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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
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 plasminogendeficient mice may be an excellent model for the development of therapeutic strategies for the treatment of this
debilitating disease.
r 1998 by The American Society of Hematology.
L
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 (PAS1) 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 (Plg2/2)
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. Plg2/2 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.
IGNEOUS 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
From the Divisions of Developmental Biology and Pathology,
Children’s Hospital Research Foundation; and the Department of
Ophthalmology, University of Cincinnati, Cincinnati, OH.
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.
r 1998 by The American Society of Hematology.
0006-4971/98/9105-0035$3.00/0
1616
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 activaBlood, Vol 91, No 5 (March 1), 1998: pp 1616-1624
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PLASMINOGEN DEFICIENCY AND CONJUNCTIVITIS
tor (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, 58-TCTGCCCAAGACCACTTTAAGATGATT-38) 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, 58-GTGCGAGGCCAGAGGCCACTTGTGTAGCG-38) that together yield a 300-bp PCR product. The
wild-type uPA allele was detected with primers complementary to exon
11 (uPAEx11-1, 58-GCGATTCTGGAGGACCGCTTATCT-38 and uPAEx11-3, 58-ATTGAATCCAGTCCAGGAAGTGTGAGACCC-38) that
together yield a 141-bp PCR product. The targeted uPA allele was
detected by using primers uPAEx11-3 (see above) and PGK-Neo 58 that
together yield a 160-bp PCR product.
The incidence of ligneous conjunctivitis was assessed in Plg2/2 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 Plg2/2 mice of both backgrounds (C57B1/6J,
n 5 13; 129/Black Swiss, n 5 18) and Plg1/2 and Plg1/1 littermate
controls (n 5 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 Plg2/2
mice and 27 Plg1/2 and Plg1/1 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 Plg2/2 (n 5 17) and Plg2/2/Fib2/2 (n 5 11) mice. These
mice were generated from a cross between inbred Plg2/2 C57B1/6J33
and inbred Fib2/2 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 5 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 Fib2/2
mice34 and application of nonimmune rabbit serum in place of the
primary antibody in Plg2/2 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
1617
wash step was repeated eight times with the OD280 of the third wash
being less than 0.02. Bound material was eluted from the lysineSepharose by a 30-minute incubation (with continuous mixing) with an
equal volume of buffer B containing 0.4 mol/L e-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 SDSpolyacrylamide 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 Plg2/2 and C57B1/6J Plg2/2 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).
RESULTS
Development of conjunctival and corneal lesions. Conjunctival lesions were grossly apparent in adult C57B1/6J Plg2/2
mice (Fig 1A), whereas no lesions resembling ligneous conjunctivitis were observed in either Plg1/2 or Plg1/1 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.
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 Plg2/2 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 Plg2/2 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,
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1618
DREW ET AL
Fig 1. Photomicrographs indicating development of conjunctival lesions in Plg2/2 (A) but not in
Plg1/2 (B) and Plg1/1 (C) mice. (D)
Exposed palpebral conjunctiva
from a Plg1/2 mouse shows normal conjunctival tissue.
Fig 2. Photomicrographs of
gross pathology of ligneous lesions in Plg2/2 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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PLASMINOGEN DEFICIENCY AND CONJUNCTIVITIS
Fig 3. Development of ligneous conjunctivitis in Plg2/2 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
Plg2/2 mice and 27 littermate control mice of an inbred C57Bl/6J
background until 151 days of 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 Plg2/2 mice was already significantly lower than that
of littermate control mice [weight of 60-day-old Plg2/2 and
Plg1/1 mice was 18.5 6 0.8 g (n 5 16) and 23.5 6 0.6 g
(n 5 14), respectively (mean 6 SEM, P 5 .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 5 .002; Kendall Rank Correlation Coefficient).
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 Plg2/2 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 5 .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 Plg2/2 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
Plg2/2 mice of both C57B1/6J and 129/Black Swiss genetic
backgrounds (median age, 80 days) showed a complete absence
1619
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 Plg1/2 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 Plg2/2 mice of each genetic background (129/Black
Swiss, 2.4 6 0.5 mg/mL; C57B1/6J, 2.8 6 0.7 mg/mL).
To investigate if Fib deficiency protects Plg mice from the
development of ligneous conjunctivitis, a group of 11 Plg2/2/
Fib2/2 mice in a C57BL/6J background and 17 Plg2/2 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 Plg2/2/Fib2/2 mice. In contrast,
7 of 17 Plg2/2 littermates showed ligneous lesions (P 5 .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 Plg2/2 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 PAS1, 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 Plg2/2 mice (bottom, Fig
4E) or with Fib-specific antisera in the conjunctivae of Fibdeficient mice (data not shown). Acute inflammatory infiltrates
with a predominance of neutrophils frequently accompanied
conjunctival lesions (Fig 4F). Occasionally, CD41 and CD81
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 PAS1 material (Fig 4G)
or in lesions exhibiting epithelial reduplication around similar
amorphous deposits (Fig 4H).
Marked abnormalities were also evident in the microscopic
analyses of the cornea. Stromal vascularization, increased
cellularity, and deposition of PAS1 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
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DREW ET AL
Fig 4.
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PLASMINOGEN DEFICIENCY AND CONJUNCTIVITIS
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.
DISCUSSION
Plg2/2 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 Plg2/2 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 Plg2/2 mice with increasing age.29,30
Conjunctival lesions were a feature of adult Plg2/2 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
Plg2/2 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 Plg2/2 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 Plg2/2 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 Plg2/2 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 Plg2/2 mice.29,32 Although no data have been
collected regarding the frequency or timing of lesion develop-
1621
ment in mice with combined uPA/tPA deficiency, we have
observed conjunctival and corneal lesions in these mice that
histologically resemble those of Plg2/2 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 Plg2/2 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 Plg2/2 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 Plg2/2 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 Plg2/2 mice may both stimulate inflammatory
responses and impede timely re-epithelialization. Indeed, as
reported previously for incisional skin wounds in Plg2/2 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 Plg2/2 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
;
Fig 4. Photomicrographs of (a) normal palpebral conjunctiva from a Plg1/2 mouse in contrast with (b through h) Plg2/2 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 PAS1 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 PAS1 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 1003 (panels b and e), 2003 (panels a, c, d, g, and h) and 4003 (panel f).
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1622
DREW ET AL
Fig 5.
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PLASMINOGEN DEFICIENCY AND CONJUNCTIVITIS
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 Plg2/2 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 Plg2/2 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 Plg2/2
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
1623
expectancy, as well as potentially significant differences in
Plg-independent proteolytic pathways, there is no reason to
expect the Plg2/2 humans and Plg2/2 laboratory mice to be
phenotypically identical. Nevertheless, there are many notable
similarities between Plg2/2 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 Plg2/2 mice.5,17,18,26,29,30,39
Two recent reports describe impaired wound healing in two
Plg2/2 patients25,26 and retarded postnatal growth in one patient,25 both of which have been well documented in Plg2/2
mice.29,30,40 Also, as noted in two patient case studies, hydrocephalus has been observed in Plg2/2 mice, but the phenotype
seems to be very rare (unpublished results, October 1997). One
striking phenotypic feature of Plg2/2 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 Plg2/2 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 Plg2/2 mouse as an appropriate model for human
ligneous conjunctivitis. Furthermore, Plg2/2 mice may be
useful in the development of clinical strategies to treat or
prevent the life-threatening pathological conditions associated
with ligneous conjunctivitis.
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.
;
Fig 5. Photomicrographs of corneal lesions in Plg2/2 mice. (a) Stromal vascularization (arrowheads) in regions underlying extended plaques;
(b) Extensive cellular infiltration and anterior deposition of PAS1 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 1003 (panels c and d), 2003 (panels a, b, e, f, and g) and 4003 (panel h).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1624
DREW ET AL
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1998 91: 1616-1624
Ligneous Conjunctivitis in Plasminogen-Deficient Mice
A.F. Drew, A.H. Kaufman, K.W. Kombrinck, M.J.S. Danton, C.C. Daugherty, J.L. Degen and T.H. Bugge
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