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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1616-1624
Ligneous Conjunctivitis in Plasminogen-Deficient Mice
By
A.F. Drew,
A.H. Kaufman,
K.W. Kombrinck,
M.J.S. Danton,
C.C. Daugherty,
J.L. Degen, and
T.H. Bugge
From the Divisions of Developmental Biology and Pathology,
Children's Hospital Research Foundation; and the Department of
Ophthalmology, University of Cincinnati, Cincinnati, OH.
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ABSTRACT |
Ligneous conjunctivitis is a rare form of chronic pseudomembranous
conjunctivitis that is associated with systemic membranous pathological
changes. A probable link between plasminogen and ligneous
conjunctivitis has been indicated by the recent diagnoses of
plasminogen deficiency in five patients suffering from ligneous conjunctivitis. The current study reports that plasminogen-deficient mice develop conjunctival lesions indistinguishable from human ligneous
conjunctivitis in both appearance and histology. Both human and mouse
lesions contain acellular material rich in fibrin, and aberrant or
disrupted epithelium. The incidence of lesion development in mice
increases with age and is strongly influenced by genetic background.
Interestingly, ligneous conjunctivitis was not observed in
plasminogen-deficient mice simultaneously lacking fibrinogen. This
study provides direct evidence that plasminogen deficiency is one cause
of ligneous conjunctivitis and suggests that plasminogen-deficient mice
may be an excellent model for the development of therapeutic strategies
for the treatment of this debilitating disease.
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INTRODUCTION |
LIGNEOUS CONJUNCTIVITIS is a rare form of
chronic membranous conjunctivitis of unknown origin. Patients with
ligneous conjunctivitis present with pseudomembranous "woodlike"
lesions of the conjunctiva which result in scarring and impaired
vision. White, yellowish, or red nodules form on the tarsal and bulbar
conjunctivae, which can result in eversion of the eyelid.1
Secondary corneal involvement including corneal melting, scarring, and
development of extensive sessile lesions has also been
reported.2,3
This disorder occurs most frequently in young children, with a slight
predominance in females, and an autosomal recessive inheritance pattern
is apparent in some cases.1,3,4 Increased childhood
mortality rates accompany ligneous conjunctivitis due to associated
systemic pathological changes,5-7 although elderly patients
have been described.3 The chronic phase of this condition may persist for years, or spontaneously remit and recur. Treatment with
antibiotics, antifungals, or immunosuppressants has been largely
ineffective6,8,9 and attempts to identify a microbial causal agent have had little success.6,10 Surgical excision of conjunctival lesions is invariably followed by rapid reformation and
often worsens the condition.1,2,7,11 Furthermore, several
reports indicate that in rare cases, conjunctival surgery can initiate
the formation of ligneous lesions.12-14 However, De Cock et
al15 have reported remission of ligneous conjunctivitis in
some patients after excision of the membranes followed by cauterization and application of topical heparin. The pathological changes associated with this condition are not restricted to the eye. Rather, lesions in
other mucous membranes such as the nasopharynx, larynx, tongue, trachea, middle ear, gingiva, peritoneum, vagina, and cervix may occur
in association with, or in place of, ocular
manifestations.5,11,16-22 Occlusive hydrocephalus has also
been noted in some patients with ligneous
conjunctivitis.3,7,10
Histological examination of the membranous conjunctival lesions shows
extensive epithelial ulceration with hyperplasia and extension of the
epithelial layer into the substantia propria in the form of cysts and
glandlike structures.2,7 Mucopolysaccharide exudate
frequently overlies large areas of ulceration.3,11 Lesions
contain large, sparsely cellular deposits of eosinophilic, periodic
acid Schiff-positive (PAS+) amorphous material with
adjacent acute and/or chronic inflammatory cell infiltrates
composed of neutrophils, T cells, macrophages, B cells, and mast
cells.3,11-13,17,23 Neovascularization and deposition of
plasma proteins such as immunoglobulin and albumin are frequently
present, whereas lipid, amyloid, and keratin are generally not
detectable.2,17 The amorphous deposits contain fibrillar
material, consistent with fibrin, and stain intensely for fibrin by
immunohistochemistry.3,23,24
Ligneous conjunctivitis has been associated with plasminogen (Plg) in
recent reports describing five unrelated patients with severe Plg
deficiency.25-27 Plg is an abundant plasma protein which is
the zymogen precursor of the serine protease, plasmin, the key
fibrinolytic enzyme.28 Plg-deficient
(Plg /) mice were recently generated to define in
greater detail the physiological roles of plasmin(ogen) in
vivo.29,30 These mice develop to term and generally survive
to adulthood, but the phenotypic consequences of Plg deficiency are
severe and life expectancy is short. Plg/ mice
experience widespread thrombotic occlusions within terminal vessels,
organ damage, and wasting. Fibrin-rich ulcerative lesions develop
throughout the gastrointestinal, respiratory, and female genital
tracts.29,31 The current study reports that Plg deficiency in mice results in the development of fibrin-rich conjunctival lesions
that are indistinguishable from human ligneous conjunctivitis.
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MATERIALS AND METHODS |
Generation of cohorts of inbred and outbred mice.
Gene-deficient mice were generated in accordance with National
Institutes of Health recombinant DNA guidelines, and study protocols
were approved by the Children's Hospital Research Foundation Institutional Animal Care and Use Committee. All mice used in these
studies were maintained in parallel by the same caregivers. Mice with
single and combined deficits in Plg and fibrinogen (Fib) were genotyped
as described previously.31 The genotypes of mice with
combined tissue-type plasminogen activator (tPA) and urokinase (uPA)
deficiencies32 were established by polymerase chain
reaction (PCR), using ear biopsy DNA as a template. The wild-type tPA
allele was detected with primers complementary to exon 10 (tPAEx10-131) and exon 11 (tPAEx11-1,
5 -TCTGCCCAAGACCACTTTAAGATGATT-3) that together yield a 350-bp PCR
product. The targeted tPA allele was detected by using tPAEx11-1 (see
above) and a primer complementary to the PGK-Neo cassette inserted into
the disrupted tPA gene (PGK-Neo, 5-GTGCGAGGCCAGAGGCCACTTGTGTAGCG-3)
that together yield a 300-bp PCR product. The wild-type uPA allele was
detected with primers complementary to exon 11 (uPAEx11-1,
5-GCGATTCTGGAGGACCGCTTATCT-3 and uPAEx11-3,
5-ATTGAATCCAGTCCAGGAAGTGTGAGACCC-3) that together yield a 141-bp PCR
product. The targeted uPA allele was detected by using primers
uPAEx11-3 (see above) and PGK-Neo 5 that together yield a 160-bp PCR
product.
The incidence of ligneous conjunctivitis was assessed in
Plg/ mice in a mixed 129/Black Swiss
background29 and in mice backcrossed for six generations to
C57B1/6J mice (Jackson Laboratories, Bar Harbor, ME).
Groups of older Plg/ mice of both backgrounds
(C57B1/6J, n = 13; 129/Black Swiss, n = 18) and
Plg+/ and Plg+/+ littermate controls
(n = 24) were anesthetized with ketamine/xylazine/acepromazine (4:1:1), and eyelids were everted with forceps and examined with a
dissecting microscope.
In later detailed studies, a prospective cohort of 17 C57B1/6J
Plg/ mice and 27 Plg+/ and
Plg+/+ littermate control mice, aged between 51 to 70 days
at the beginning of the observational period, were observed until 133 to 152 days of age. Mice were inspected weekly by an investigator
unaware of the genotypes of the mice. Conjunctival lesion development was recorded along with general health parameters, including weight and
development of rectal prolapse.
In a separate study, the development of ligneous conjunctivitis was
investigated in Plg/ (n = 17) and
Plg//Fib/ (n = 11) mice. These
mice were generated from a cross between inbred Plg/
C57B1/6J33 and inbred Fib/ C57B1/6J
mice.34 Mice were age-matched with an age range of 60 to
172 days and a median age of 109 days. Lesions were detected in
anesthetized mice by eversion of eyelids and viewed with a dissecting
microscope. The presence of conjunctival lesions was explored in mice
lacking both uPA and tPA in a retrospective evaluation of arbitrarily
selected mice that had been sacrificed, formalin-fixed, and stored
(n = 6).
Histopathology.
Eyes, eyelids, and surrounding skin were excised and fixed en bloc in
neutral-buffered formalin (Sigma, St Louis, MO). Tissues were
paraffin-embedded and sectioned at a thickness of 4 µm. Longitudinal, sagittal sections were taken at 180-µm intervals for sequential analysis. Tissue sections were routinely stained with hematoxylin and
eosin, PAS stain, or Leder stain.35
Immunohistology.
Fibrin(ogen) was detected with a polyclonal rabbit anti-mouse
fibrinogen serum (diluted 1:1,000) by using the Vectastain Elite ABC
Kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine (DAB;
Sigma) substrate. Negative controls for staining consisted of parallel
staining of conjunctival tissue from Fib/
mice34 and application of nonimmune rabbit serum in place
of the primary antibody in Plg/ mice. Mouse
immunoglobulin was detected with biotinylated anti-mouse IgG (Vector)
and mouse CD4 and CD8 were detected with GK1.5 (monoclonal anti-mouse
CD4, ATCC) and 2.43 (monoclonal anti-mouse CD8, ATCC) and the
Vectastain Elite Kit.
Detection of Plg protein in plasma.
Plasma Plg was purified by lysine-Sepharose chromatography. Citrated
plasma samples (60 µL) were diluted in an equal volume of buffer B
(1.5 mmol/L potassium phosphate; 8 mmol/L sodium phosphate; 14 mmol/L
sodium chloride; 3 mmol/L potassium chloride, pH 7.3) and combined with
60 µL (settled volume) of lysine-Sepharose 4B (Pharmacia Biotech,
Piscataway, NJ). The suspensions were incubated at room temperature for
30 minutes with continuous mixing. Sepharose beads were collected by
centrifugation at 3,000g for 30 seconds and resuspended in 1 mL
of buffer B. This wash step was repeated eight times with the
OD280 of the third wash being less than 0.02. Bound
material was eluted from the lysine-Sepharose by a 30-minute incubation
(with continuous mixing) with an equal volume of buffer B containing
0.4 mol/L  -amino caproic acid. The eluates were diluted in sample
buffer containing 2% sodium dodecyl sulfate (SDS) for gel
fractionation and subsequent Plg detection by Western blotting.
Proteins were fractionated by SDS-polyacrylamide gel electrophoresis
(12% acrylamide), transferred to Immobilon P membranes (Millipore,
Bedford, MA), and Plg was detected with sheep anti-rat Plg antiserum
(kindly provided by Dr E. Reich, SUNY, Stony Brook, NY) that
cross-reacts with mouse plasminogen as described
previously.29 Bound primary antibody was detected by using
the Vectastain ABC (peroxidase) kit (Vector) and the ECL
chemiluminescence system (Amersham, Arlington Heights, IL).
Fib enzyme-linked immunosorbent assay (ELISA).
Plasma Fib levels of 129/Black Swiss Plg/ and
C57B1/6J Plg/ mice were determined with an
Fib-specific ELISA (Asserachrom-Fibrinogen; Diagnostica Stago, France)
by using purified mouse Fib as a standard. Purified mouse Fib was a
gift from Dr D.I. Simon (Brigham and Women's Hospital, Boston, MA).
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RESULTS |
Development of conjunctival and corneal lesions.
Conjunctival lesions were grossly apparent in adult C57B1/6J
Plg/ mice (Fig 1A),whereas no lesions resembling ligneous conjunctivitis were observed in
either Plg+/ or Plg+/+ littermate control
mice (Fig 1B through D). The earliest lesions appeared as subtle
thickenings of the lower or upper eyelid. These lesions were
accompanied by a whitish, irregular surface of the palpebral
conjunctiva, an increase in vascularization of the lid, and external
accumulations of mucus (Fig 2A). Corneal
defects such as stromal haze and epithelial irregularities were
occasionally present in mice with early conjunctival lesions (Fig 2B),
but lesions always occurred in the palpebral conjunctiva prior to corneal involvement.
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| Fig 1.
Photomicrographs indicating development of conjunctival
lesions in Plg/ (A) but not in Plg+/
(B) and Plg+/+ (C) mice. (D) Exposed palpebral
conjunctiva from a Plg+/ mouse shows normal
conjunctival tissue.
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| Fig 2.
Photomicrographs of gross pathology of ligneous lesions
in Plg/ mice, showing mild and severe manifestations.
(A) Thickened eyelid representing an early or mild lesion. Note that
the observed cataract formed postmortem. (B) Stromal haze and
epithelial defect; (C) pedunculated lesion in the upper lid, with
ulceration and hemorrhage in the lower lid; (D) plaque extending above
the corneal surface; (E) neovascularization of the cornea extending
from the limbus into the plaque (arrows); and (F) corneal
neovascularization around the circumference of a plaque (arrows).
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Lesions were progressive in nature, developing over a period of several
weeks into extensive, pedunculated plaques (Fig 2C). Advanced lesions
occurred in the lid margin, along the entire palpebral and bulbar
conjunctivae, and on the cornea. Palpebral conjunctival alterations
resulted in hypertrophied, everted eyelids with ulceration and
hemorrhage. Scarring and hair loss were also commonly associated with
advanced lesions. Ulceration and necrosis were observed in the cornea,
and large lesions that extended 1 to 2 mm above the corneal surface
developed in some mice (Fig 2D). Stromal opacification and
neovascularization extending from the limbus were apparent throughout
the lesion (Fig 2E and F). Occasionally, foreign bodies such as
cage-bedding material were seen embedded in lesions.
Frequency of lesion development.
To define the age- and gender-dependence of ligneous lesion formation,
a prospective cohort of highly inbred C57B1/6J Plg/
mice was monitored for ocular lesion development over a period of
several months. Changes in body weight and development of rectal lesions (a common feature of adult Plg/ mice) were
also recorded. Nine of 17 mice (53%) developed conjunctival lesions
during the observational period (Fig 3).The median age of onset of lesions was in young adulthood, at 151 days.
Gender was not a significant factor in the development of conjunctival lesions; lesions developed in four of eight female mice (50%; median
age, 151 days) and five of nine male mice (56%; median age, 124 days)
during the observational period. Development of lesions was
predominantly bilateral. Thus, occurrence of a lesion in either eye was
associated with a high incidence of involvement of the other eye. All
conjunctival lesions developed after 60 days of age. At this age, the
mean weight of inbred C57B1/6J Plg/ mice was already
significantly lower than that of littermate control mice [weight of
60-day-old Plg/ and Plg+/+ mice was 18.5 ± 0.8 g (n = 16) and 23.5 ± 0.6 g (n = 14), respectively (mean ± SEM, P = .0001, Student's t-test)].
Rectal lesions developed in 12 of 17 mice (71%) with a median age of
126 days. Of the nine mice in the cohort that developed conjunctival
lesions, eight also developed rectal lesions during the same period. A
significant correlation was found between the age of onset of
conjunctival lesions (median age, 151 days) and rectal lesions (median
age, 126 days; P = .002; Kendall Rank Correlation
Coefficient).
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| Fig 3.
Development of ligneous conjunctivitis in
Plg/ mice, as a function of age. The development of
ligneous lesions of the conjunctiva was followed in a prospective
cohort of mice consisting of 17 Plg/ mice and 27 littermate control mice of an inbred C57Bl/6J background until 151 days
of age.
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The genetic background of mice had a marked effect on both the
frequency of lesion development and the severity of the lesions. In a
separate study, a random selection of older mice from two genetic
backgrounds of Plg/ mice were examined for ocular
lesion formation, including inbred C57B1/6J mice (median age, 190 days;
range, 132 to 324 days) and outbred 129/Black Swiss mice29
(median age, 235 days; range, 201 to 397 days). Thirteen of thirteen
(100%) C57B1/6J mice included in this selection had conjunctival
lesions at the time of examination. A significantly lower incidence of
ligneous lesions, 3 of 18 mice (17%), and milder lesions were observed
in the 129/Black Swiss background (P = .0001, Chi-square
analysis).
To investigate the difference in susceptibility to ligneous lesion
development between the two genetic backgrounds, hematologic parameters, including plasma Fib and Plg antigen levels, were investigated. Consistent with earlier results in 129/Black Swiss Plg/ mice documenting that hepatic Plg mRNA, plasma
Plg protein, and Plg activity were undetectable in assays sensitive to
at least three orders-of-magnitude below normal,29 parallel
analyses of plasma collected from adult Plg/ mice of
both C57B1/6J and 129/Black Swiss genetic backgrounds (median age, 80 days) showed a complete absence of Plg antigen, regardless of genetic
background (data not shown). In contrast, Plg antigen was easily
detected and was at similar levels in the plasma of
Plg+/ mice of both genetic backgrounds (data not shown).
Through use of a specific ELISA, parallel assays of plasma Fib-related
antigen levels indicated that circulating Fib antigen was not
appreciably different in Plg/ mice of each genetic
background (129/Black Swiss, 2.4 ± 0.5 mg/mL; C57B1/6J,
2.8 ± 0.7 mg/mL).
To investigate if Fib deficiency protects Plg mice from the development
of ligneous conjunctivitis, a group of 11 Plg//Fib/ mice in a C57BL/6J
background and 17 Plg/ littermates were inspected.
The median age in both groups was 109 days and the range was 60 to 172 days. Conjunctival lesions were detected in none of eleven
Plg//Fib/ mice. In contrast, 7 of
17 Plg/ littermates showed ligneous lesions
(P = .014, Chi-square test). Additionally, formalin-fixed
cadaveric mice in a hybrid 129/C57BL/6J genetic background and with
combined uPA and tPA deficiency were retrospectively investigated for
conjunctival lesion development. Of six mice examined, ligneous lesions
were observed in two mice (median age, 139 days; range, 134 to 139 days). These observations were confirmed histologically. Thus, ligneous
conjunctivitis seems to be a consequence of failed Plg activation
resulting in impaired fibrin clearance.
Microscopic analyses of conjunctival and corneal lesions.
Consistent with the conjunctival and corneal defects observed grossly
in Plg/ mice, microscopic analyses of sectioned
tissues revealed abnormal tissue organization. Extensive disruption of
the conjunctival epithelium was evident in all lesions, with associated
hypertrophy, disorganization, and reduplication consistent with
chronic, recurrent ulceration and attempted re-epithelialization (Fig
4). Amorphous, eosinophilic
deposits were associated with areas of disrupted epithelium. These
deposits were largely acellular and PAS+, indicating
mucopolysaccharide (Fig 4C and D). These areas were shown to contain
appreciable amounts of fibrin(ogen) (Fig 4E) and immunoglobulin (data
not shown) by immunohistochemistry. No staining was observed with
normal rabbit serum in Plg/ mice (bottom, Fig 4E) or
with Fib-specific antisera in the conjunctivae of Fib-deficient mice
(data not shown). Acute inflammatory infiltrates with a predominance of
neutrophils frequently accompanied conjunctival lesions (Fig 4F).
Occasionally, CD4+ and CD8+
lymphocytes were identified in the infiltrates by immunohistochemistry (data not shown). Tissue disorganization was highly evident in lesions
containing "islands" of epithelial cells (including goblet cells)
surrounded by PAS+ material (Fig 4G) or in lesions
exhibiting epithelial reduplication around similar amorphous deposits
(Fig 4H).
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| Fig 4.
Photomicrographs of (a) normal palpebral conjunctiva from
a Plg+/ mouse in contrast with (b through h)
Plg/ mice. (b) Large eosinophilic deposits with low
cellular density constitute the majority of the area occupied by
lesions. (c) Disrupted epithelium was associated with (d) amorphous
PAS+ material (in the same lesion). (e) The amorphous
material stained with an anti-mouse Fib antiserum (top) but not with
normal rabbit serum (bottom; arrowheads indicate lesion area). (f) An
acute inflammatory infiltrate with prominent neutrophils (arrows) is adjacent to amorphous, eosinophilic material. (g) Epithelial cells, including PAS+ goblet cells, existing as a discreet
cluster (*) inside the eyelid show the degree of disorganization of the
epithelium. (h) Reduplication of the epithelium (denoted by "e")
occurred adjacent to eosinophilic deposits. Panels b, c, and h are
hematoxylin and eosin preparations; panels a, d, and g are PAS
preparations; panel f is a Leder stain. Original magnifications are
100× (panels b and e), 200× (panels a, c, d, g, and h) and 400×
(panel f).
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Marked abnormalities were also evident in the microscopic analyses of
the cornea. Stromal vascularization, increased cellularity, and
deposition of PAS+ material were frequently associated with
corneal lesions (Fig 5A and
B). Ulceration or destruction
of the anterior epithelium and stroma were noted (Fig 5C) with
extensive, protruding plaques overlying the cornea (Fig 5D).
Fibrin(ogen) was found to be a significant component of such corneal
plaques (Fig 5E). Corneal epithelium was frequently absent, necrotic,
or hypertrophied. Epithelium accompanying severe corneal lesions was
occasionally observed to form aberrant wedge-shaped projections,
adjacent to amorphous fibrin-containing deposits, indicating that
impaired re-epithelialization is a feature of these diseased tissues
(Fig 5F). Formation of a pupillary or a retrocorneal membrane was
observed in several mice, consistent with chronic intraocular
inflammation (Fig 5G). This aberrant membrane stained intensely for
fibrin(ogen) (Fig 5H). The formation of extensive fibrin(ogen)-rich
plaques overlying the cornea, with associated epithelial ulceration,
hypertrophy, and disorganization are consistent with an ongoing process
of tissue injury, repair, and partial epithelialization.
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| Fig 5.
Photomicrographs of corneal lesions in
Plg/ mice. (a) Stromal vascularization (arrowheads)
in regions underlying extended plaques; (b) Extensive cellular
infiltration and anterior deposition of PAS+ material,
yet relatively normal posterior stroma with only mild inflammatory
infiltration; (c) corneal ulceration; (d) An extensive corneal plaque
which contained (e) abundant fibrin(ogen) matrix; (f) Formation of a
wedge-like projection of epithelial cells was identified underlying but
not penetrating the shoulder region of a plaque. The acellular,
eosinophilic material above the "wedge" appeared to bisect the
epithelial layer, leaving a thin layer of necrotic squamous cells
exposed. (g) A pupillary membrane (arrows) formed in mice with severe
corneal lesions. (h) This aberrant membrane stained intensely for
fibrin(ogen) (arrows) by immunohistochemistry (DM denotes Descemet's
membrane). Panels a, c, d, f, and g are hematoxylin- and eosin-stained,
panel b is PAS-stained, and panels e and h are fibrin(ogen)
immunohistochemistry. Original magnifications are 100× (panels c and
d), 200× (panels a, b, e, f, and g) and 400× (panel h).
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DISCUSSION |
Plg/ mice spontaneously develop palpebral and bulbar
conjunctivitis. Conjunctival lesions occurred with a high penetrance, often with bilateral involvement and with no evidence of a gender bias.
The development of conjunctival lesions increased as a function of age
and was associated with a general decline in health (indicated by a
significant deviation from normal body weight and the development of
rectal prolapse)29 in Plg/ mice. The
conjunctival lesions described closely resembled human ligneous
conjunctivitis in gross appearance, distribution, and histologic
features, including lesion composition and organization.2,3 The current studies show that Plg deficiency alone is sufficient for
the development of ligneous conjunctivitis in mice and that lesion
development may be another manifestation of the many progressive disorders which befall Plg/ mice with increasing
age.29,30
Conjunctival lesions were a feature of adult Plg/
mice in each of two genetic backgrounds evaluated, although mice with a
mixed 129/Black Swiss background were significantly less susceptible to
conjunctival lesion formation than the C57B1/6J inbred cohort followed
in this study. Thus, additional genetic factors seem to contribute to
lesion incidence and severity in Plg/ mice. The
secondary genetic factors that contribute, in combination with Plg
deficiency, to the risk for developing conjunctival lesions are
presently unknown, but differences in plasma Plg and/or Fib levels in Plg/ mice were excluded. However, this does
not preclude background-specific differences in the regulation of the
coagulation system, or differences in the availability and efficiency
of Plg-independent pathways for fibrin clearance. Whatever the factors
are in Plg/ mice that contribute to the risk of
conjunctival and corneal lesions, it is clear that the loss of Fib
prevents the development of these lesions. This finding strongly
implies that diminished fibrinolysis and fibrin clearance in
Plg/ mice is mechanistically related to conjunctival
lesion development.
Mice deficient in both Plg activators (uPA and tPA) have previously
been reported to suffer from similar pathological manifestations as
Plg/ mice.29,32 Although no data have
been collected regarding the frequency or timing of lesion development
in mice with combined uPA/tPA deficiency, we have observed conjunctival
and corneal lesions in these mice that histologically resemble those of
Plg/ mice. This indicates that ligneous
conjunctivitis can be caused by more than one genetic disorder, and
disorders other than Plg and Plg activator deficiency may also be
encountered in ligneous conjunctivitis patients. It should also be
noted that patients may develop ligneous conjunctivitis without any
major underlying genetic deficit; a case report of a patient being
treated for menorrhagia with tranexamic acid, a plasmin inhibitor,
describes that systemic inhibition of plasmin was associated with the
development of ligneous lesions in the conjunctiva, which regressed on
cessation of treatment.19 Taken together, these data
suggest that similar pathological processes may lead to the formation
of ligneous lesions when plasmin-dependent events are interrupted in
both humans and mice.
Conjunctival lesions of Plg/ mice are invariably
associated with epithelial disruptions. Minor wounding or disruption of
the epithelium may be the initiating event in the formation of ligneous lesions. In normal human and mouse skin, injury results in the rapid
deposition of fibrin to control blood loss and to provide a provisional
extracellular matrix to support the formation of granulation tissue and
re-epithelialization.36 The cellular organization of wound
fields requires migration of keratinocytes, endothelial cells,
fibroblasts, and other cell types through the fibrin-rich matrix and
hence, pericellular proteolysis. In Plg/ mice, a lack
of plasmin-mediated proteolysis is a major impediment to healing in
skin wound fields,37 characterized by impaired keratinocyte
migration from the wound edges. Remarkably, these defects can be
ameliorated by simultaneous Fib deficiency.31 Based on the
presence of local epithelial defects and persistent fibrin deposits
within conjunctival lesions of Plg/ mice, together
with the findings that defective wound repair and the deleterious
formation of ocular lesions are corrected, in parallel, by simultaneous
Fib deficiency, it seems likely that compromised tissue repair
contributes to conjunctival lesion formation in these mice. The
presence of persistent fibrin deposits adjacent to regions of
epithelial disruption in the conjunctivae of Plg/
mice may both stimulate inflammatory responses and impede timely re-epithelialization. Indeed, as reported previously for incisional skin wounds in Plg/ mice,37 epithelial
cells within conjunctival lesions were frequently observed at the
periphery of the fibrinous deposits, seemingly unable to penetrate the
fibrin-rich matrix in the absence of plasmin-mediated proteolysis.
Thus, conjunctival lesions formed in Plg/ mice may
result from minor epithelial disruption, chronic inflammation, and a
failure to resolve local tissue damage.
Clinical case studies in the literature provide additional support for
the hypothesis that tissue injury is an initiating event in the
formation of ligneous lesions in the conjunctiva. Several reports
document induction of lesions shortly after surgical procedures12-14 and one case identifies a dog bite to the
eyelid one month before ligneous lesion development.3
Foreign bodies have been identified within human ligneous lesions and
the aggravating effect of this material may have caused injury and
inflammation sufficient to initiate lesion formation in these
individuals.3 Although the mice in the current
study were not subjected to invasive procedures, foreign bodies
consisting of cage-bedding material were occasionally identified
embedded in plaques. Scratching, or abrasion of foreign bodies against
the conjunctival epithelium, may initiate development of ligneous
lesions by disrupting the epithelium and underlying tissue, resulting
in the accumulation of fibrin which persists in the absence of plasmin.
Repetitive minor trauma and the progressive distortion of normal
structure may set up a cycle of repetitive injury and abortive healing, resulting in the progression of these lesions.
Tissue injury may also result from microvascular thrombotic occlusion.
As a de novo event, this seems unlikely because tissues in many
anatomic locations are unaffected. However, lesion distribution in
Plg/ mice tends to occur in areas where microtrauma
would be suspected. It is possible that microtrauma, secondary to
mechanical tissue stress or stretching in such areas as the rectum or
conjunctiva, may induce microthrombosis that results in localized
tissue ischemia as an early event in the evolution of such lesions.
Whatever the nature of the initiating event, concurrent diminution of
plasmin-mediated proteolysis appears to be essential for ligneous
lesion development.
Occurrence of lesions in the palpebral conjunctiva was always observed
before corneal involvement. It is likely that involvement of the cornea
is secondary to conjunctival lesion development, and may result from
abrasion of ulcerated palpebral lesions against the corneal epithelium
during eye closure and/or drying caused by impaired eye
closure. Corneal injury induced by epithelial scraping with a blade in
Plg/ mice results in impaired wound healing,
persistent fibrin-containing matrix, and corneal
opacification.38 Corneal caps of the type seen in human
ligneous conjunctivitis may develop in Plg/ mice
after repetitive surgical removal of the epithelium, but this has not
yet been explored.
Recent reports in the literature have identified several patients with
severe Plg deficiency.25-27 In addition to developing ligneous conjunctivitis, these patients sustained other clinically recognized disorders, including hyperviscosity of tracheobronchial and
nasopharyngeal secretions, bronchopneumonia, gingival hyperplasia, and
hydrocephalus. Given the obvious differences between humans and mice in
anatomy, physiology, diet, environmental challenges (eg, pathogen
exposure), and life expectancy, as well as potentially significant
differences in Plg-independent proteolytic pathways, there is no reason
to expect the Plg/ humans and Plg/
laboratory mice to be phenotypically identical. Nevertheless, there are
many notable similarities between Plg/ mice and
humans, and more may be recognized as additional detailed studies are
performed in each species. For example, pathological disturbances
involving mucous membranes have been previously reported in both human
ligneous conjunctivitis patients and in Plg/
mice.5,17,18,26,29,30,39 Two recent reports describe impaired wound healing in two Plg/
patients25,26 and retarded postnatal growth in one
patient,25 both of which have been well documented in
Plg/ mice.29,30,40 Also, as noted in two
patient case studies, hydrocephalus has been observed in
Plg/ mice, but the phenotype seems to be very rare
(unpublished results, October 1997). One striking
phenotypic feature of Plg/ mice that has not yet been
explored in patients with confirmed Plg deficiency is the development
of thrombotic occlusions in terminal microvasculature, particularly in
the gastrointestinal tract. Detailed histological evaluation of patient
tissues collected at biopsy or autopsy will be useful in establishing
whether this feature is shared between species. However, with regard to
the hemostatic consequences of Plg deficiency, it is notable that presently there is no evidence of occlusive thrombotic events in large
vessels of either Plg/ mice or
humans.25,26,29,30
Complete remission of ligneous conjunctivitis, including dissolution of
fibrin and resolution of pseudomembranes, has been reported in a male
infant after replacement therapy with lys-Plg.26 Increases
in plasma D-dimer formation after addition of Plg indicated an
otherwise intact capacity for fibrinolysis in this patient. Fibrin-containing deposits reappeared after cessation of treatment. Whether the therapeutic benefit of Plg administration will be equally
apparent in other patients remains to be established. Rapid Plg
clearance may be a sufficient limitation of this
approach.25 Systemic antithrombotic drugs may prove to be
an effective alternative but this has not yet been explored.
The current studies show that Plg deficiency results in the development
of ligneous conjunctivitis in mice. These studies identify the
Plg/ mouse as an appropriate model for human ligneous
conjunctivitis. Furthermore, Plg/ mice may be useful
in the development of clinical strategies to treat or prevent the
life-threatening pathological conditions associated with ligneous
conjunctivitis.
| |
FOOTNOTES |
Submitted June 11, 1997;
accepted October 27, 1997.
Supported by a Career Development Award from Research to Prevent
Blindness, New York, NY (A.H.K.) and Grant No. HL47826 from the
National Institutes of Health (J.L.D.). This study was performed during
the tenure of an Established Investigatorship (J.L.D.) from the
American Heart Association (93002570).
Address reprint requests to T.H. Bugge, PhD, Division of
Developmental Biology, Children's Hospital Research Foundation, 3333 Burnet Ave NRB 2018A, Cincinnati, OH 45229.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We are grateful to Dr Peter Gartside for assistance with statistical
analyses, and Heidi Schiman and Chonnettia Jones for assistance with
histological analyses and the Fib ELISA, respectively.
| |
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Abstract
Introduction
Abstract