Mechanism of macrophage recognition of SH-oxidized erythrocytes: recognition of glycophorin A on erythrocytes by a macrophage receptor for sialosaccharides

BB ql

ELSEVIER

Biochimica et Biophysica Acta 1223 (1994) 47-56

Biochi~& et BiophysicaA~ta

Mechanism of macrophage recognition of SH-oxidized erythrocytes: recognition of glycophorin A on erythrocytes by a macrophage receptor for sialosaccharides Masatoshi Beppu, Takuya Takahashi, Takahiro Hayashi, Kiyomi Kikugawa

*

Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan Received 25 January 1994

Abstract

Mouse erythrocytes treated with diamide, an SH-oxidizing agent, attach to mouse resident peritoneal macrophages in the absence of serum. The mechanism by which macrophages recognize the SH-oxidized erythrocytes was investigated. Although phosphatidylserine-liposomes inhibited the macrophage recognition, there was no detectable phosphatidylserine on the outer surface of diamide-treated erythrocytes. It is unlikely that phosphatidylserine, that has been proposed to be a determinant in the recognition of some pathologic erythrocytes by macrophages, is involved in the recognition of diamide-treated erythrocytes. Sialyl lactose and glycophorin A effectively inhibited the macrophage recognition, while lactose and neuraminidase-treated glycophorin A did not. Disialoganglioside GDla, but not monosialoganglioside GMI, partially inhibited the recognition. Trypsinized erythrocytes, in which majority of glycophorin A glycopeptides were expected to be removed from the cell surface, poorly attached to macrophages after diamide treatment. Therefore, it is likely that an interaction between glycophorin A on diamide-treated erythrocytes and a macrophage receptor for sialosaccharides is involved in the recognition. Similar inhibition specificity was observed in the macrophage recognition of erythrocytes treated with periodate, an oxidant that induces disulfide-dependent erythrocyte changes causing macrophage recognition, and of erythrocytes treated with SH-blocking agents, N-ethylmaleimide and p-chloromercuribenzoic acid, that were also found to be susceptible to macrophage recognition in the absence of serum. These results suggest that the macrophages recognize sialosaccharide chains of glycophorin A molecules on SH-oxidized or SH-blocked erythrocytes through a receptor for sialosaccharides. Key words: Macrophage recognition; Sialosaccharide receptor; Macrophage lectin; SH oxidation; Glycophorin A; (Erythrocyte); (Mouse)

1. Introduction

One of the important functions of macrophages to maintain homeostasis is to recognize and remove damaged or effete cells [1,2]. However, little is known about the type of cell damage leading to macrophage recognition and about the mechanism of the recognition. One of possible causes of cell damage in vivo is oxidative stress [3]. Oxidative stress, which arises under such conditions as exposure to active oxygen species

Abbreviations: NEM, N-ethyimaleimide; PCMB, p-chloromercuribenzoic acid; PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; PA, phosphatidic acid. * Corresponding author. Fax: +81 426 752605. 0167-4889/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4889(94)00065-M

and vitamin E deficiency, can cause oxidative damage of cell components [4-8]. Evidence for occurrence of in vivo oxidative damage of cell components has been demonstrated in aged erythrocytes [9,10] and pathologic erythrocytes [11-15]. SH groups are readily oxidized and in vivo occurrence of SH oxidation of membrane proteins has been suggested in pathologic eyrthrocytes such as glucose-6phosphate dehydrogenase deficiency [16], fl-thalassemia [17], and sickled erythrocytes [18]. We have previously shown that mouse erythrocytes that underwent oxidation of free SH groups in vitro by SH-oxidizing agents or periodate were susceptible to binding to mouse resident peritoneal macrophages [19]. The finding suggested that SH-oxidized erythrocytes occurring in vivo are recognized by macrophages as targets to be

48

M. Beppu et al. /Biochimica et Biophysica Acta 1223 (1994) 47-50

removed. The oxidized erythrocytes were not recognized upon treatment with dithiothreitol [19], indicating that the SH-oxidation-induced membrane changes may be reversed by restoration of free SH groups of the cells. Various changes of the membrane properties have been known to be induced by SH-oxidation of erythrocytes. Of the reported changes, loss of membrane phospholipid asymmetry [20,21], i.e., exposure of phosphatidylserine (PS) on the outer surface of the membrane, may be relevant to the macrophage recognition since erythrocytes displaying an exogenously supplied PS analog on the outer surface are recognized by macrophages [22,23]. In this report, we have investigated the mechanism of macrophage recognition of SH-oxidized erythrocytes. First, we examined the possibility of PS as a determinant of the macrophage recognition, and found it was unlikely in our system. Instead, it was found that macrophages recognize sialosaccharide chains of the SH-oxidized cell membrane presumably through a receptor for sialosaccharides. Furthermore, we found that modification of erythrocytes by SH-blocking agents also results in macrophage recognition, and the recognition is mediated through the same receptor as that for SH-oxidized erythrocytes. 2. Materials and methods

2.1. Materials Diazene dicarboxylic acid bis(N,N'-dimethylamide) (diamide), N-acetylneuraminic acid, N-acetyl-Dgalactosamine, N-acetylneuramin lactose (a mixture of N-acetylneuraminyl-ot2-3 lactose (85%) and N-acetylneuraminyl-a2-6 lactose(15%)), mannan, bovine fetuin, lactoferrin (human milk), at-acid glycoprotein (human), monosialoganglioside (GM1), disialoganglioside (GDla), trypsin (bovine pancreas, N-tosyl-Lphenylalanine chloromethylketone-treated) (EC 3.4.21.4), trypsin inhibitor (soybean), and phospholipase A 2 (bee venom) (EC 3.1.1.4) were purchased from Sigma. Sodium metaperiodate, N-ethylmaleimide (NEM), p-chloromercuribenzoic acid (PCMB), D-glucose, D-galactose, D-mannose, L-fucose, and lactose were obtained from Wako Pure Chemical Industries. PS (bovine brain), and phosphatidic acid (egg, PA) were the products of Serdary Research Laboratories. Phosphatidylcholine (egg yolk, PC) and phosphatidylethanolamine (egg, PE) were from Nippon Oil and Fats and Avanti Polar Lipids, respectively. N-AcetylD-glucosamine, bovine submaxillary mucin, Pronase (Actinase E), neuraminidase (Vibr/o cholerae) (EC 3.2.1.18), and fetal calf serum were obtained from Tokyo Kasei Kogyo, Worthington, Kaken Seiyaku, Behringwerke, and Gibco Laboratories, respectively. Band 3 oligosaccharides, prepared by hydrazinolysis of

human band 3 glycoprotein and then N-acetylation, were generously donated by Drs. Toshiaki Osawa and Kazuo Yamamoto (University of Tokyo). Glycophorin A was isolated from human erythrocytes according to the method of Merchesi and Andrews [24], and Furthmayr et al. [25]. Sialic acid content of glycophorin A, lactoferrin, bovine submaxillary mucin, a l-acid glycoprotein, and fetuin used was 20.5, 0.7, 13.6, 20.1, and 9.2%, respectively, as determined by the method of Warren [26] after mild acid hydrolysis. Lysophosphatidylcholine (lysoPC) and lysophosphatidylserine (lysoPS), and lysophosphatidylethanolamine (lysoPE) were prepared by hydrolyzing PC, PS, and PE, respectively, using bee venom phospholipase A 2 according to the method of Okuyama and Nojima [27] with minor modifications. Concentrations of phospholipids and lysophospholipids were estimated by phosphorus concentrations determined by the method of Gerlach and Deuticke [28]. 2.2. Liposomes Liposomes were prepared as described below. A mixture of the desired phospholipids (20/zmol) in 6 ml of chloroform/ethanol (5 : 2, v/v) was placed in a flask. The solvent was removed in a rotary evaporator at about 40°C under reduced pressure. The dried lipids were dispersed with a vortex mixer in 3 ml of 10 mM Tris-HC1 buffer (pH 7.4) containing 0.14 M NaCI followed by sonication at 10°C. After centrifugation (650 × g, 5 min), the supernatant of the liposome suspension was recovered, and the cono~ntration of phospholipids was estimated by phosphorus concentration. Three types of liposomes, PC-liposomes, PS-liposomes and PE-liposomes, were prepared. PC-liposomes were prepared from a mixture of PC and PA with a molar ratio of 10 : 0.4, PS-liposomes from a mixture of PC, PS and PA with a molar ratio of 7:3:0.4, and PE-liposomes from a mixture of PC, PE and PA with a molar ratio of 7 : 3 : 0.4. 2.3. Cells Macrophages were obtained from the peritoneal cavity of 7-12-week-old ddY male mice. Resident peritoneal cells were washed and the macrophage monolayers were prepared on round glass coverslips (18-mm diameter) as described previously [19] and used after overnight culture in RPMI 1640 medium supplemented with 50 U / m l penicillin, 50 /~g/ml streptomycin and 10% heat-inactivated fetal calf serum in 5% CO 2. Erythrocytes were obtained from ddY male mouse blood collected on the day of the use by cardiac puncture using acid citrate dextrose as an anticoagulant. Erythrocytes were washed four times with isotonic saline and resuspended in a buffer consisting of 12.5

M. Beppu et al. / Biochimica et Biophysica Acta 1223 (1994) 47-56

mM Na2HPO4-NaH2PO4/100 mM KC1/50 mM NaCI/44 mM sucrose (pH 8.0, buffer A) or in 10 mM acetate buffer containing 0.14 M NaCl (pH 6.0, buffer

B). 2.4. Modification of erythrocytes An erythrocyte suspension (20% hematocrit) in buffer A was mixed with an equal volume of freshly prepared solutions of diamide, NEM, and PCMB in the same buffer, and incubated at 37°C for 30 min for the modification with diamide, and at 37°C for 15 or 30 min for the modification with NEM and PCMB. For modification with periodate [19], an erythrocyte suspension (20% hematocrit) in buffer B was mixed with an equal volume of freshly prepared sodium metaperiodate solution (2 mM) in the same buffer, and incubated at 0°C for 15 min in the dark. The modified cells were washed four times with an ice-chilled buffer A at pH 7.4 by centrifugation (375 x g, 7 min) at 0-4°C, resuspended in RPMI 1640 medium supplemented with 20 mM Hepes (pH 7.2), 50 U / m l penicillin and 50 /zg/ml streptomycin (RPMI-Hepes medium) to make a 2% cell suspension, and assayed for macrophage recognition.

2.5. Quantitation of phospholipid classes on the surface of SH-oxidized erythrocytes Phospholipid calsses on the surface of SH-oxidized erythrocytes were quantified using phospholipase A 2 according to the method described [29] with minor modifications. The SH-oxidized erythrocytes were suspended in 10 mM Tris-HCl (pH 7.4) containing 0.1 M KC1, 50 mM NaCI, 1 mM CaCI 2, 0.25 mM MgCI2, 44 mM sucrose to make a 20% suspension, and a solution of bee venom phospholipase A 2 (8000 U / m l ) in the same buffer was added at a final concentration of 8 U/ml. After incubation at 37°C for 1 h, cells were centrifuged (650 x g, 5 min). To assess the degree of hemolysis, absorbance of hemoglobin of the supernatant was measured at 523 nm, an isosbestic point of human oxyhemoglobin and methemoglobin (E = 7880 [30]). After washing the cells with 50 mM Tris-HCl containing 5 mM EDTA (pH 7.4), lipids were extracted from the cells according to the method of Rose and Oklander [31]. Total phospholipids were estimated from the amount of phosphorus. The phospholipids in the lipid extract (600/zg as phospholipids) were separated by two-dimensional thin-layer chromatography on a Kieselguhr 60HR (Merck) plate (20 × 20 cm, 1 mm thick), with a solvent system consisting of CHC13/ CH3OH/NH3(25%)/H20(90:54:5.5:5.5, v/v) in the first dimension, and C H C I 3 / C H 3 O H / C H 3 C O O H / H 2 0 (90 : 40 : 12 : 2, v/v) in the second dimension [29]. The lipid spots on the plate were located by staining

49

with iodine vapor, and phospholipids (PC, PE and PS) and lysophospholipids (lysoPC, lysoPE and lysoPS) were identified by comparison with the spots of similarly separated standards. Each spot was scraped from the plate and transfered to a test tube and phopholipid phosphorus content was determined. The lysophospholipid content was regarded as the amount of corresponding phospholipid on cell surface.

2.6. Intracellular GSH and membrane SH groups of SH-blocked erythrocytes Measurement of intracellular GSH content of the SH-blocked erythrocytes and free SH groups of the SH-blocked cell membrane were made as described previously [19].

2. 7. Adhesion and phagocytosis by macrophages A 2% erythrocyte suspension (200 /~1) was loaded on the macrophage monolayer. For observation of adhesion, cells were incubated at 37°C for 1 h, then nonadhering erythrocytes were removed by gentle washing with Dulbecco's phosphate-buffered saline (buffer C), and the cells were fixed with 1.25% glutaraldehyde in buffer C. The percentage of macrophages that bound one or more erythrocytes was determined under phase-contrast microscopy (macrophage adhesion). For observation of phagocytosis, cells were incubated at 37°C for 3 h. After removal of nonadherent erythrocytes by washing, adherent erythrocytes were lysed and the cells were stained with Giemsa's solution as described previously [32]. The number of macrophages ingesting one or more erythrocytes was determined (macrophage phagocytosis). Representative data of at least three independent experiments were shown as the mean + S.D. of triplicate assays.

2.8. Inhibition of macrophage adhesion to modified erythrocytes For inhibition by liposomes, the macrophage monolayer was incubated with a liposome suspension (0.2 ml/coverglass) in RPMI-Hepes medium at 37°C for 30 min. The monolayer was washed with Ca 2+-, MgZ+-free Dulbecco's phosphate-buffered saline (buffer D), and a 2% suspension of the modified erythrocytes in RPMIHepes medium (200 ~1) was loaded on the monolayer. After incubation at 37°C for 1 h, the percentage of macrophages that bound one or more erythrocytes was determined as described above. For inhibition by saccharides or glycoproteins, macrophage monolayer was incubated with a 2% suspension (200 tzl) of the modified erythrocytes containing saccharides or glycoproteins at 37°C for 1 h, and processed as described above. The inhibition study by gangliosides was per-

50

M. Beppu et al. / Biochimica et Biophysica Acta 1223 (1994) 47-56

formed according to the method of Riedl et al. [33] with minor modifications. The macrophage monolayer was incubated with a ganglioside solution (200/xg/ml, 0.2 ml/coverglass) in RPMI-Hepes medium on ice for 30 min. The monolayer was washed with buffer D and processed as described above. The data for the inhibition studies with saccharides, glycoproteins and gangliosides were expressed as % of control assay, where macrophage adhesion in the absence of inhibitors was taken as 100.

3. Results

3.1. Mechanism of macrophage recognition of SHoxidized erythrocytes Mouse erythrocytes treated with diamide are recognized by mouse resident peritoneal macropohages in the absence of serum [19]. Usually, up to 80% of the macrophages bind the modified erythrocytes depending on the conditions of the modification, but the adherence is rarely accompanied by phagocytosis [19]. As a possible mechanism by which macrophages recognize diamide-treated erythrocytes, we investigated whether PS, which exclusively resides in the inner leaflet of erythrocyte membrane [34], is exposed on the cell surface and recognized by macrophages as suggested [22,23]. Sickled erythrocytes, which have high

5O

§20 0

0.05

Liposome concentration (mM of total phospholip~'s)

0.5

Fig. 1. Effect of phospholipid liposomes on macrophage recognition of diamide-treated erythrocytes. Mouse erythrocytes incubated with ( ) or without ( . . . . . . ) diamide (1 raM) at 37°C for 30 rain were washed, resuspended in RPMI-Hepes medium, and loaded on the macrophage monolayers preincubated with the indicated concentrations of PS (o)-, PC (e)-, and PE (,x)-iiposomes: Macrophage adhesion was assayed as described in Section 2. Results shown are the mean + S.D. of triplicate assays.

Table 1 Phospholipids on mouse erythrocyte surface hydrolyzed by phospholipase A 2 Erythrocyte

Lyso PC Lyso PS Lyso PE (% of total PC) (% of total PS) (% of total PE)

Control 41.4 Diamide-treated 40.7 (40.1) a

1.2 0.0 (0.0) a

10.4 12.5

Mouse erythrocytes were incubated with or without (control) diamide (1 mM) at 37°C for 30 min. After washing, cell surface phospholipids were hydrolyzed by bee venom phospholipase A2, and the amount of respective lysophospholipid and phospholipid was determined as described in Section 2. Only 1.8, and 2.6% of hemolysis took place by the enzyme treatment of control and diamide-treated erythrocytes, respectively. a Cells incubated for additional 1 h at 37°C in RPMI-Hepes medium after the diamide treatment.

avidity for adherence to monocytes [35], display PS in the outer membrane leaflet [36], and its adherence to monocytes is inhibited by preincubation of monocytes with PS-liposomes [37]. We tested whether PS-liposomes inhibit adherence of diamide-treated erythrocytes to macrophages. When mouse erythrocytes were treated with diamide, approx. 40% of macrophages bound the erythrocytes while 10% of macrophages bound control cells (Fig. 1). Adherence of diamidetreated erythrocytes to macrophages was reduced to the level of adherence of control erythrocytes by preincubation of the macrophages with PS-liposomes, while PE- and PC-liposomes were not effective. Adherence of control erythrocytes was little changed by either type of liposome. Whether PS is exposed on the outer surface of erythrocytes by SH oxidation with diamide was then examined. Mouse erythrocytes treated with diamide were treated with bee venom phospholipase A 2, and each phospholipid class on the cell surface was detected as respective lysophospholipid (Table 1). In control cells, only little part of membrane PS was hydrolyzed to lysoPS, indicating absence of PS on the outer surface of the erythrocytes. Diamide treatment did not affect the proportion of hydrolyzed PC, PS, and PE. Incubation of diamide-treated erythrocytes in RPMI-Hepes medium for additional period, as is done in the macrophage recognition assay, gave similar results (Table 1, parentheses). Hence, it is unlikely that macrophages recognize PS of diamide-treated mouse erythrocytes. Inhibition by PS-liposomes of macrophage recognition of diamide-treated erythrocytes may not be due to blocking of the putative 'PS receptor' [22,37] of macrophages, but to blocking another receptor that 'cross-reacts' with PS. We then investigated other possibilities for the mechanism of macrophage recognition of diamidetreated erythrocytes. It is known that macrophages recognize aged erythrocytes and xenogeneic erythrocytes, and some reports have shown that these cells are recognized by lectin-like receptors [38-44]. To examine

M. Beppu et al. / Biochimica et Biophysica Acta 1223 (1994) 47-56

whether saccharide recognition is involved in the macrophage recognition of diamide-treated erythrocytes, inhibition studies with various saccharides were conducted. Although monosaccharides did not inhibit the macrophage recognition at the concentration as high as 50 mM, N-acetylneuramin lactose effectively inhibited even at lower concentrations (5 and 20 mM) (Table 2). Since lactose was noninhibitory at 50 mM, sialyl residues of the trisaccharide appeared to be essential. Of the glycoproteins tested, glycophorin A, a major sialoglycoprotein of erythrocyte membrane, exhibited potent inhibitory activity (Table 2). Its activity was dose dependent (Fig. 2), and was lowered when treated with neuraminidase (Table 3). It is therefore likely that macrophages recognize diamide-treated erythrocytes through their surface receptor recognizing sialosaccharide chains, oq-Acid glycoprotein was not inhibitory (Table 2) although its sialic acid content (20.1%) was as high as that of glycophorin A (20.5%). Treatment of glycophorin A with Pronase resulted in a decrease in its inhibitory activity (Table 3). Thus, the macrophage receptor may recognize better highly clustered sialosaccharide chains since saccharide chains of glycophorin A are clustered in N-terminal region of its polypeptide [45]. On the erythrocyte surface, not only glycoproteins but also glycolipids contain sialic acids, and we tested

Table 2 Effect of saccharides and glycoproteins on macrophage recognition of diamide-treated erythrocytes Saccharide or glycoprotein

D-Glucose D-Galactose D-Mannose L-Fucose N-Acetyl-D-glucosamine N-Acetyl-o-galactosamine N-Acetylneuraminic acid Lactose N-Acetylneurami lactose .. Glycophorin A Band 3 oligosaccharides cq-Acid glycoprotein Fetuin Lactoferrin Bovine submaxillary mucin Mannan

Macrophage adhesion (% of control assay) (mM) 50 50 50 50 50 50 50 50 5 20 (mg/ml) 0.1 0.1 a 0.1 0.1 1 1 1

100 8O .O

•

40

~o 2o v

0

i

0

1

3.35:1.3 109.05:0.3 104.1 + 4.3 110.05:2.9 106.7 + 3.2 102.8 + 5.6 107.8 + 2.4

Mouse erythrocytes incubated with diamide (1 mM) at 37"C for 30 min were washed and assayed for macrophage adhesion in the presence of indicated concentrations of saccharides or glycoproteins. In the absence of the inhibitors (control assay), 68.6-82.8% of macrophages bound the modified erythrocytes. Values given are the mean + S.D. of triplicate assays. a Concentration as neutral sugars.

10

100

Glycophorin A (p,g/ml) Fig. 2. Effect of glycophorin A on macrophage recognition of diamide-treated erythrocytes. Mouse erythrocytes incubated with (e) or without (<3) diamide (1 mM) at 37°C for 30 min were washed, resuspended in RPMI-Hepes medium, and assayed for macrophage adhesion in the presence of indicated concentrations of glycophorin A.

the effect of gangliosides, sialic acid-containing glycolipids, on the macrophage recognition of diamidetreated erythrocytes. As shown in Table 4, pretreatment of a macrophage monolayer with disialoganglioside GDla, NeuAc a2-3Gal /31-3GalNAc /314(NeuAc a2-3)Gal/31-4Glc-Cer, before addition of Table 3 Effect of neuraminidase and Pronase treatment of glycophorin A on its inhibitory activity to the macrophage recognition of diamidetreated erythrocytes Glycophorin A (0.1 mg/ml)

98.4 + 8.7 112.9+ 8.3 100.1 + 5.2 111.0+ 2.9 117.8 5:1.6 106.0 + 3.8 104.8 + 0.6 97.4 5:15.3 54.05:5.8 35.05:5.9

51

Expt. A a Control Neuraminidase-treated Expt. B b Control Pronase-treated

Macrophage adhesion (% of control assay) 9.1 + 0.3 70.5 + 11.2 7.8-I- 0.9 112.1+ 7.4

Mouse erythrocytes incubated with diamide (1 mM) at 37°C for 30 min were washed and assayed for macrophage adhesion in the presence of neuraminidase- or Pronase-treated glycophorin A. In the absence of the inhibitors (control assay), 50.1% (Expt. A) and 72.9% (Expt. B) of macrophages bound the modified erythrocytes. Values given are the mean + S.D. of triplicate assays. a Glycophorin A (1 mg/ml) was incubated with or without neuraminidase (50 m U / m l ) in buffer C at 37°C for 96 h, and then heated at 100°C for 5 rain to inactivate the enzyme. Chloroform was present (1 p,l/ml) as a preservative during the incubation. No proteolytic break down of glycophorin A took place by this treatment as confirmed by SDS-polyacrylamide gel electrophoresis of 1251labeled glycophorin A followed by autoradiography. b Glycophorin A (3 mg/ml) was incubated with or without Pronase (0.5 mg/ml) in buffer C at 37°(2 for 48 h, and then heated at 100°C for 5 rain to inactivate the enzyme. Chloroform was present (1 p,l/ml) as a preservative during the incubation. Small amount of precipitates formed by the heating was removed by centrifugation.

52

M. Beppu et al. / Biochimica et Biophysica Acta 1223 (1994) 47-56

Table 4 Effect of gangliosides on macrophage recognition of diamide-treated erythrocytes Ganglioside (200 ~ g / m l )

Macrophage adhesion (% of control assay)

GMI GD1a

101.4+2.0 85.1 + 1.8

A o~

Q~ cJ 0

Mouse erythrocytes incubated with diamide (1 mM) at 37°C for 30 min were washed and assayed for macrophage adhesion using the macrophage monolayer preincubated with GM1 or GD1a (200/~g/ml) at 0°C for 30 min. The monolayers were washed before the assay. In the absence of the inhibitors (control assay), 80.3% of macrophages bound the modified erythrocytes. Values given are the mean + S.D. of triplicate assays.

•-= ~

diamide-treated erythrocytes, weakly but significantly prevented the macrophage recognition, while monosialoganglioside GMt, Gal/31-3GalNAc /31-4(NeuAc a2-3)Gal/31-4Glc-Cer, was ineffective. This indicates that ganglioside can be a ligand for the macrophage receptor for diamide-treated erythrocytes depending on sialic acid content. It is possible that glycophorin A molecules of diamide-treated erythrocytes are recognized by macrophages. To investigate this possibility, effect of removal of saccharide-chain-rich peptide region of glycophorin A from erythrocyte surface on the macrophage recognition was examined. Erythrocytes were briefly treated with trypsin, which treatment is known to hydrolyze mainly glycophorin A polypeptide and release its glycopeptides from human erythrocyte surface [46,47]. As shown in Table 5, subsequent treatment of the trypsinized cells with diamide did not result in an increased macrophage recognition. Pretreatment of erythrocytes with neuraminidase partially prevented an increase in macrophage recognition by diamide treat-

Table 5 Effect of pretreatment of erythrocytes with trypsin and neuraminidase on the macrophage recognition of the erythrocytes subsequently treated with diamide

First

Second

None None Trypsin Trypsin Neuraminidase Neuraminidase

none diamide none diamide none diamide

Macrophage adhesion (% of total macrophages) 48.3 + 68.5+ 32.2+ 34.7 + 36.6 + 47.0+

11.2 2.1 0.8 12.1 7.0 0.1

A suspension of mouse erythrocytes (10%) was incubated with or without neuraminidase (25 m U / m l ) or trypsin (100/Lg/ml) in buffer C at 37°C for 30 rain. The digestion with trypsin was terminated by addition of soybean trypsin inhibitor at the final concentration of 200 ~g/ml. After washing the cells with buffer C four times at 0*C, the cells were subjected to diamide treatment (1 raM, 37*(2, 30 rain) or control incubation, and assayed for macrophage adhesion. Values given are the mean + S.D. of triplicate assays.

40

E~ 20

0

o2 ~

Treatment

B

100

lOC

0.25 N E M (raM)

0.5

C

I

,

0

I

I

0.25 PCMB (rnM)

0.5

D /

/ // ./ / / s

b~,~ ...... -U"

/

T..~z !

i

0

0.25 NEM

(raM)

/

/

/

z_

!

0.5

0

,

I

I

0.25

0.5

PCMB

(raM)

Fig. 3. Effect of NEM (A) and PCMB (B) on membrane SH content and intracellular GSH concentration of erythroeytes, and adhesion and phagocytosis of NEM (C)- and PCMB (D)-treated erythrocytes by macrophages. Mouse erythrocytes were incubated with the indicated concentrations of NEM or PCMB at 37°C for 15 min. Free SH groups of the membrane and intracellular GSH concentration were measured as described in Section 2. The amount of membrane SH groups and GSH concentration of the control erythroeytes were 119 nmol/mg protein and 12 nmol/mg hemoglobin, respectively. After washing, the modified cells were resuspended in RPMI-Hepes medium, and assayed for adhesion and phagocytosis by the monolayers of macrophages. Results shown are the mean + S.D. of triplicate assays.

ment. These observations suggest that macrophages recognize glycophorin A, possibly its sialosaccharide chains, on the surface of diamide-treated erythrocytes through a lectin-like receptor.

3.2. Macrophage recognition of SH-blocked erythrocytes and the mechanism of the recognition Mouse erythrocytes were treated with SH-reagents, NEM or PCMB, both of which specifically block free SH groups without producing disulfides. Treatment of mouse erythroeytes with 0.05-0.5 mM NEM at 37"C for 15 min resulted in the loss of the membrane free SH groups and intraeellular GSH (Fig. 3A). GSH was completely diminished at the NEM concentrations of 0.25 mM and above, but about 50% of the SH groups of the membrane proteins remained free at the NEM concentration of 0.5 raM. Treatment with PCMB also

M. Beppu et al. / Biochimica et BiophysicaActa 1223 (1994) 47-56 resulted in the loss of the m e m b r a n e free SH groups and intracellular G S H (Fig. 3B). These modified erythrocytes attached to the mouse resident peritoneal m a c r o p h a g e monolayers in the absence of serum at the reagent concentrations of 0.5 m M for the both cases (Figs. 3C and D). T h e r e was no significant increase in the phagocytosis of the N E M - or PCMB-treated erythrocytes. These results indicate that simply blocking free SH groups of erythrocytes leads to the macrophage recognition, and suggest that the modification responsible for the m a c r o p h a g e recognition in the SH-oxidizing agent-treated erythrocytes is the blocking of free SH groups by disulfide formation rather than cross-link production. The specificity of the m a c r o p h a g e receptor involved in the SH-blocked erythrocytes was investigated in two respects; specificity to PS and sialosaccharides. As was the case for diamide-treated erythrocytes, PS-liposomes inhibited m a c r o p h a g e recognition of N E M treated erythrocytes effectively, while PC-, and PEliposomes did not (Fig. 4). N-acetylneuraminic acid and N-acetylneuramin lactose markedly inhibited recognition of N E M - t r e a t e d erythrocytes at the concentrations of 50 m M and 5 mM, respectively, while

70

60

§ 40 E

Table 6 Effect of saccharides, glycophorinA, and gangliosides on macrophage recognition of NEM-treated erythrocytes Saccharide or glycoeonjugate Macrophage adhesion (% of control assay) 15.3:t: 1.1 N-Acetylneuraminic acid (50 mM) 89.6:1:18.0 Lactose (50 mM) N-Acetylneuramin lactose (5 mM) 13.85:5.7 6.1 5:3.4 Glycophorin A (control) a Glycophorin A (neuraminidase treated) a 74.5 5:8.0 GM1(200/a,g/ml) b 95.75:4.9 G D I a (200 Izg/ml) b 38.4 5:11.8 Mouse erythrocytes incubated with NEM (1 mM) at 37°C for 30 min were washed and assayed for macrophage adhesion in the presence of saccharides or glycophorin A, or after pretreatment of the macrophage monolayers with gangliosides. In the absence of the inhibitors (control assay), 44.1-47.5% of macrophages bound the modified erythrocytes. Values given are the mean + S.D. of triplicate assays. a Glycophorin A with or without neuraminidase treatment was prepared as described in the legend to Table 3. b Macrophage monolayers were preincubated with or without the gangliosides as described in the legend to Table 4.

lactose was ineffective even at 50 m M (Table 6). Glycophorin A was a potent inhibitor for the recognition, but neuraminidase treatment lowered its activity (Table 6). Furthermore, GD1 a but not GM1 inhibited the recognition (Table 6). Thus, the recognition specificity of the macrophages for N E M - t r e a t e d erythrocytes is similar to that for diamide-treated erythrocytes. It is likely that macrophages recognize the SH-oxidized and SH-blocked erythrocytes with the same receptor. NAcetylneuraminic acid, N-acetylneuramin lactose, and GD~ a, which were not or weakly inhibitory in the macrophage recognition of diamide-treated erythrocytes (Tables 2 and 4), were effective inhibitors for the recognition of N E M - t r e a t e d erythrocytes (Table 6). This may reflect the difference in the binding potency of the modified cells to the macrophage receptor.

3.3. Mechanism o f macrophage recognition of periodatetreated erythrocytes

PE .....,.. - - ' ' ' ' ~

8 "5

53

PS

2°f 10

:~

0

I

!

0.05

0.5

Liposome concentration (raM of total phospholipids) Fig. 4. Effect of phospholipid liposomes on macrophage recognition of NEM-treated erythrocytes. Mouse erythrocytes incubated with ( ) or without (. . . . . . ) NEM (1 mM) at 37°C for 30 rain were processed for assay for macrophage adhesion as described in the legend to Fig. 1. (o), PS-liposomes; (e), PC-liposomes;(zx), PE-liposomes.

Mouse erythrocytes treated with periodate are recognized by mouse resident peritoneal macrophages in the absence of serum [19,48]. Usually about 70% of the macrophages recognize the treated cells. We have previously shown that the m e m b r a n e alteration of the periodate-treated erythrocytes that is responsible for inducing the macrophage recognition is caused by oxidation of SH groups of the cells [19]. A possibility that the macrophage recognition of periodate-treated erythrocytes also involves sialosaccharide recognition was examined. In the inhibition study using monosaccharides and glycoproteins (Table 7), it was found that N-acetylneuraminic acid and glycophorin A signifi-

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cantly inhibit the m a c r o p h a g e recognition of the periodate-treated cells, while o t h e r m o n o s a c c h a r i d e s and glycoproteins are ineffective or m u c h less effective. T h e result suggests that sialosaccharide recognition is at least partially involved in the m a c r o p h a g e recognition of p e r i o d a t e - t r e a t e d erythrocytes, although the possibility of involvement of receptors with o t h e r specificity can not be excluded. It is known that sialic acid is readily oxidized, provided that the hydroxyls at positions C 7 - C 9 are unsubstituted, by mild periodate t r e a t m e n t as d o n e here to g e n e r a t e aldehyde function at a position C7 or C8 [49]. A part of sialyl residues of m o u s e erythrocytes are 9-O-acetylated ones which are resistant to this treatment, and these residues may be involved in the recognition. However, it is also possible that the oxidized forms of periodate-sensitive sialyl residues are still recognized by the m a c r o p h a g e receptor. T o test this possibility, glycophorin A, whose sialyl residues are exclusively 5-N-acetylneuraminic acid, was treated with periodate, and its inhibitory effect on the m a c r o p h a g e recognition was examined. As shown in Table 8, period a t e - t r e a t e d glycophorin A was still an effective inhibitor of m a c r o p h a g e recognition of p e r i o d a t e - t r e a t e d erythrocytes. Successive t r e a t m e n t of the oxidized glyc o p h o r i n A with borohydride, which reduces the aldehyde function, did not destroy the activity. T h e results suggest that oxidized sialyl residues, as well as unoxidized ones, on the p e r i o d a t e - t r e a t e d erythrocytes are

Table 7 Effect of saccharides and glycoproteins on macrophage recognition of periodate-treated erythrocytes Saccharide or glycoprotein Macrophage adhesion (% of control assay) D-Glucose D-Galactose D-Mannose L-Fucose N-Acetyl-D-glucosamine N-Acetyl-D-galactosamine N-Acetylneuraminic acid Glycophorin A a 1"Acid glycoprotein Fetuin Lactoferrin Bovine submaxillary mucin

(mM) 50 50 50 50 50 50 50 (mg/ml) 0.1 0.1 1 0.1 1 0.1 1 0.1 1

105.6+ 3.2 105.9+ 4.0 97.1 + 7.8 107.4_+ 2.2 93.4 + 4.1 102.4 + 0.9 49.5 +_ 2.2 44.3 + 7.7 96.1 + 10.9 99.8+_ 1.9 97.1 5:6.3 96.7+ 5.3 85.5 ± 4.2 84.05:3.8 79.1 5:7.5 91.3+ 8.2

Mouse erythrocytes incubated with NalO 4 (1 mM) at 0°C for 15 min were washed and assayed for macrophage adhesion in the presence of indicated concentrations of saccharides or glycoproteins. In the absence of the inhibitors (control assay), 62.3-73.1% of macrophages bound the modified erythrocytes. Values given are the mean + S.D. of triplicate assays.

Table 8 Effect of periodate treatment of glycophorin A on its inhibitory activity to the macrophage recognition of periodate-treated erythrocytes Glycophorin A (0.1 mg/ml)

Macrophage adhesion (% of control assay)

Control a Oxidized with NaIO4 b Oxidized with NaIO4, then reduced with NaBH 4 c

54.8 _+14.6 19.6+_ 8.6 39.8_+ 7.2

Mouse erythrocytes incubated with NalO 4 (1 mM) at 0°C for 15 min were washed and assayed for macrophage adhesion in the presence of oxidized or oxidized and reduced glycophorin A. In the absence of inhibitors (control assay), 76.2% of macrophages bound the modified erythrocytes. Values given are the mean +S.D. of triplicate assays. a Glycophorin A processed as a control. b Glycophorin A (1 mg/ml) was incubated with NalO4 (2 mM) in buffer B at 0°C for 15 min. The reaction was terminated by adding 0.17 M ethylene glycol, and dialyzed against buffer C. c Glycophorin A (1 mg/ml) was processed as described in b. Before the dialysis, an aliquot of a solution of NaBH 4 in a dilute NaOH solution was added at the final concentration of 50 mM, and then dialyzed against buffer C.

recognized by the m a c r o p h a g e r e c e p t o r responsible for the recognition of the p e r i o d a t e - t r e a t e d cells.

4. D i s c u s s i o n

In the present study, the m e c h a n i s m by which m a c r o p h a g e s recognize SH-oxidized erythrocytes was investigated. PS of erythrocytes is known to reside exclusively on inner surface of the lipid bilayer [34]. This asymmetric distribution of PS is p r o p o s e d to be maintained by aminophospholipid translocase, a specific carrier protein that transports aminophospholipids from outer surface to inner surface of the cell m e m b r a n e [50]. T h e two r e p o r t e d observations that SH oxidation of h u m a n erythrocytes resulted in exposure of PS [20,21], and that PS insertion into the outer surface of h u m a n erythrocytes resulted in m a c r o p h a g e recognition of the cells [22,23] led us to investigate the possibility of PS involvement in the m a c r o p h a g e recognition of SH-oxidized erythrocytes. T h e present results indicate that no detectable PS exposure o c c u r r e d by SH oxidation u n d e r the conditions employed, and involvement of PS is unlikely. Competition studies using sialosaccharides in the m a c r o p h a g e recognition of the SH-oxidized erythrocytes indicated that the erythrocytes were recognized t h r o u g h the r e c e p t o r for sialosaccharides. Main determinants on the ei'ythrocytes recognized are probably located in glycophorin A because: (1) glyeophorin A strongly inhibited the binding o f these cells to macrophages, (2) majority o f sialic acid in mouse erythrocyte m e m b r a n e are expected to be c o n t a i n e d in glycophorin A as is the case for h u m a n cells, and (3)

M. Beppu et al. / Biochimica et Biophysica Acta 1223 (1994) 47-56

removal of surface glycopeptides, possibly from cellsurface region of glycophorin A, by limited typsinization prevented the macrophage recognition of the SHoxidized cells. Macrophage receptors or macrophage lectins for sialoglycoconjugates have been reported in several assay systems [33,44,51-58]. Sialoadhesin (originally named sheep erythrocyte receptor) [54-56], a sialic acid-binding receptor of mouse macrophages, is expressed on stromal tissue macrophages but is poorly or not on peritoneal macrophages [54]. Although in vitro culture of peritoneal macrophages in the presence of homologous serum leads to its expression, it usually takes two days [55]. Thus, it is unlikely that this receptor is expressed on the macrophages used in this study. 'Ganglioside receptor' [33,51-53], found on rat resident peritoneal and alveolar macrophages but not on stromal tissue macrophages, may be related to the receptor of mouse resident peritoneal macrophages for the SH-oxidized erythrocytes because the sugar specificities of these receptors are similar. L-Selectin, an adhesive protein of leukocyte surface having a specificity for sialylated and fucosylated oligosaccharides including sialyl-Lewis x and sialyl-Lewis a [57,58], could be a candidate for the receptor for the SH-oxidized erythrocytes. However, considering its limited specificity, this molecule is less likely to be involved in the recognition of the SH-oxidized erythrocytes in this study. Further characterization of the receptor activity observed here is necessary to clarify its relevance to the reported silosaccharide receptors. Although no detectable PS was present on diamidetreated erythrocytes, PS liposomes inhibited the binding of the erythrocytes to macrophages. Considering net negative charge of PS, it is possible that PS liposomes blocked the receptor for sialosaccharides. Sialosaccharide recognition was also suggested to be involved in the macrophage recognition of periodatetreated erythrocytes. Periodate-oxidized glycophorin A, and periodate-oxidized and borohydride-reduced glycophorin A were active in inhibiting the binding of periodate-treated cells to macrophages. Since sialic acid residues of both of the modified glycophorin A retain carboxyl function, the negative charge of sialic acid residues due to the carboxyl function may be important in the interaction of silosaccharides and the receptor. High sialic acid-content may not be an exclusive requisite for the ligand for the sialosaccharide receptor since sialic acid-rich glycoprotein al-acid glycoprotein was not an effective inhibitor for the receptor. There may be some structural requirements for glycoconjugates to be its ligands. In addition to SH-oxidized erythrocytes, SH-blocked erythrocytes were also found to be recognized by the macrophages. Similarly to SH-oxidized erythrocytes, the recognition of SH-blocked erythrocytes was inhibited by sialosaccharides. The finding suggests that loss

55

of free SH groups by disulfide formation is responsible for the induction of macrophage recognition of SHoxidized erythrocytes. Lines of evidence indicate that SH oxidation of membrane proteins occurs in vivo. For example, disulfide-mediated protein aggregates were found in the membrane of erythrocytes from patients with glucose-6-phosphate dehydrogenase deficiency with chronic hemolytic disease [16], and free SH content of erythrocyte membrane of /3-thalassemia was shown to be lower than that of normal erythrocytes [17]. Band 4.1 protein of sickled erythrocyte membrane contains cysteic acid, an oxidized product of cystein, which is not present in band 4.1 of normal cells [18]. Sickled erythrocytes are known to be susceptible to removal from circulation [59], and it is possible that the mechanism by which macrophages recognize SHoxidized erythrocytes is involved in the removal of sickled cells in vivo. One of the questions arising from the present results is that why macrophages recognize the sialosaccharide chains on SH-oxidized erythrocytes while they do not recognize those on unoxidized cells. Considering that saccharide chains of glycoproteins are likely to be usually exposed on cell surface, one possible explanation is that the membrane glycoproteins may form clusters (or aggregates) when SH groups of the membrane proteins are oxidized, and as a result, they serve high density and thus high affinity saccharide ligands. We have shown that human erythrocytes oxidized with iron catalysts [60] or diamide [61] or modified with NEM [61] were recognized by autoantibody present in normal plasma. The antibody recognized sialosaccharide chains of band 3 glycoprotein [62]. The alterations of the membrane glycoproteins of the SH-modified erythrocytes inducing macrophage recognition are very likely to be common to those inducing the autoantibody binding. Analysis of the alterations of these glycoproteins is important for the elucidation of the mechanism of macrophage recognition of oxidatively damaged cells.

Acknowledgements This work was supported in part by a Grant-in-Aid from the Tokyo Biochemical Research Foundation. We thank M. Fujimaki for assistance in part of this work.

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