Biol Cell (1991) 73, 35-42

35

© Elsevier, Paris

Original article

Human keratinocyte membrane lectins: characterization and modulation of their expression by cytokines Dominique Cerdan i** Catherine Grillon m**, Michel Monsigny 1, G6rard Redziniak 29 Claudine Kieda I, i Ddpartement de biochimie des glycoconjuguds, Centre de biophysique moldculaire, CNRS, 1, rue Haute, 45071 Orldans Cedex 2; 2PCD Biologie, PB 58, 45804 Saint-Jean-de-Braye Cedex, France (Received 9 April 1991 ; accepted 16 September 1991)

Summary - in an attempt to identify cell surface molecules involved in recognition phenomena between cells such as keratinocytes and melanocytes and putatively target biological responses modifiers to keratinocytes, we undertook the detection of cell surface sugar specific receptors: membrane lectins. Keratinocyte membrane leedns were found to bind synthetic glycoproteins (neoglycoproteins) carrying either a-L-fucosyl or a-L-rhamnosyl residues. Fluorescence microscopy observations indicate that cultured keratinocytes are able to bind these two neoglycoproteins while frozen sections of human skin labelled with neoglycoprotein.coated covaspheres show that the selectivity of the binding to keratinocytes is restricted to a-L-rhamnosyi-BSA. Keratinocytes were adapted to grow on collagen; harvesting conditions allowing the analysis of keratinocytes by flow cytometry are described. This technique allows the quantification of the binding at 4°C, and the estimation of the endocytosis of F-, neoglycoproteins: F-, ~-L-Rha-BSAand F-, a-L-Fuc-BSA were efficiently internalized. Thereafter, a-L-rhamnose-substituted liposomes containing 5-(6)carboxyfluorescein were prepared in order to follow the delivery of the fluorescent dye into cells. This was measured both by flow cytometry and by spectrofluorimetry. The expression of surface lectins was checked upon action of cytokines (ILma, ILhB, IL2 and TNF) which are known as biological response modifiers of keratinocytes. kerstinocyte I lectin I eytoldne I expression

Introduction

Keratinocytes are the main cell type among the various cell populations in the skin. Keratinocytes insure fundamental protective functions. Among those, body prevention against UV radiations is achieved by the co-operation of melanocytes and keratinocytes. Melanocytes produce melanin vesicles: melanosomes. Keratinocytes recognize melanosomes and internalize them in such a way that melanosomes in the cytoplasm allow a protection, as shown by melanosome redistribution upon UVA irradiation [27, 37, 63]. Such a behaviour, in normal skins, depends on highly specific and efficient intercellular interactions which allow one melanocyte to interact with up to 30 keratinocytes by interdigitating dendrites through which melanosomes are transferred [12, 13]. Melanosomes are supposedly uptaken by keratinocytes through specific membrane recognition molecules which are highly regulated by various dermal growth factors, hormones and cytokines [10, 11, 18, 22, 25, 49, 64].

* Correspondence and reprints ** Present address: lnstitut de Chimie des Substances Naturelles, CNRS, 91190 Gif-sur-Yvette, France Abbreviations: BSA, bovine serum albumin; IL, interleukin; TNF, tumor necrosis factor; UV, ultra-violet; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate buffered saline, pH 7.4; FITC, fluoresceinylisothiocyanate; IP, propidium iodide; FBS, foetal bovine serum; RhDPPE, rhamnosyl-dipalmitoyl phosphatidyl choline; DPPC, dipalmitoyl phosphatidyl choline; chol, cholesterol; DCP, dicetyl phosphate; CF, carboxyfluorescein; FSC, forward scatter; SSC, side scatter; FACS, fluorescence activated cell sorter.

To get an insight into the mechanism by which melanocytes and keratinocytes do correspond with each other and in order to understand how one can interfere with these recognition phenomena, either to help melanogenesis and melanosome uptake or to control keratinocyte activity and growth, we undertook the study of keratinocyte cell surface receptors. More precisely, our study was focused on the search for sugar binding molecules of keratinocytes. Membrane lectins (sugar specific receptors) [41, 43] are putative candidates as specific cell interaction molecules, they have been shown to be involved in lymphocyte/endothelial cell interaction during homing (for reviews see [8, 43, 57]) as well as in tumor cell/pulmonary cell recognition ([29] for review see [43]). Expression of cell surface lectins is very much dependant upon biological phenomena such as maturation [53], activation [20], or differentiation [1, 21]. About keratinocytes, special attention has to be paid to the particular status of the skin cells which produce numerous cytokines involved in inflammation processes [16, 24, 39] in skin repair and in local cutaneous immunological reactions including: i) B and T lymphocyte activation related to antigen presentation by specialized accessory cells (Langerhans cells) [33, 35]; ii) natural killer cells [38] as well as; iii) antiviral activity [45]. Cell surface properties of keratinocytes are linked to their biological status, as shown by the modulation of glycoconjugate expression during keratinocyte differentiation, evidenced by the binding of exogenous lectins to the cell surface [31, 46-48]. Furthermore, cell surface glycoproteins are involved in adhesion processes [9]. In this paper, we identify specific sugar receptors (endogeneous lectins) on the surface of keratinocytes, and

36

D Cerdan et al

describe some of their biological properties assessed by fluorescence microscopy, flow cytometry or spectrofluorimetry using fluorescein substituted neoglycoproteins [44, 52] as tools. In addition, the expression of keratinocyte cell surface lectins in relation with several effectors was investigated on the basis of identified sugar specificity. Finally, we prep p e d liposomes containing synthetic glycolipids to allow their recognition by cell surface lectins of keratinocytes and to define putative carriers specific for keratinocytes.

Materials and methods Materials Dulbecco's minimum essential medium (DMEM), fungizone and trypsin were from Gibco (Paisley, UK). Ham FI2 medium, penicillin and streptomycin were from Eurobio Laboratories (Paris, France). Foetal bovine serum was from Flow Laboratories (Ayrshire, UK). Type I collagenase, trypsin inhibitor, propidium iodide, phosphatidylethanolamine were from Sigma Chemical Co (Saint Louis, MO, USA). Type I collagen from bovine Achille's tendon was from Serva (Heidelberg, Germany). Ethylenediamine tetraacetic acid (EDTA) was from Merck (Darmstadt, Germany). Monensin and pronase (Grade B) were from Calbiochem (La Jolla, CA, USA) and a stock solution (25 raM) was prepared in ethanol before use. Bovine serum albumin (BSA) and ACA 54 Ultrogel were from IBF-Biotechnics (Villeneuve-la-Garenne, France). Murine epidermal growth factor was from Boehringer (Mannheim, Germany). Fluorescent microspheres MX were from Covalent Technology Corp (Ann Arbor Ml, USA).

Cell culture Keratinocytes from a human squamous carcinoma line (SCL-I) [6], generously given by Dr Y Milner (The Hebrew University, Jerusalem, Israel) were grown in tissue culture dishes in Dulbeco's minimum essential medium (DMEM) and Ham Fl2 media (i: 1, v/v) containing 10e/0 (v/v) heat inactivated foetal bovine serum, 2 mM L-glutamine, 2.5 ~g/ml fungizone, penicillin (100 U/mi), streptomycin (100 t~g/ml). Cells were maintained at 37°C in a humidified atmosphere of 7e/0 CO~, 93% air.

Preparation of collagen coated plates Type I collagen from bovine Achille's tendon (2 mg/mi) was solubilized in a I M NaOH aqueous solution at 100°C for 15 to 20 min. Before use, the alkaline solution of collagen was extensively dialyzed at 4°C against double distilled water. Aliquots of the collagen solution (0.1 mg/cm 2) were added to each well of a 12-flat bottomed well tissue.culture multiwell plate, 2.2 cm diameter from Corning (New York, USA) or Petri dishes (10 cm diameter), (Falcon, Becton and Dickinson, Grenoble, France). Culture plates were placed around a Bunsen flame and heat-dried, sheltered from contamination, in a laminar flow hood. Collagencoated plates were kept at 4°C.

Keratinocyte culture on collagen coated plates Confluent keratinocyte cultures were trypsin.treated (0.05070 trypsin/0.02% EDTA in PBS, w/v), collected in complete culture medium and washed in culture medium without serum. Cell suspension was plated (5 104 cells/cm2) on collagen-coated culture dishes in keratinocyte culture medium at 37°C for 24 h before use so that the final cell density reached 9 104 cells/cmL Keratinocytes were harvested by treatment with at t.ype I collagenase solution. Contaminating trypsin-like activity was inhibited for preservation of membrane receptors. To estimate the importance of trypsin-like activity the following conditions were

used: i) collagenase 0.1% in complete PBS (c-PBS): PBS containing 1 mM Ca 2+ and 0.5 mM Mg2+ (without inhibitor); ii) collagenase 0.1% in c-PBS plus 0.2~o BSA (w/v) (c-PBSBSA); iii) collagenase 0.1% in complete culture medium (FBS 10%); iv) collagenase 0.1% in c-PBS plus 0.001% of trypsin inhibitor (ovomucoid). Cell harvest was routinely achieved by using a I mg/ml collagenase solution in c-PBS-BSA (37°C, 10 rain).

Neoglycoprotein synthesis and fluorescein labeling Neoglycoproteins were prepared by reaction of glycosidophenylisothiocynate with bovine serum albumin (BSA) as previously described [44, 52]. Neoglycoproteins were fiuoresceinylated by reaction with fluoresceinyl-isothiocyanateisomer I (FITC-I), as previously described [52]. The fluorescein-labeled neoglyco. proteins (F-neoglycoproteins) were purified by gel filtration on a column of Ultrogel GF 05 (IBF-Biotechnics) in butanol/water (5:95) [52] and then freeze dried. All neoglycoproteins used contained 23 :l: 3 sugar units. The neutral sugar content of a neoglycoprotein was determined by using a resorcinoi sulfuric acid micromethod [42]. The average number of fluorescein residues bound to a neoglycoprotein molecule (F/N ratio) was determined from the absorbance at 495 nm after proteolytic digestion with pronase [40] and was found to be 2.5 + 0.5.

Neoglycolipid synthesis Neoglycolipids were prepared by reaction of rhamnopyranosylphenylisothiocyanate [44] with phosphatidylethanolamine. 72/~mol of phosphatidylethanolamine (50 mg) were solubilized in 20 ml of an ethanol/carbonate buffer 0.2 M pH 9.0 mixture at 50°C, under stirring, 144/zmoI of rhamnopyranosyIphenylisothiocyanate, solubilized in a minimal volume (I ml) of the same solvent at 50°C, was added and the solution was kept at 50°C for 3 h. The mixture was then incubated overnight at 4°C to allow neoglycolipid precipitation. The neoglycolipid (RhDPPE) was collected upon centrifugation at 40000 g for 20 rain, washed with distilled water, spun down at 40000 g for 20 rain, homogenized in distilled w~/ter by ultrasounds, freeze dried and kept at -20°C.

Lipid vesicles preparation Lipid vesicles (iiposomes) were prepared according to the method described by Bangham et at [4], slightly modified to obtain smaller liposomes. Lipids were dipalmitoyl phosphatidyl choline (DPPC), cholesterol (chol), dicetyl phosphate (DCP) and the sL-rhamnopyranosylphenylthiourea derivative of dipalmitoyl phosphatidyl ethanolamine (RhDPPE). The molar proportions of the compounds, in glycosylated lipogames were: RhDPPE/DPPC/DCP/chol, 1:4 :I :4; in liposeines: DPPC/DCP/chol, 5:1:4. I00 mg of each lipid mixture were solubilized in a chloroform/methanol mixture (7: I, v/v). Solvents were evaporated at 55°C under reduced pressure and lipids were dried under water-free nitrogen. Then, I0 ml of a 200 mM 5-(6)carboxyfluorescein aqueous solution [62] were added. The suspension was stirred for 20 h in the dark at room temperature, cooled to 4°C and sonicated three times for 2 rain. Liposomes were purified by gel filtration on an ACA 54 Ultrogel (2.5 × 16 cm) column in PBS and then kept at 4°C.

Lectin detection Fluorescent neoglycoproteins containing one of the following sugar residues: N-acetyl-~-D-glucosaminyl (F-,GIcNAc.BSA), s-Lrhamnosyl (F-,Rha-BSA), 6-phospho-~-D-mannosyl (F-,6PMan-BSA), 6-phospho-p-D-galactosyl (F-,6P-GaI-BSA), a-Dgalactosyl (F-GaI-BSA), lactosyl (F-, Lac-BSA), 0~-D-glucosyl CF-,Glc-BSA), ~-D-mannosyl (F-,Man-BSA), ~-t.-fucosyl (F-, Fuc-BSA), N-acetyl-~-D-galactosaminyl (F-,GalNac-BSA) were used. Binding experiments were performed by incubating 2 IOs cells

Keratinocyte membrane lectins expression for 1 h at 4°C in the prese~*ceof 100/~g/ml of fluorescent neoglycoproteins in c-PBS containing 0.2% BSA (c-PBS-BSA). For endocytosis experiments incubation lasted for 90 min at 37°C. In all cases, after incuba~on, cells were harvested by type I collagenase, collected by cen~:rifugation at 500 g for 10 min at 4°C, washed in cold c-PBS-BSA and resuspended in c-PBS. Cells, incubated at 37°C to allo~ internalization, were post-treated at 4°C for 30 rain in the presence of 50/AM monensin [40, 44]. Cell fluorescence intensities were analyzed by using a FACS 440, fluorescence activated cet; sorter (see below).

Cytokine-induced modulation of membrane lectins A cell suspension was plated on collagen-coated culture dishes in keratinocytes culture medium containing cytokines: human recombinant (Hr) ILt~; Hr ILt-~; Hr TNF (National Institute for Biological Standards and Controls, London) and Hr IL, (Boehringer Mannheim, Germany) were used at 5 10-t° M, 2.$10 -I° M [51l, 2.9 10-i° M [26], 2 10-t° M [36] respectively, for 15 h at 37°C before labelling. Such concentrations were determined as optimal after testing from a five.fold lower range to a five-fold higher range.

Lipid vesicle binding to keratinocytes Keratinocytes cultured on collagen-coated plates were washed and preincubated for 30 rain at 37°C in c-PBS-BSA and then incubated with c-PBS-BSA containing various concentrations of liposomes or glycosylated liposomes for I h at 37°C. Inhibition of glycosylated liposome binding to keratinocytes was achieved by preincubation with c-PBS-BSA containing 1 mg/ml neoglycoproteins for 1 h at 37°C. Then, a five-fold diluted suspension of liposomes or glycosylated liposomes was added for 1 h at 37°C.

37

In situ lectin detection on human skin sections Human skin sections (10 to 15 ~m in thickness) were made with a microtome (Slee, London) at -60°C. Lectin detection was achieved with neoglycoprotein-coated green fluorescent microspheres MX (0.5/~m diameter): glycosyl-BSA-MX. Microspheres (109) were washed with PBS, then re.suspended and incubated in a 10-mg/ml neoglycoprotein solution in PBS at room temperature. Before use, microspheres were washed twice in PBS, spun down at 100000 g, resuspended in c-PBS-BSA (109 particules/ml) and sonicated. Coated microspheres (10s) were incubated on human skin sections (on microscope slides) at room temperature for 90 min under gentle stirring. After incubation, skin sections were washed with c-PBS-BSA. Observations were made with a Zeiss epifluorescence microscope.

Results Cell culture o f keratinocytes on collagen Various parameters were investigated with regards to cell culture conditions on collagen and to cell harvesting by type I collagenase. The optimal cell density was determined to be 5 104 cells/cm'; higher densities made collagenase action less efficient. Culture on collagen film for less than 20 h allows a rapid harvesting. The time required to efficiently release cells upon collagenase action ranges from 10 to 20 min, at 37°C; a longer time decreases cell viability. The contaminating trypsin-like activity present in type I collagenase had to be blocked to maintain cell receptor integrity. Best results were obtained by using BSA 0.2% (w/v) in c-6PBS as a proteinase activity competitor (fig 1). To reach a high cell viability ( > 95%), experimental steps must follow the sequence: incubation at 37°C for 30 min in c-PBS-BSA to allow removal of inhibitors; in-

Flow cytometry analysis Cells were analyzed by using a FACS 440 (Fluorescence Activated Cell Sorter, Becton and Dickinson, Sunnyvale, CA, USA). Four parameters were simultaneously recorded for each cell at a rate of 800 cells/s: forward (small angle, < 12°) scattered light signal (FSC), side (90 ° angle) scattered light signal (SSC), and two fluorescence signals (FL~, FL,). Dead cells were excluded on the basis of red fluorescence emission of DNA-intercaled propidinm iodide [54]. The 488-nm line of an argon ion laser (Spectra Physics, Moutain View, CA, USA) was used as excitation beam (laser power 300 mW). Fluorescence signals were collected using appropriate optical filters (BP 530 + 30 nm) for fluorescein emission and (LP > 625 nm) for propidium iodide emission. Before analysis, cells which had been incubated at 37°C to allow internalization were post-treated at 4°C for 30 min in the presence of 50/,tM monensin.

A Collagenase B Collogenose/PBS-BSA

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.

C Collogenase/complete medium (I0~ FBS) D Collagenaee plus trypsin inhibitor 0.001

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1

o

1 Ul

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Fluorescence microscopy of cultured keratinocytes Keratinocyte suspension was plated at a density of 2.7 104 cells/cm 2 on sterile round cover slips (14 mm diameter, Polylabo, Strasbourg, France), placed in culture multiwell plates (Linbro 24 flat bottomed wells), in keratinocyte culture medium at 37°C. After 24 h, cells were incubated with one of the above cited fluorescent neoglycoproteins for I h at 4°C or for 90 min at 37°C in the presence of a 100 ~g/ml fluorescent marker solution in c-PBS-BSA followed by a 30-min incubation at 4°C in the absence or in the presence of 50/~M monensin. After incubation, cells were washed twice in c-PBS-BSA. Observations were made with a Zeiss epifluorescence microscope. Photographs were taken on 400 ASA Kodak Ektachrome films.

A BC

D

GIcNAc

ABCD Rha

A B C D GoI6P

A BC

D

Man

Fig 1. Effect of various inhibitors of tryptic-fike activities in type I coilagenase on the binding of neoglycoproteins to keratinocytes. Keratinocytes were cultured for 24 h on collagen, then they were harvested by a type I collagenase solution (1 mg/ml) in c-PBS (A) or in 0.2070 BSA in c-PBS (B) or in culture medium (C) or in c-PBS containing 0.001070 ovomucoid, a trypsin inhibitor (D). • Cells were incubated with 100/~g/ml of fluoresceinylated neoglycoproteins: F-,GIcNAc-BSA; F-,Rha-BSA; F.,6P-Gai-BSA; F-,Man-BSA, for I h at 4°C. The cell fluorescence intensity was measured by flow cytometry. Data are from a typical experiment, made in triplicate; SD = _+ 10°70.

D Cerdan et al

38 r---i Incubation at 4"C E ~ Incubation at 37"C I Incubation at 3 T C then treatment with mononsin

5°t ,

1

200

-~ too I+J

g

Fig 2. Binding and endocytosis of fluoresceinylated neoglycoproteins assessed by flow cytofluorometry. Keratinocytes were cultured for 15 h on collagen, and then incubated for 1 h at 4°C and/or 90 min in the presence of 100/,tg/ml of a neoglycoprotein in c-PBS-BSA. Cells were harvested upon treatment with type I collagenase in c-PBS-BSA, washed and resuspended in cold c-PBS. The cell fluorescence intensity was measured by flow cytometry. Endocytosis was estimated after a 30-min postincubation at 40C in the presence of 50 ~M monensin. Data are mean values from three experiments in triplicate.

cubation in the presence of F-neoglycoproteins; collagenase release from the plate; harvesting and analysis. FIB 3, Fluorescence microscopy of keratinocytes cultured on

Cell surface lectin-mediated internalization of fluorescein. substituted neoglycoproteins Some neoglycoproteins bind to keratinocytes and are in. ternalized as shown in figure 2. In terms of relative fluorescence intensity, F-,Rha-BSA was the most efficient at 4°C. Cell associated fluorescence intensity increased upon incubation at 37°C and further increased upon monensin posttreatment at 4°C indicating that an endocytotic process occurs [40]. Monensin is a proton/sodium N a + / H + specific ionophore which equilibrates the external and the internal pH of endosomes or lysosomes in intact cells. A fluorescence microscopy picture of keratinocytes after incubation in the presence of F-, Rha-BSA is shown in figure 3. F-,GaI6P-BSA, F-,Fuc-BSA and F-, Lac-BSA are endocytosed too, but to a lesser extent. Upon binding, other neoglycoproteins such as F-,Man-6P-BSA, F-,GalBSA (as well as F-,Man-BSA and F-,GaINAc-BSA, data not shown in fig 2), led to an increase in the fluorescence intensity upon incubation at 370C but monensin posttreatment did not produce any effect. This may indicate that internalization, if it occurs, does not involve acidic compartments. The proportion of cells able to internalize neoglycoproteins was 50 + 5% for F-,Rha-BSA and F-,Fuc-BSA, but only 25 :l: 5% for other neoglycoproteins such as F-,Gal 6P-BSA or F-,Lac-BSA. This may be due to the facts that cells are not synchronized and are present in various differentiation stages; although flow cytometry data indicate that fluorescence intensity is not related to the size of the cells. Consequently, the binding of a neoglycoprotein such as F-,Rha-BSA, does not reflect the stage of differentiation in the case of keratinocytes.

round cover slips and reacted with F-,Rha-Bsa (100/~g/ml) at 37°C. (2 exc: 495 nm, 2 m: 520 nm), x 250. r--1 No stimulotlon R~ I L l = mm t L l p i IL2 200 tkl U

150

,oo

50,

0

' z:

--

o.' "6

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Fig 4. Cytokine-induced modulation of the binding and endocytosis of fluoresceinylated neoglycoproteins by keratinocytes. Keratinocytes were plated on collagen in keratinocyte culture medium in the absence or in the presence of cytokines (HrlLr¢, HrlLrj3 , HrlL22, HrTNF), used at optimal concentrations, as determined, at 37°C. After 15 h, cells were incubated in c-PBS-BSA containing a given fluoresceinylated neoglycoprotein 100/~g/ml for 1 h at 4°C for 90 min at 37°C. Cells were harvested upon treatment with type I collagenase in c-PBS-BSA, washed and resuspended in cold c-PBS. The cell fluorescence intensity was measured by flow cytometry.

39

Keratinocyte membrane lectins expression

Cytokine-dependent modulation of membrane sugar receptor expression Various cytokines were tested for their effects on membrane sugar receptor expression. In preliminary experiments, the cytokines were used at various concentrations within a 25-fold range; the optimal concentrations were selected to draw figure 4. Interleukin-10t (ILI~) increased significantly the endocytosis of F-,Gal6P-BSA; tumor necrosis factor (TNF) increased F-,Man6P-BSA and F-,Fuc-BSA endocytosis, but slightly decreased F-,RhaBSA endocytosis (fig 4). The influence of the cytokines on the proportions of cells able to bind neoglycoproteins was assessed by flow cytometry which did not indicate a noticeable change on the labeled subpopulations. IL2 decreased F-,Rha-BSA endocytosis and did not induce any change with other neoglycoproteins. IL~-~ had almost no effect on membrane sugar receptor expression. Such data show a separate behaviour of sugar specific receptors on keratinocyte plasma membrane and consequently indicate that neoglycoproteins identify and specifically label various endogenous lectins. Furthermore, cytokines are able to modulate separately the expression of the various sugar specific receptors on the keratinocytes plasma membrane.

Targeting of glycosylated liposomes to cell surface lectins of keratinocytes The neoglycoprotein carrying 0t-rhamnosyl residues being the most efficiently recognized and internalized by keratinocytes, glycoconjugates containing this sugar should be suitable as specific carriers. Accordingly, rhamnosylated liposomes were more efficiently taken up than sugar free liposomes upon incubation at 37°C (fig 5). Quantification of such an uptake was possible because both sugar-free and glycosylated liposomes were loaded with high concentrations of 5-(6)carboxyfluorescein [62] which can light up upon dilution. The sugar specificity of these interactions was assessed in inhibition experiments (fig 6) using neoglycoproteins. Among the various neoglycoproteins tested, Rha-BSA and Fuc-BSA were the most efficient inhibitors of the binding of rhamnosylated liposomes to keratinocytes, at 37°C. GIcBSA, GaI6P-BSA and Lac-BSA were less efficient at 37°C. A--A A--A

900,

rhamnosylated Ilposomes at 37"C Ilposomes ut 37"C

800, 700. -

5O

"t 30

2o

I

°:t ' I 1

1

0.. ID

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3

o

Inhibitory neoglycoprotein

Fig 6. Inhibition of glycosylated liposome interaction with keratinocytes. Cultured keratinocytes were preincubated in c-PBSBSA containing 1 mg/ml of a nenglycoprotein for 1 h at 4°C. Then, lipid vesicles or rhamnosylated vesicles were added at 0.5 mM of cholesterol equivalent, corresponding to 0.125 mM rhamnose, then incubated for I h at 37°C. Cells were harvested and analyzed as described in figure 4 captions.

Other neoglycoproteins have no effect on rhamnosylated liposomes uptake. The inhibition values obtained are expressed as percentages, relative to the maximum fluorescence intensity values. In these experiments, the concentration of rhamnosyl residues embedded in liposome bilayer is 0.125 raM, while the sugar concentration due to 1 mg/ml inhibitory neoglycoprotein is 0.250 mM + SD = 10%0.Consequently, the inhibition values obtained at 37°C, rangiag around 30 + 3%, are highly significant.

Sugar spec~c receptors detection and localization by fluorescence microscopy on human skin sections Frozen sections of human skin were labeled by neoglycoprotein.coated microspheres and observed by fluorescence microscopy. As shown in figure 7, ~-L-Rha-BSA-coated microspheres (fig 7a) were selectively localized in the epidermal zone while the dermal zone was poorly labeled. Conversely, 6P~-o-Man-BSA-coated microspheres labeled internal area of the derm (fig 7b). Therefore, neoglycoproteins are suitable to selectively label specific cells in the skin and sugar receptors are good candidates to target active molecules towards keratinocytes.

600, 500.

Discussion

400. 300. 200,

~

~

_

--A ~

A

100.

0.05

J

o

e

0.1

0.2

0,4

0,5[Chol] mM

SD+5

Fig 5. Keratinocytes targeting by glycosylated liposomes. Keratino~ytes were cultured for 15 h on collagen, and then incubated in c-PBS-BSAcontaining various concentrations of liposomes (A--~) or rhamnosylated liposomes (A--A) for 1 h, at 37°C. Cells were harvested and analyzed as described in figure 4 captions.

With the aim of characterizing putative cell-surface sugarbinding proteins (membrane lectins) of human keratinocytes, we developed a method to allow the flow cytometry analysis of cell-associated fluorescence from adherent keratinocytes. Spread keratinocytes were incubated in the presence of fluorescent markers, and then released from the plate by using collagenase in the presence of 0.2%o serum albumin acting as proteinase activity competitor and protecting both membrane proteins and bound neoglycoproteins. Under such conditions cell associated fluorescence could be assessed on single cells by flow cytometry. Keratinocytes possess sugar-specific receptors, called

40

D Cerdan et

al

Fig 7. Fluorescence microscopy of human skin section incubated with a. 0c-L-Rha-BSA.coatedmicrospheres; b. 6P-~-D-Man-I~A. coated microspheres. (,I ~c _ 495 nm), × 250. Scale: I cm - I0/~m,

membrane lectins, as shown by fluorescent neoglycoprotein binding: the most efficient neoglycoproteins were those containing ~-L-rhamnose, ~-L-fucose and p-Dgalactose-6.phosphate. Flow cytometry as well as fluorescence microscopy ex~riments showed that only a fraction of cultured keratinocytes is able to bind and to internalize neoglycoproteins. The uptake was ascertained by using a post-treatment in the presence of monensin. Monensin, by neutralizing acidic compartments, increases the fluorescence intensity of fhorescein. The proportion of positive cells ranges from 25 to 50% when cells are cultured on collagen as well as when cells

are grown directly on glass cover slips~ This heterogeneity may be related to various differentiation and/or maturation stages of the cells which are not synchronized. Although they are kept in culture conditions which restrain differentiation [2, 7, 15] non-transformed keratinocytes do undergo a maturation process (for a review see [61]) to some extent. Furthermore, cell membrane lectin expression may depend upon the biological state of the cells [19] which is modulated by the action of cytokines. Cytokines are known to play an important role in the skin [33, 34, 60]. For example ILl is produced by keratinocytes [32] and is responsible for: i) activation of epithelial cells and fibro-

Keratinocytemembrane lectins expression blast growth especially in the case of skin repair process [45]; ii) T lymphocyte activation and IL2 production ([17] see Kupper and Edelson [33] for a review) as well as induction of interleukine-2 receptors on T lymphocytes [28] (see [14] for a review); iii)potent chemotactic activity towards T helper cells [55] and; iv) proinflammatory activity in normal human skin [16]. Our data shwo that ILl-= induces an increased expression of 6P-galactose receptors, but has no effect on =-Lrhamnosyl-receptor expression, while IL2 is the only cytokine which lowers rhamnose-specific receptor expression. Because ILl is induced and equilibrated by UV irradiation [3, 50] and mediates several effects, the demonstration that it increases a 6-phosphogalactose receptor on keratinocyte surface is of interest. TNF is a cytotoxin which is also produced by keratinocytes [36, 56]; it may even control cutaneous T kcell lymphoma [59] and has properties similar to ILl to some extent (for a review see [5]). In contrast, its effects on keratinocytes membrane lectins are very different from that of ILIa since it increases the expression of 6 phosphomannose- and fucose-specific receptors but does not change the expression of 6 phospho-galactose-specific receptors. Such modulations must be taken into account in studies of cell/cell interactions in which intercellular adhesion molecule including membrane lectins are involved, such as in lymphocyte/endothelial cell interactions which can be inhibited by glycoconjugates (for a review see [8]). Membrane lectins, especially =-t.-rhamnose receptors, may provide good specific candidates to target molecules towards keratinocytes. Because they have a rather stable level of expression and because they are involved in the internalization of their ligand, advantage can be taken of this =-L-rhamnose specific membrane lectin to deliver biological modulators using glycosylated liposomes as carriers. As a model, carboxyfluorescein-loaded liposomes [62] containing glycosylated lipids are suitable tools to test this hypothesis. Such glycosylated liposomes may mimic natural vesicles which are putatively produced by cells such as melanocytes in the neighbourhood of keratinocytes. The preparation of glycosylated iiposomes (ie liposomes containing rhamnosylated phosphatidylethanolamine) and their use compared to sugar free liposomes allowed to demonstrate a sugar-dependent binding of rhamnosebearing liposomes to keratinocytes; the cell-associated fluorescence comes from the liposome-entrapped carboxyfluorescein. While neoglycoproteins containing either fucose or rhamnose inhibit the binding of rhamnosylated liposomes and the fluorescence of the cell-associated carboxyfluorescein, it seems that fucose-specific lectins and rhamnosespecific lectins may be distinct receptors because, using fluorescein-labeled neoglycoproteins, their expression is differentiately modulated by TNF. The cytokinemodulation of the detected membrane lectins suggests that there are, at least, three types of sugar receptors. One (rhamnose specific) which is not upregulated by any tested cytokine but is down regulated by IL2, a second one (the 6-phosphomannose- or fucose-specific) which is upregulated by TNF and a third one which is upregulated by ILia. These results open new perspectives for studying the uptake of melanosomal complexes by keratinocytes and especially for the understanding of recognition events which allow the passage from melanocyte to keratinocyte.

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References

1 Agpar JR, Cresswell P (1982) Expression of cell surface lectins on activated human lymphod cells. Eur Jlmmuno112, 570-576 2 Allen-Hoffman BL, Rheinwald JG (1984) Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytesin culture. Pro,:NatlAcadSci USA 81, 7802-7806 3 Ansel JC, Luger TA, Lowry D, Perry P, Roop DR, Mountz JD (1988) The expression and modulation of IL-I alpha in murine keratinocytes. Jlmmuno1140, 2274-2278 4 BanghamAD, Standise MM, Weissmann G (1965) The action of steroid and streptolysin S and permeability of phospholipid structures to cations. J Mol Biol 13, 253-259 5 lkutler B, Cerami A (1988) The common mediator of shock cachexia and tumor necrosis. Adv lmmunol 42, 213-231 6 Boukamp P, Rupniak HTR, Fusenig N (1985)Environmental modulation of the expressionof differentiation and malignancy in six human squamous cell carcinoma cell lines. Cancer Res 45, 5582-5592 7 BoyceST, Ham RG (1983) Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol 81, 33S-40S 8 Brandley BK, Swiedler SJ, Robbins PW (1990) Carbohydrate ligands of the LEC cell adhesion molecules. Cell 63, 861-863 9 Brysk MM, Rajaraman S, Penn P, Barlow E (1988) Glycoproteinsmodulate adhesion in terminallydifferentiated keratinocytes. Cell Tissue Res 253, 657-663 10 BurchiliSA, Virden R, Fuller BB, Thody AJ (1988) Regulation of tyrosinase synthesis by a melanocyte-stimulatinghormone in hair follicular melanocytes of the mouse. JEndocr 116, 17-23 11 Cannon JG, Tatro JB, Reichlin S (1986) ,~-Melanocyte stimulating hormone inhibits immunostimulatory and inflammatory actions of interleukin 1. J Immunol 137, 2232-2236 12 De Luca M, D'Anna F, Bondanza S, Tito Franzi A, Cancedda R (1988) Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis. J Cell Biol 107, 1919-1926 13 De Luca M, Tito Franzi A, D'Anna F, Zicca A, Albanese E, Bondanza S, Cancedda R (1988) Coculture of human keratinocytes and melanocytes: differential melanocytes are physiologicallyorganized in the basal layer of the cultured epithelium. Fur J Cell Biol 46, 176-180 14 Di Giovine FS, Duff GW, Phil D (1990) lnterleukin 1 : the first interleukin. Immunol Today 1I, 13-10 15 DoverR, Watt FM (1987) Measurementof the rate of epidermal terminal differentiation: expression of involucrin by Sphase keratinocytes in culture and in psoriatic plaques. Jlnvest Derm 89, 349-352 16 DowdPM, Camp RDR, Greaves MW (1988)Human recombinant IL-I alpha is proinflammatory in normal human skin. Skin Pharmacol I, 30-37 17 Durum SK, Schmidt JA, Oppenheim JJ (1985) lnterleukin I : an immunological perspective. Annu Rev Immunol 3, 263-287 18 Fuller BB, Lunsford JB, lman DS (1987) A-Melanocytes stimulating hormone regulation of tyrosinase in Cloudman $91 mouse melanoma cell cultures. J Biol Chem 262, 4024-4033

19 Gordon S, Perry VH, Rabinowitz S, Chung LP, Rosen H (1988) Plasma membrane receptors of the mononuclear phagocyte system. J Cell Sci Suppl 9, !-26 20 GrillonC, MonsignyM, Kieda C (1990)Changes in human T lymphocyte membrane lectins expression upon mitogenic stimulation. Carbohyd Res (in press) 21 Grillon C, Monsigny M, Kieda C (1990.)Cell surface lectins of human granulocytes: their expression is modulated by mononuclear cells and GM-CSF. Olycobiol 1, 33-38 22 Halaban R, Ghosh S, Baird A (1987) bFGF is a putative natural growth factor for human melanocytes. In Vitro Cell & Dev Biol 23, 47-52

42

D Cerdan et al

23 Haiaban R, Langdon R, Birchall N, Cuono C, Baird A, Scott G, Moellmann G, Guire JMC (1988) bFGF from human keratinocytes is a natural mitogen for melanocytes. J Cell Biol 107, 1611-1619 24 Hauser C, Saurat JH, Schmitt A, Jaunin F, Dayer JM (1986) Interleukin 1 is present in normal human epidermis. J Immunol 136, 3317-3323 25 Herlyn M, Mancianti ML, Jambrosic J, Bolen JB, Koprowski H (1988) Regulatory factors that determine growth and phenotype of normal human melanocytes. Exp Cell Res 179, 322-331 26 Hughes CCW, Male DK, Lantos PL (1988) Adhesion of lymphocytes to cerebral microvascular cells: effects of interferon-gamma tumour necrosis factor and interleukin-l. Immunol 64, 677-681 27 Kaidbey KH, Kligman AM (1978) The acute effects of long wave ultraviolet radiation on human skin. JInvest Dermatol 72, 253-256 28 Kaye JS, GiUis S, Mizel SB, Shevach EM, Malek TR, Dinarello CA, Lachman LB, Janeway CA (1984) Growth of a cloned helper T cell line induced by a monoclonal antibody specific for the antigen receptor: interleukin 1 is required for the expression of receptors for interleukin 2. J lmmunol 133, 1339-1345 29 Kieda C, Monsigny M (1986) Involvement of membrane sugar receptors and membrane glycoconjugates in the adhesion of 3LL cell subpopulations to cultured pulmonary cells. Invasion & Metastasis 6, 347-366 30 K6ck A, Urbanski A, Luger TA (1989) mRNA expression and release of tumor necrosis factor alpha by human epidermal cells. J Invest Dermatoi 92, 462A 31 Ku W, Berstein IA (1988) Lectin binding as a probe of proliferative and differentiative phases in primary monolayer cultures of cutaneous keratinocytes. Exp Cell Res 175, 298-316 32 gupper TS, Ballard DW, Chua AO, McGuire JS, Flood PM, Horowitz MC, Langdon R, Lightfoot L, Gubler U (1986) Human keratinocytes contain mRNA indistinguishablefrom monocyte interleukin I alpha and I beta mRNA ; keratinocyte epidermal cell-derived thymocyte-activating factor is identical to interleukin 1. J Exp Med 164 (6), 2095-2100 33 Kupper TS, Edelson RL (1987) The role of cytokines in the pathophysiology of T cell mediated skin disease. J Dermatol 14, 517-523 34 Kupper TS, Horowitz M, Lee F, Coleman D, Flood P (1987) Molecular characterization of keratinocytes cytokines. Jlnvest Dermatol 88, 501A 35 Kupper TS, May L, Birchalt N, Sehgal P (1988) Keratinocytes produce interleukin 6, a cytokine which can provide a second signal in the activation of T cells. J Invest Dermatol 90, 578A 36 Larsen CS, Christiansen NO, Esmann V (1988) Modulation of high affinity interleukin-2 receptors on activated human T lymphocytes by activators of protein kinase C. Scand J Immunol 28, 167-175 37 Lavker RM, Kaidbey KH (1982) Redistribution of melanosomal complex within keratinocytes following UV A irradiation: a possible mechanism for cutaneous darkening in man. Arch Dermatoi Res 272, 215-228 38 Luger TA, Uchida A, Kock A, Colot M, Micksche M (1985) Human epidermal cells and squamous carcinoma cells synthesize a cytokine that augments natural killer cell activity. J Immunol 134-4, 2477-2483 39 Luger TA, Wirth U, Kock A (1985) Epidermal cells synthesize a cytokine with interleukin 3-like properties. Jlmmunol 134-2, 915-919 40 Midoux P, Roche AC, Monsigny M (1987) Quantitation of the binding, uptake, and degradation of fluoresceinylated neoglycoproteins by flow cytometry. Cytometry 8, 327-334 41 Monsigny M, Kieda C, Roche AC (1983) Membrane glycoproteins, glycolipids and membrane lectins as recognition signals in normal and malignant cells. Bioi Cell 47, 95-110 42 Monsigny M, Petit C, Roche AC (1988) Colorimetric determination of neutral sugars by a resorcinol sulfuric acid micromethod. Anal Biochem 175, 525-530

43 Monsigny M, Roche AC, Kieda C, Midoux P, Obrenovitch A (1988) Characterization and biological implications of membrane lectins in tumor, iymphod and myelod cells. Biochimie 70, 1633-1649 44 Monsigny M, Roche AC, Midoux P (1984) Uptake of neoglycoproteins via membrane lectin(s) of LI210 cells evidenced by quantitative flow cytofluorometry and drug targeting. Biol Cell 51, 187-196 45 Morhenn VB (1988) Keratinocyte proliferation in wound healing and skin diseases. Immunol Today 9, 104-107 46 Morrison AI, Keeble S, Watt F (1988) The peanut lectin binding glycoproteins of human epidermal keratinocytes. Exp Cell Res 177, 247-256 47 Navas P, Villalba JM, Buron MI, Garcia Herdugo G (1987) Lectins as markers for plasma membrane differentiation of amphibian keratinocytes. Biol Cell 60, 225-234 48 Momura H, Hashizuka H, Higuchi M, Sasai Y (1989) Cytofluorimetric study of lectin binding of isolated guinea pig keratinocytes. Acta Histochem 87, 59-62 49 Nordlund JJ (1988) Post-inflammatory hyperpigmentation. Dermatol Clin 6, 185-192 50 Oxholm A, Oxholm P, Staberg B, Bendtzen K (1988) Immunohistological detection of interleukin I-like molecules and TNF in human epidermis before and after UVB irradiation in vivo. Br J Dermatoi 118, 369-376 51 Qwarnstrom EE, Page RC, Gillis S, Dower SK (1988) Binding internalization and intracellular localization of interleukin-I A in human diploid fibroblast. J Biol Chem 17, 8261-8269 52 ROche AC, Barzilay M, Midoux P, Junqua S, Sharon N, Monsigny M (1983) Sugar-specific endocytosis of glycoproteins by Lewis Lung carcinoma cells. J Cell Biochem 22, 131-140 53 Roche AC, Midoux P, Bouchard P, Monsigny M (1985) Membrane lectins on human monocytes. Maturationdependent modulation of 6-phosphomannose and mannose receptors. FEBS Lett 193, 63-68 54 Sasaki DT, Dumas SE, Engelman EG (1987) Discrimination of viable and non-viable cells using propidium iodide in two colors immunofluorescence. Cytometry 8, 413-420 55 Sauder DN, Monick MM, Hunningghake GW (1985) Epidermal cell-derived activating factor (ETAF) is a potent T-cell chemoattractant. J Invest Dermatol 85, 431-433 56 Sauder DN, Wong D, Kenzie RM, Stetsko D, Harnish D, Tron V, Nickoloff B, Arsenault T, Harley CB (1988) The pluripotent keratinocyte: molecular characterization of epidermal cytokines. J Invest Dermatol 90, 605A 57 Stooiman LM (1989) Adhesion molecules controling lymphocyte migration. Cell 56, 907-910 58 Stoolman LM, Rosen SD (1983) Possible role for cell-surface carbohydrate-binding molecules in lymphocyte recirculation. J Cell Biol 96, 722-729 59 Takahashi M, Mogi Y, Goto Y, Tsushima N, Takahashi Y, Fujikawa K, Watanabe N, Kohgo Y, Sugiyama S, Niitsu Y (! 989) A case of cutaneous T cell lymphoma improved with local administration of tumor necrosis factor. Rinshoketsueki 30, 2152-2156 60 Valdimarsson H, Baker BS, Jonsdottir I, Fry L (1986) Psoriasis: a disease of abnormal keratinocyte proliferation induced by T lymphocytes. Immunol Today 7, 256-259 61 Watt FM (1988) Keratinocyte cultures: an experimental model for studying how proliferation and terminal differentiation are co-ordinated in the epidermis. J Cell Sci 90, 525-529 62 Weinstein JN, Yoshikami S, Henkart P, Blumenthal R, Hagins WA (1977) Liposome-cell interaction: Transfer and intracellular release of a trapped fluorescent marker. Scier.ce 195, 489-491 63 Wolff K (1973) Melanocyte-keratinocyte interactions in vivo: the fate of melanosomes. Yale J Biol Med 46, 384-396 64 Yaar M, Peacocke M, Bhawan J, Gordon JL, Southmayd JR, Gilchrest BA (1988) Human keratinocytes contain nerve growth factor: A posfible inducer of melanocyte dentricity and tropism. J. Invest l~ermatol 90, 619A 65 Yednock TAg Rosen SD (1989) Lymphocyte homing. Adv Immunol 44, 313-378