, 1976.
14. Nowack H, Gay S, Wick G, Becker U, and Timpl R:
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Preparation and use in immunohistology of an-
human and keratoconus corneas and normal tibodies specific for types I and III collagen and pro-
sclera. It appears that a normal complement collagen. J Immunol Methods 12:117, 1977.
of corneal stromal collagens is present in 15. Rennard SI, Berg R, Martin GR, Foidart J-M, and
normal proportions in keratoconus. Thus, if Gehron Robey P: Enzyme-linked immunoassay
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keratoconus is a true collagen disease (i.e.,
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