Edible mushroom (Agaricus bisporus) lectin inhibits humanretinal pigment epithelial cell proliferation in vitro

DAVID KENT, FRCOphtha,b; CARL M. SHERIDAN, PhDa; HEATHER A. TOMKINSON, BSca; SARAH J. WHITE, MScc;PAUL HISCOTT, PhDa,b; LUGANG YU, PhDd; IAN GRIERSON, PhDa,b

The retinal pigment epithelium (RPE) plays a major role in the development of the anomalous retinal scarring responsetermed proliferative vitreoretinopathy. The present study was undertaken to investigate whether agaricus bisporuslectin inhibited human RPE proliferation in vitro. Fluorescein isothiocyanate-labeled agaricus bisporus lectin was usedto study binding of lectin to cultured human RPE. The effect of a 24-hour exposure of agaricus bisporus lectin on RPEproliferation was measured using (methyl-3H)-thymidine incorporation into DNA. Toxicity studies were assessed usingmorphologic evaluation, trypan blue exclusion, and a cell viability assay. Agaricus bisporus lectin bound to RPE cellsand was inhibited by preincubation of lectin with asialomucin. Agaricus bisporus lectin caused a dose-dependentinhibition of RPE proliferation (one-way ANOVA, F ¼ 94.470, p < 0.001) that was partially reversible on removal of thelectin. Compared with controls, cells remained viable and no morphological changes or trypan blue staining wasnoted in RPE exposed to agaricus bisporus lectin. Human RPE binds agaricus bisporus lectin and inhibits proliferationwithout apparent cytotoxicity. It therefore merits consideration as a potential antiproliferative agent in theprevention and treatment of proliferative vitreoretinopathy and other nonocular anomalous wound healingprocesses. (WOUND REP REG 2003;11:285–291)

Proliferative vitreoretinopathy (PVR), the anomalous

wound healing response seen in association with rhegma-

togenous retinal detachment (RRD), continues to exact a

significant toll in conventional ophthalmic surgical prac-

tice.1,2 RRD occurs when fluid from the vitreous cavity

passes through a retinal break resulting in separation of the

neurosensory retina (NSR) from the retinal pigment

epithelium (RPE). PVR is characterized by the formation

of ectopic sheets of fibrocellular tissue within the vitreous

and/or on either side of the detached retina. Contraction of

these sheets or membranes can cause a more complicated

form of detachment termed combined traction-rhegmato-

genous detachment. PVR remains the most common cause

of failed retinal reattachment surgery.2,3 Furthermore, as

newer microsurgical techniques, such as macular reloca-

tion, emerge and gain currency in everyday practice, the

challenge of PVR is set to remain a formidable one.4 The

ABL Agaricus, bisporus lectin

FCS Fetal calf serum

FITC Fluorescein isothiocyanate

NSR Neurosensory retina

PBS Phosphate buffered saline solution

PVR Proliferative vitreoretinopathy

RPE Retinal pigment epithelium

RRD Rhegmatogenous retinal detachment

TF Thomsen FriedenreichFrom the Unit of Ophthalmologya, Department ofMedicine, University of Liverpool, St. Paul’s EyeUnitb, Royal Liverpool University Hospital, Liver-pool, Biostatisticsc, Department of Psychiatry, St.George’s Hospital Medical School, London, andUnit of Glycobiologyd, Department of Medicine,University of Liverpool, Liverpool, United King-dom.

Presented in part at the Fourth Joint Meeting of theEuropean Tissue Repair Society and the WoundHealing Society, Baltimore, MD, USA.

Reprint requests: David Kent, FRCOphth, Unit of Oph-thalmology, Department of Medicine, DaulbyBuildings, University of Liverpool, Liverpool L693GA, UK. Email: dkent@liv.ac.uk.

Copyright � 2003 by the Wound Healing Society.ISSN: 1067-1927 $15.00 + 0

285

treatment of PVR is surgical and can be combined with

pharmacological adjuvants with the aim of modifying the

aberrant tissue response to allow successful and perma-

nent reattachment of the retina. However, these adjuvants

generally tend to be ineffective or toxic in the therapeutic

range,5 though recent combination therapy certainly shows

promise.6 Pivotal in the development of PVR is the ability of

de-differentiated RPE cells to proliferate.7 Normally situ-

ated as a mitotically inactive monolayer beneath the NSR

throughout life, in disease these RPE cells assume a wound

repair phenotype similar to fibroblasts.8 Blocking or

modifying this key RPE proliferative response could

provide a potential therapeutic target that could enhance

the overall prognosis of surgical management.

Lectins are ‘‘carbohydrate-binding proteins of nonim-

mune origin that agglutinate cells or precipitate polysac-

charides or glycoconjugates.’’9 The lectin of the edible

mushroom, agaricus bisporus, which binds the cell mem-

brane located Thomsen Friedenreich (TF) antigen, galact-

osyl b1–3N-acetyl-galactosamine (Galb1–3GalNAc), is a

reversible noncytotoxic inhibitor of epithelial cell prolifer-

ation.10,11 Recently we have shown this lectin to be

antiproliferative to bovine RPE in vitro.12 The purpose of

the present study was to assess whether agaricus bisporus

lectin (ABL) was similarly antiproliferative and noncyto-

toxic to human RPE cells in vitro.

MATERIALS AND METHODSAll reagents were of analytical grade, all lectin and

fluorescein isothiocyanate (FITC)-conjugated lectin were

obtained from TCS Biologicals, Buckingham, UK, and

(Methyl-3H)-thymidine from Amersham International,

Amersham, Buckinghamshire, UK. All concentrations for

each experiment were used in triplicate while experiments

themselves were also performed in triplicate. Normal

human eyes were obtained from the local eye bank at the

Royal Liverpool University Hospital (Liverpool, UK).

RPE culturingHuman RPE cells were cultured as previously described.13

Briefly, cells were cultured with Ham’s F10 media

containing glutamine, fungizone, penicillin and streptomy-

cin, and 20% fetal calf serum (FCS; all from Gibco Europe,

Ltd., Paisley, Scotland). The cultures were maintained at

37�C in the presence of 5% CO2 and air and reached

confluence within 3–5 days. Cells between the fifth and

tenth passage were used for all experiments, which were

conducted in F10 media with only 2% FCS (the lowest

concentration of FCS that allowed RPE cells to remain

viable) to minimize neutralization of ABL by serum

glycoproteins.14,15 Confirmation of human RPE purity was

routinely performed by immunohistochemical labeling of

the cells with a wide-spectrum anticytokeratin monoclonal

antibody (clone K8.13, ICN Biomedicals Ltd., High Wyco-

mbe, UK) that has been shown to stain the human RPE cell

population.13 Routine morphological comparison between

the different RPE cell passages was performed using

phase-contrast microscopy.

Lectin histochemistryCells were seeded on eight chamber tissue culture slides

(LabTeks, Nalge Nunc International, Glasgow, UK) at a

density of 7.5 · 103 per chamber and incubated at 37�C in

5% CO2 until they reached confluence. They were then

washed twice with phosphate buffered saline solution

(PBS) before the addition to selected wells of 30 lg/ml

FITC-conjugated ABL in the presence of medium contain-

ing 2% FCS. Controls consisted of the preincubation of the

FITC-conjugated ABL with 10 mg/ml asialomucin for

5 minutes before addition to the wells. Asialomucin is a

glycoprotein that contains the TF antigen. It binds to ABL

and prevents it from interacting with cells. The tissue

culture slides were then incubated at 37�C in 5% CO2 for

24 hours. The medium was then discarded and the wells

were washed twice with PBS. After fixing the slides with

10% paraformaldehyde, they were mounted in fluorostab

(ICN, Basingstoke, UK) and photographed (Polyvar,

Reichert-Jung, Austria).

Morphological evaluation and trypan blue stainingHuman RPE were seeded in 24-well plates (Corning

Costar, High Wycombe, UK) at a concentration of

2 · 104 cells/well. After 1 day, the preconfluent cells were

washed three times with F10 media without serum

(to remove the serum transferred with the maintenance

medium). ABL was then added in concentrations of 20 and

60 lg/ml in F10 media with 2% FCS. Controls were kept in

F10 with 2% FCS without ABL. Cell morphology was

evaluated daily for 3 days by phase-contrast microscopy.

Representative wells were selected each day and stained

with 2% trypan blue for 5–10 minutes. Stained and

unstained cells were counted in each well.

Cell viability determinationEvaluation of cell viability/cytotoxicity was performed on

cultured cells grown on tissue culture slides. Cells were

seeded at a density of 7.5 · 103 per chamber in media with

20% FCS. They were incubated at 37�C in 5% CO2 and fed

every third day until subconfluent. They were then washed

twice with PBS before the addition of lectin at concentra-

tions of 60 lg/ml in media with 2% FCS. Control wells

consisted of cells incubated in 2% FCS without lectin. After

3 days wells were washed twice with PBS before the

WOUND REPAIR AND REGENERATIONJULY–AUGUST 2003286 KENT ET AL.

addition to each well of 50 ll each of 4 lM calcein AM and

2 lM ethidium homodimer in PBS, the live-dead reagents

(Molecular Probes Europe BV, Leiden, The Netherlands).

After 45 minutes at room temperature, the cells were

observed immediately using a fluorescence microscope.

Proliferation assayCells were seeded into 24-well plates at a density of

1 · 104/well in 0.5 ml of F10 media containing 20% FCS for

48 hours. After incubation all wells were gently washed

twice with 0.5 ml PBS using a large aperture plastic pipette.

Then ABL, at concentrations of 20, 40, and 60 lg/ml, was

added in 0.5 ml of 2% medium and incubated for 24 hours.

Twenty microliters of 0.5 lCi/ml (methyl-3H)-thymidine

was added to each well and incubated for 1 hour at 37�C.

After washing the cells twice with PBS, the proteins and

DNA were precipitated by the addition of 5% trichloroace-

tic acid at 4�C for at least 30 minutes. The precipitate was

then washed once with 5% trichloroacetic acid to remove

any unattached or unincorporated (3H)-thymidine and

twice with 0.5 ml/well of 95% ethanol at 4�C to remove

the remaining trichloroacetic acid. Drying at room tem-

perature was then carried out for 2 hours. After this, the

precipitate was solubilized with 0.5 ml/well 0.2 M NaOH

and the plate was left at room temperature for at least

2 hours. The dissolved precipitate was then transferred to

scintillation vials (0.3 ml per vial), followed by the addition

of 1 ml Optima Gold MV scintillation cocktail (Packard,

Panbourne, UK) and the cell-associated radioactivity was

determined with a Packard scintillation counter.

To assess potential recovery from inhibition of prolif-

eration, cells were seeded at a concentration of 1 · 104/well

in F10 media and 20% FCS in 24-well plates. After 24 hours

the cells were washed twice with PBS and a concentration

of either 20, 40, or 60 lg/ml ABL was added in the presence

of media containing 2% FCS. After a further 24 hours,

unbound lectin was removed by washing once with FCS

and twice with PBS. Cells were then cultured in media with

20% FCS for an additional 4 days and sample wells were

counted in triplicate daily. Cells were washed once with

PBS followed by FCS before trypsinization with 460 ll of

phosphate-buffered trypsin/EDTA at 37�C for 10 minutes.

Neutralization was achieved by the addition of 40 ll of FCS.

Final cell counts were obtained using a Coulter counter.

RESULTSLectin histochemistry showed that FITC-conjugated ABL

bound to human RPE cells (Figure 1). The staining was

abolished by preincubation of the conjugated lectins with

10 mg/ml asialomucin. Following a 1-hour exposure to

ABL, the cell membranes were noted to be fluorescent. By

24 hours the FITC-conjugated ABL was noted to be

intracellular with a discrete and bright yellow speckled

fluorescence that was most prominent around the nucleus

without any evidence of obvious nuclear internalization, an

appearance that did not change over the next 24 hours.

Toxicity of human RPE-bound ABL in vitroCell morphology was observed for several days with 20 and

60 lg/ml concentrations of ABL in the presence of 2% FCS.

Morphologically, no differences were noted either between

the different cell passages or between controls and lectin-

exposed cells during the 3 days of incubation with ABL in

FIGURE 1. Epifluorescent micrographs showing that FITC ABL binds

to cultured RPE cells. FITC-conjugated ABL (30 lg/ml) in the

presence of medium containing 2% FCS was added to each well.

Controls consisted of the preincubation of the FITC-conjugated

ABL (30 lg/ml) with asialomucin (10 mg/ml) for 5 minutes before

addition to the wells. (A) Note the bright and discrete fluorescence

throughout the cytoplasm that is most marked in the peri-nuclear

region. (B) Control shpwing that ABL binding is abolished by

preincubation of labeled lectin with asialomucin (scale bar 3 lm).

WOUND REPAIR AND REGENERATIONVOL. 11, NO. 4 KENT ET AL. 287

24-well plates (Figure 2). Compared to controls, precon-

fluent cells remained de-differentiated in appearance. An

overlying precipitate was noted in the medium of the

lectin-treated cells as described elsewhere.16 This may

represent agglutination of some of the serum components

known to bind ABL.14,15 Staining with trypan blue revealed

more than 95% viability of RPE cells after 3 days of

incubation at concentrations of 20 and 60 lg/ml ABL and in

the controls. Viability staining with the live-dead assay

showed no difference between controls and the cells

treated with 60 lg/ml ABL for the numbers of dead cells

present (Figure 3).

Nontoxic levels of ABL inhibits RPE proliferationThe addition of ABL to in vitro RPE cells for 24 hours

caused a statistically significant (one-way ANOVA;

F ¼ 94.470, p < 0.001) and dose-dependent inhibition of

proliferation (Figure 4). All doses (20–60 lg/ml) were

significantly different from the controls and from each

other. ABL in the range 20–60 lg/ml produced inhibition of

thymidine incorporation in RPE ranging from 26.5% to

83.4%. An increase in cell number was shown for all ABL

concentrations after removal of ABL from the medium

(Figure 5). However, cells exposed to higher lectin

concentrations exhibited a slower proliferation rate. Thus,

4 days after the removal of ABL, the control cells

(incubated without ABL) had reached a density of 6.5 ·104 ± 0.63 cells/well whereas cells incubated with 60 lg/ml

ABL had reached 3 · 104 ± 0.1 cells/well (p < 0.05).

DISCUSSIONPVR, an anomalous wound healing condition, is charac-

terized by the development of fibrocellular membranes

within the vitreous and on the retinal surfaces and occurs

in association with RRD. It remains a formidable adversary

in modern vitreoretinal surgery. Despite advances in

microsurgical techniques and improvements in pharmaco-

logical adjuvants,6 PVR continues to exact a significant toll

with respect to both visual and overall ocular morbidity.

Lectins, naturally occurring carbohydrate-binding glyco-

proteins, have long been used to explore cell membranes

and distinguish different cell types, including their exten-

sive use to investigate changes in glycoconjugate expres-

sion in both normal and diseased retina.17–23 For example,

the distribution and composition of certain glycoconju-

gates, including the TF antigen, within the interphotore-

ceptor matrix suggests that these glycoconjugates may be

potential candidates for mediating normal attachment

between the NSR and the RPE.23

We have recently reported the binding characteristics

of ABL in the normal human retina.24 We found that the

A

B

C

FIGURE 2. Phase-contrast micrographs of trypan blue staining of

preconfluent human RPE cells cultured in 24-well plastic plates after

a 3-day incubation. (A) Medium alone; (B) in the presence of

20 lg/ml ABL; and (C) 60 lg/ml ABL. Note that in vitro, RPE cells

appear fibroblastic in appearance. Cells did not stain with trypan

blue and no morphological differences were noted between the

three culture conditions. Note that in the absence of ABL cells are

almost confluent, while with increasing concentrations of lectin

fewer numbers of cells are present due to the antiproliferative

effect of ABL (scale bar 15 lm).

WOUND REPAIR AND REGENERATIONJULY–AUGUST 2003288 KENT ET AL.

normal RPE monolayer did not appear to express the TF

antigen. In addition, we also confirmed the previous

observations of expression of the TF antigen in the

interphotoreceptor matrix and the inner limiting mem-

brane, but not in any other cell types within the NSR.22,25

This is an important finding as it suggests the effect of ABL

may be relatively specific to de-differentiated RPE in PVR

with sparing of uninvolved retinal tissue. Further confir-

mation of this potential binding specificity has been

observed elsewhere and showed that only following RRD

did peanut lectin, which also binds the TF antigen, bind to

RPE.18 Expression of this glycoconjugate may denote a

higher rate of metabolic activity or may be a marker of cell

proliferation similar to that seen in transformed cells.26 In

the present study we were able to demonstrate binding by

ABL to in vitro RPE. As cultured RPE assume the

de-differentiated phenotype of RPE cells involved in

PVR, our findings would be consistent with those of Bopp

and coworkers who proposed that expression of the TF

antigen acts as a marker for these de-differentiated cells.18

It has also been suggested that the strong reaction product

seen in these macrophage-like RPE for peanut lectin is

similar to that observed in blood-borne macrophages and

that these de-differentiated RPE cells may therefore share

similar biological functions with systemic macrophages

such as phagocytosis.27

Proliferation by de-differentiated RPE is fundamen-

tal in the development and progression of the fibrocel-

lular membranes seen in PVR. The proliferation assay

used in this study provides an effective in vitro tool for

the investigation of this key process.28 Previously we

have shown that ABL binds to bovine RPE and inhibits

both in vitro RPE-mediated contraction and RPE

proliferation.12 The present study clearly show that

HRPE- Inhibition of proliferation

0

20

40

60

80

100

ug/ml ABL

%p

rolif

erat

ion

0204060

FIGURE 4. Inhibition of human RPE cell proliferation by ABL. Thymi-

dine incorporation by cultured human RPE cells exposed to ABL for

24 hours. Cell proliferation in medium without lectin was set to

100%. Columns represent the mean of three experiments, each

performed in triplicate. Error bars represent the SD.

FIGURE 5. Recovery following the ABL effect on human RPE cells

determined by cell counting. Cells were seeded in lectin-free

medium on day 0 and lectin was added on day 1 for 24 hours

before a further 4 days incubation in medium without lectin. Lines

represent the mean of three experiments, each performed in

triplicate. Error bars represent the SD.

FIGURE 3. Epifluorescent photomicrographs of cell viability assay

showing cells incubated with 60 lg/ml ABL for 3 days that were

then incubated with a mixture of calcein AM and ethidium

homodimer for 45 minutes and then immediately photographed

(A and B). Live cells accumulate calcein AM, which is enzymat-

ically converted by esterase activity to calcein, which fluoresces

green. Ethidium enters cells with damaged membranes, binds to

nucleic acids, and fluoresces red in dead cells (arrows). Similar to

controls, all cells stained almost uniformly green, indicating that the

majority of cells were viable (scale bar 5 lm).

WOUND REPAIR AND REGENERATIONVOL. 11, NO. 4 KENT ET AL. 289

ABL has similar dose-dependent antiproliferative effects

on human RPE cells. It is possible that the reduced

reproliferation rate following lectin removal was due to

residual binding by ABL to the cells. Moreover, this

study indicates that ABL is not toxic to human RPE at

the concentrations employed.

Several studies have shown the antiproliferative

effects of ABL in dose ranges similar to the present work

on a number of cell types, including various fibroblast

subtypes and a number of different cancer cell lines and

lymphocytes.10,11,16 Although the precise mechanism of

action is unknown, Yu and coworkers have demonstrated

that ABL must be internalized to exert its antiproliferative

action in HT 29 colon cancer cells.29 The perinuclear

accumulation of ABL in these cells, similar to that which

we observed in RPE cells, led Yu and colleagues to

postulate that ABL could inhibit proliferation by interfering

with nuclear pores, perhaps by blocking nuclear protein

import. Future work is therefore needed to elucidate the

exact mechanism of action of ABL and precisely establish

the role of the TF antigen in proliferation. Furthermore, in

common with other studies, the optimum dose and the

precise duration of exposure of cells to ABL to achieve

adequate antiproliferative effects will need to be deter-

mined.30,31

In summary, this study shows the role played by the

TF antigen in the proliferation of RPE cells. Blocking this

antigenic site with ABL produced a dose-dependent

inhibition of cellular proliferation. These effects were

partially reversible, dose-dependent, and noncytotoxic.

Indeed, ABL may represent a means of specifically

controlling RPE proliferation in PVR without retinal

toxicity. In conclusion, our observations suggest that

ABL merits further investigation as a potential surgical

adjuvant in the management of PVR and other anomalous

nonocular tissue repair processes.

ACKNOWLEDGMENTSThese studies were supported by the Wellcome Trust (DK),

Dunhill Medical Trust (CS), and the British Council for the

Prevention of Blindness (DK, HAT).

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