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1 From the Unit of Ophthalmology, Department of Medicine and St. Paul’s Eye Unit, University of Liverpool, UK; 2 The Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; the 3 Department of Ophthalmology, University of Erlangen–Nürnberg, Erlangen, Germany; and the 4 Unit of Glycobiology, Department of Medicine, University of Liverpool, UK.
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Abstract |
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METHODS. Using cultured bovine RPEs, immunohistochemistry was used to study ABL binding. Morphologic and trypan blue exclusion techniques were used for toxicity studies. The effect of ABL on RPE proliferation was investigated by [methyl-3H]–thymidine incorporation. The effect of ABL on RPE-mediated matrix contraction was evaluated with RPE-populated three-dimensional collagen matrices.
RESULTS. ABL bound to RPE cells. This binding was inhibited by asialomucin. No change in RPE morphology or trypan blue exclusion compared with controls was observed in RPEs incubated with 5 to 60 µg/ml ABL for 3 days. Twenty-four-hour incubations of RPEs with ABL significantly inhibited RPE proliferation in a dose-dependent way, 40 µg/ml ABL inhibited proliferation by 83% (SE 14, P < 0.05). ABL showed a dose-dependent significant inhibition of RPE-mediated collagen matrix contraction over 3 days, with 93% inhibition compared with controls by 40 µg/ml lectin (P < 0.05). The inhibitory effect of ABL on proliferation and gel contraction was partly reversible after eliminating ABL from the culture medium.
CONCLUSIONS. Bovine RPE cells bind ABL, and preliminary evaluations suggest that levels of ABL that are nontoxic to the cells potently inhibit RPE proliferation and RPE-mediated matrix contraction. ABL deserves further investigation as a potential inhibitor of RPE proliferation and cell-mediated matrix contraction in anomalous reparative processes such as proliferative vitreoretinopathy and as a laboratory tool for RPE behavioral studies.
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Introduction |
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Lectins are ubiquitous carbohydrate-binding non-immunoglobulin proteins. They bind noncovalently to carbohydrates and are readily purified from a wide variety of sources. A range of carbohydrates occurs on all cell surfaces, and lectins have been used to explore cell membranes and to distinguish different cell types, because cells express distinct carbohydrates that can be detected by specific lectins.2 Furthermore, lectins binding to cell surface carbohydrates may affect the behavior of the cell. Thus, lectins such as peanut agglutinin and the lectin of the edible mushroom (Agaricus bisporus lectin; ABL), which both bind to the carbohydrate structure galactosyl ß-1,3-N-acetyl-galactosamine, modulate the proliferation of malignant epithelial cells.3 Peanut agglutinin increases colonic carcinoma cell division, whereas ABL inhibits proliferation of these cells.3 Moreover, ABL inhibits proliferation of a range of other cells including Tenon’s capsule fibroblasts, and it inhibits contraction of collagen matrices by Tenon’s fibroblasts.4 These effects occur without apparent cytotoxicity.3 4 Because cell-mediated membrane contraction and proliferation are key RPE activities in PVR, we evaluated the effect of ABL on these RPE activities in vitro, having first determined whether ABL binds to (or affects the viability of) cultured RPEs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPE Culture
Bovine RPEs were obtained and cultured as previously
described.5
Established cultures were maintained with
minimal essential medium (MEM) containing glutamine and fungizone,
penicillin and streptomycin, and 15% newborn calf serum (NCS) (GIBCO
Europe Ltd., Paisley, UK). The cultures were kept at 37°C in the
presence of 5% CO2 and air. The RPEs reached
confluence within 1 to 2 weeks, and subcultures between the fourth and
seventh passages were used in the present study. The purity of the
cultures was confirmed on the basis of cytokeratin staining as
described previously.5
All experiments were conducted in MEM and 2% NCS. Some serum components in NCS bind ABL (e.g., IgA, fetuin),6 neutralizing its activity, but 2% serum was required to maintain RPE viability.
Lectin Histochemistry
Bovine RPEs were grown on eight chamber tissue culture glass
slides (LabTeks; Nalge Nunc International, Life Technologies Limited,
Glasgow, UK) as reported previously.5
Peroxidase- or
fluorescein isothiocyanate (FITC)–labeled ABL was added to the
chambers at a concentration of 30 µg/ml for 1 or 8 hours,
respectively. Controls consisted of preincubation of the labeled ABL
with 10 mg/ml asialomucin for 5 minutes before the addition of the
labeled lectin to the cells and preincubation of the chambers with 100
µg/ml unconjugated ABL for 1 hour before adding the conjugated
lectin. The slides were washed with phosphate-buffered saline (PBS),
and either peroxidase was developed with diamine benzidine or slides
were mounted after fixation with methanol for 7 minutes at 20°C.
Slides were dehydrated, mounted, and evaluated using DIC optics
(Polyvar, Reichert–Jung, Austria) or epifluorescent photography,
respectively.
Morphologic Evaluation and Trypan Blue Staining
RPEs were seeded in 24-well plates (Corning Costar, High Wycombe,
UK) at a concentration of 2 x 104
cells/well. After 1 day, the cells were washed three times with MEM
without serum (to remove the serum transferred with the maintenance
medium). ABL was added in concentrations ranging from 5 to 60 µg/ml
in MEM with 2% NCS. Controls were kept in MEM with 2% NCS. Three
wells were used for each concentration, and the experiment was repeated
twice. Cell morphology was evaluated daily for 3 days by phase contrast
microscopy. Representative preparations were selected each day and
stained with 2% trypan blue for 5 to 10 minutes. Stained and unstained
cells were counted in each well.
Cell Proliferation Assay
Cells were seeded in 24-well plates at a density of 1
x104/well. After incubation for 48 hours, the
wells were washed three times with PBS after which ABL was added (20,
30, 40 µg/ml in 0.5 ml MEM with 2% NCS). Cells were incubated for a
further 24 hours. The cells then received a 1-hour pulse with 0.5
µCi/well [methyl-3H]–thymidine. Each well
was washed twice with PBS before cell precipitation with 0.5 ml/well of
5% trichloroacetic acid at 4°C. The precipitate was washed once with
5% trichloroacetic acid at 4°C and twice with 0.5 ml/well of 95%
ethanol at 4°C and left to dry at room temperature. After
solubilization in sodium hydroxide (NaOH), 0.3 ml of the precipitate
was added to 1 ml Optima Gold MV scintillation cocktail (Packard,
Pangbourne, UK), and the cell-associated radioactivity was determined
using a Packard scintillation counter.
Recoverability of inhibition of proliferation was evaluated by cell counting. Cells were seeded in 24-well plates as described above. After 24 hours, the cells were washed twice with PBS, and ABL at concentrations between 20 and 40 µg/ml was added in 2% NCS/MEM. After a further 24 hours, the lectin was removed by washing once with NCS and twice with PBS. Cells were then incubated for up to 7 days with 15% NCS/MEM and sample wells counted in triplicate daily. Cell counts were performed by washing the cells with PBS, trypsinizing with phosphate-buffered trypsin/EDTA at 37°C for 10 minutes, and counting in a hemocytometer.
Collagen Matrix Contraction
For collagen matrix contraction experiments, the method published
by Mazure and Grierson7
was adapted to 24-well plates.
Briefly, rat tail type I collagen (Sigma, Poole, UK) was dissolved in
0.1% (vol/vol) acetic acid in sterile distilled water. RPEs were
counted after harvesting from maintenance cultures and then resuspended
in MEM at a volume of 1.23 ml containing 5.76 x
106 cells, sufficient for one 24-well plate. The
cell suspension was mixed with 4.91 ml of 5 mg/ml collagen and with
2.86 ml of concentrated serum-free MEM containing glutamine,
antibiotics, and NaOH. The collagen-cell mixture was then transferred
in 350-µl aliquots to 24-well plates, ensuring that the matrix
covered the bottom of the wells. The solution polymerized rapidly when
incubated at 37°C in the presence of 5% CO2,
thus trapping the cells (at a density of 2.4 x
105 RPEs per matrix) within the three-dimensional
matrix. The matrices were detached from the edges and allowed to float
in the wells by the addition of 1 ml of MEM with 2% NCS. Mushroom
lectin was added at concentrations of 5, 10, 20, 30, and 40 µg/ml
medium, and the preparations were incubated at 37°C in 5%
CO2 in air for at least 3 days. The surface area
of each matrix was recorded photographically daily. Four wells were
used for each concentration, and experiments were repeated three times.
After 3 days, medium containing ABL was removed and gels were washed
twice and then were incubated for an additional 4 days with medium
containing 15% of serum.
Statistical Analysis
ANOVA was used to explore between-group significance. Duncan’s
multiple comparison test was used to detect homogeneous subsets.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inhibition of Contraction of RPE-Populated Collagen Matrices by
ABL
ABL caused a significant concentration-dependent inhibition of
contraction (one-way ANOVA, P < 0.001, F = 14;
Fig. 4
). Duncan’s test for multiple comparisons indicated that 5 to 10
µg/ml ABL was moderately inhibitory (12% matrix contraction at day
3) and that 20 to 40 µg/ml ABL caused greater inhibition
(6.2%—2.5% contraction at day 3; P < 0.05).
Calculating inhibition as [1 - (test contraction/control
contraction)] x 100, ABL in the range 5 to 40µg/ml produced
inhibition of 43% to 93% (Fig. 4A)
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The binding and uptake of ABL by RPE cells do not appear to alter RPE viability in vitro. RPEs incubated for 3 days with ABL concentrations up to 60 µg/ml (the highest studied level) show normal trypan blue exclusion and no discernible light microscopic change in morphology compared with control cultures. In addition, our data indicate that the effects of ABL on RPE proliferation and cell-mediated matrix contraction are recoverable after removal of the lectin from the media. Taken together, these results suggest that ABL has little or no toxicity for cultured RPEs. Indeed, evidence from studies of other cell types suggests that ABL generally is noncytotoxic at doses up to 200 µg/ml.3
Despite the apparent lack of effect of ABL on RPE viability in vitro, the lectin markedly inhibits RPE proliferation and RPE-mediated collagen matrix contraction in vitro even at concentrations of less than 30 µg/ml. The mechanism by which ABL modulates cell activities in general is not clear, although there is evidence that it interferes with nuclear protein import.9 Whatever the mechanism of action, the lectin is known to influence the behavior of a variety of cell types. For example, the proliferative activity of colonic and breast carcinoma cells and mammary and Tenon’s fibroblasts3 4 is, like that of RPEs, inhibited by ABL. However, in contrast to our observation that ABL inhibits RPE-mediated collagen matrix contraction and to a finding that ABL inhibits matrix contraction by Tenon’s fibroblasts,4 a previous study involving dermal fibroblasts reports no effect of ABL on matrix contraction.10 The latter investigation used serum at a concentration five times the level we used; because some serum components bind ABL (e.g., IgA),6 the ABL may have been functionally neutralized by the high serum concentration.
Inhibition of ABL activity by serum components could limit the potential antiproliferative and anticontractile use of the lectin (e.g., for PVR) in the presence of blood. Hemorrhage or leakage of plasma components may occur during PVR formation or surgery.11 On the other hand, it is possible that ABL binding to RPEs is augmented in PVR. PVR is a complication of retinal detachment, and evidence from PNA binding studies suggests that TFa expression by RPEs is augmented in retinal detachment.8 Thus, ABL may represent a means of specifically controlling RPE activities in PVR without retinal toxicity. Indeed, given the lectin’s potential dual action on RPE proliferation and RPE-mediate membrane contraction and its dose-dependent titratability of effect on RPE behavior, the lectin deserves further investigation as a possible agent for the management of PVR and other anomalous wound repair disorders. Moreover, the lectin may represent a new laboratory agent with which to study further the relationship between RPE behavior and retinal pathology generally.
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Footnotes |
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Commercial relationships policy: P.
Corresponding author: Hartmut Wenkel, Department of Ophthalmology, University of Erlangen–Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany. E-mail: h.wenkel{at}augen.med.uni-erlangen.de
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References |
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