Adhesion-mediating molecules of human monocytes

CELLULAR

IMMUNOLOGY

113,278-289

Adhesion-Mediating

(1988)

Molecules of Human Monocytes

MANUEL PATARROYO,* JACQUELINE PRIETO,* PATRICK G. BEATTY,~ EDWARDA. CLARK,$ANDCARLG.GAHMBERG$ *Department of Immunology, Karolinska Institute, Stokholm, Sweden; tFred Hutchinson Cancer Research Center and #Department of Microbiology, University of Washington, Seattle, Washington 98195; $Department ofBiochemistry, University of Helsinki, Helsinki, Finland Received July 7, 1987; acceptedDecember 8, 1987

Adhesionof monocytesto eachother and to T cells and substrates is increased by phorbol esters.In the presence of these compounds monocyte aggregation was almost completely inhibited (>90%) by monoclonal antibody 60.3. This antibody recognizes GP90 (CD1 8), a leukocyte surfaceglycoprotein which is separately and noncovalently associatedto either GP 160(CD1 la), GPl55 (CD1 lb), or GP130 (CD1 lc). Anti-LFA-1 antibody (CD1 la) was only partially inhibitory (35%) while antibodies 60.1 (CD1 1b) and anti-Leu-M5 (CD1 lc) had a minimal inhibitory effect (10%). Antibody LB-2 recognizing a single glycoprotein distinct from the GP90-GP160 complex and expressedon activated B and T cells, monocytes, and vascular endothelial cells was partially inhibitory (22%). Monoclonal antibodies anti-C3bR (CD35), T29/33 (CD45, leukocyte commonantigen 200), TA-I (CD1 la), OKMl (CD1 lb), FlO-44-2 (brain-leukocyte antigen), OKM5 (monocyte-endothelial cell antigen) and to classI or classII molecules exerted no inhibition on the monocyte aggregation. Fab fragments of antibody 60.3 efficiently inhibited not only monocyte aggregation in the absence or presence of phorbol esters but also adhesion of these cells to autologous or allogeneic T lymphocytes and, to a lesser extent, to plastic surfaces. It is thus concluded that GP90, either alone or associatedto the larger glycoproteins, and LB-2 antigen mediate monocyte adhesion. o 1988 Academic PISS, IK.

INTRODUCTION Monocytes and macrophages play a major role in the generation of immune and inflammatory responses.These mononuclear phagocytes are known to interact physically with other cell types such as lymphocytes and vascular endothelial cells. Clusters of macrophages and T lymphocytes are formed in response to soluble antigens (1) and adherence of monocytes to endothelium is required during their egressfrom blood to extravascular tissues(2). Adhesion of mononuclear phagocytes to immature leukocytes and to tumor cells mediates modulation of myeloid colony formation and tumor cell lysis, respectively (3,4), and adherence of monocytes to inert surfacessuch as plastic, a procedure extensively used in purification of these cells, influences their size and surface antigen expression (5). Monocytes aggregatein vitro and the process is enhanced by treatment with interferon-y (6,7). Phorbol esters,such as tetradecanoyl phorbol acetate (TPA) and phorbol dibutyrate (P(Bu)& also induce aggregation of mononuclear phagocytes (8) and modulate, as well as in many other cell types, their function, proliferation, and maturation (9). Similarily, these activators of protein kinase C (10) were found to induce 278 0008-8749/88$3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved

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aggregation of blood lymphocytes in the absence of antigen or serum components ( 1l- 14). Analysis of the intercellular binding indicated an active participation of the cells and the existence of cell surface proteins that mediate adhesion, but not cell stimulation (15). Since some of the adhesion-mediating molecules may modulate the process, only the intercellular adhesive ligands are referred to as cell adhesion (binding) molecules, or CAMS. Monoclonal antibody 60.3 directed against a leukocyte common antigen (16) blocks phorbol ester-induced adhesion, but not stimulation, of purified T and B cells and granulocytes and hence identifies an adhesion-mediating molecule, a putative leukocytic CAM (Leu-CAM) ( 17- 19). In unfractionated blood mononuclear cells, phorbol estlersinduce not only lymphocyte-lymphocyte adhesion but also lymphocyte-monocyte adhesion ( 13) and interactions between these cells result in interleukin-2 (IL-2) production, a phenomenon which is inhibited by antibody 60.3 (20). This antibody recognizes GP90 (CD 18), a leukocyte surface glycoprotein which is separately and noncovalently associated to either GP 160 (TA- 1/LFA- 1, CD 11a), GP155 (OKMl/Mol/Macl, CD1 lb),orGP130(Leu-M5,CDl lc). Thus,GP90constitutes the common /3 chain of three heterodimers (17-22). Monoclonal antibody LB-2 recognizes another adhesion molecule, GP84, which is mainly present on activated B cells and other mononuclear leukocytes (23, 24). Purified monocytes react with antibodies 60.3 and LB-2 and antibodies with the same specificity as antibody 60.3 precipitate the four members of the protein complex from these cells (16, 24, 25). In the present study we have analyzed the effect of monoclonal antibodies to each of these proteins on monocyte adhesion to one another, to a plastic surface, and to T cells. MATERIALS AND METHODS Monoclonal antibodies and other reagents. The antibodies used are described in Table 1. Antibodies to C3bR, Leu-MS, and HLA-DR were obtained from Becton Dickinson (Mechelen, Belgium). Antibodies T29/33 and TA- 1 were purchased from Hybritech ((San Diego, CA); OKMl and OKM5 from Ortho Diagnostic Systems (Raritan, NJ); W6/32 and MAS 072 from Sera-Lab (London, UK). Anti-LFA- 1 (IOT 16) was frolm Serotec (Bicester, Oxon, UK). Monoclonal antibodies 60.3,60.1, FlO44-2, and LB-2 were produced as previously described (16,3 1,33,23). All antibodies were used as purified IgG, except W6/32 and MAS 072 which were used as ascites fluid. The antibodies were dialyzed against water for 24 hr at 4°C lyophilized, and dissolved in phosphate-buffered saline (PBS) to obtain a stock concentration of 400 pg/ml. Fab fragments of antibody 60.3 were prepared by papain digestion (17) and their purity was checked by SDS-PAGE analysis. Their antibody activity and lack of Fc portion were probed by indirect immunofluorescence and reactivity with antimouse whole Ig but not with anti-mouse IgG Fc. 4/3-Phorbol 12,13-dibutyrate (Pi; Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) and stored at -20°C. Pi was added to cells to a final concentration of 60 nM. The final concentration of DMSO was lessthan 0.05% and did not affect cell adhesion. Monocytsesand other leukocytes. Monocytes were isolated from heparinized blood of healthy donors. After Ficoll (Pharmacia, Uppsala, Sweden)-Isopaque (Nyegaard & Co., Norway) separation (38) monocytes were isolated from other mononuclear cells by adherence to tissue culture flasks. The cells were removed from the plastic

280

PATARROYO ET AL. TABLE 1 Characteristics of Monoclonal Antibodies and Their Reactivity with Human Monocytes

Monoclonal antibodies

Murine I& subclass

Anti-C3bR T29/33 TA-1 Anti-LFA- 1 (IOT 16) OKMl

I&b

60.1 Anti-Leu-MS F- 1O-44-2 60.3 OKM5 LB-2 MAS 012 W6/32 Anti-HLA-DR

Idha

Cell surface antigen (CD) C3b receptor (CD35) Leukocyte common antigen (CD45) Lymphocyte/monocyte antigen (CD I 1a) Lymphocyte function associatedantigen- 1 (CD1 la) Monocyte/granulocyte antigen (CD1 1b) Monocyte/granulocyte antigen (CD 11b) Macrophage/monocyte antigen (CD1 lc) Brain-granulocytelymphocyte antigen Leukocyte common antigen (CD 18) Monocyte/platelet antigen Activated B cell antigen Monocyte antigen (CD 14) HLA-A, B, C shared determinant Class I antigen HLA-DR nonpolymorphic Class II antigen

Molecular weight of immunoprecipitate proteinb (Da) 205.000 200.000

% Antigen pos. cells/ intensity’

Reference

94+/+++ 99++/+++

26 21

160.000/90.000

90++

28

160.000/90.000

98++

29

155.000/90.000

95++

30

155.000/90.000

98++

31

130.000/90.000

89+

32

105.000

99+++

33

90.000/ 160.000/ 155.000/130.000 88.000 84.000 55.000 43.000/12.000

99+++

16

88++ 86+ 90++ 99+++

34 23 35 36

34.000/28.000

80++

37

’ The CD names of the antigens are in parentheses. bApparent molecular weight under reducing conditions; boldface numbers designate polypeptides certainly known to contain the epitope. ’ Mean of at least two experiments; labeling intensity: +, weak; ++, intermediate; +++, strong.

surface by 1 hr incubation on ice in PBS- 10 mM EDTA (ethylenediaminetetraacetic acid, Kebo, Stockholm, Sweden). This cell population consisted of more than 90% monocytes according to the presence of the cell surface markers CD14 (monocytespecific) and CD 11b (monocyte-associated) (Table 1) and was homogeneous in morphology (Fig. 2~). A T-cell-enriched population was obtained from the non-plasticadhering cells after passagethrough a nylon-wool column (17). The cells were resuspended in RPM1 1640 medium (Grand Island Biological Co., Grand Island, NY) containing 0.5% human albumin (KabiVitrum, Stockholm, Sweden). Measurement of monocyte adhesion. To measure aggregation of monocytes, cell suspension aliquots of 0.5 ml (10’ cells/ml) in 24-well tissue culture plates (Costar, Cambridge, MA) were rotated in a gyratory shaker (Model G2, New Brunswick, Edison, NJ) at 100 rpm at 37°C. After a 10-min incubation to allow warming of the cells, Pi (60 nM) was added. Twenty minutes later, the cell suspension was briefly

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inspected via an inverted microscope and suspendedby pipeting 10 times. After this, the percentage of aggregatedcells was determined by means of a hemocytometer with a Labour K microscope (Leitz) at 400X. The suspension was also inspected before treatment, and a control sample was incubated without P(Bu)*. A minimum of 5 X lo* cells were counted in each sample. Aggregation was determined indirectly by counting thle number of single cells in a determined area and subtracting this value from the corresponding total number of cells in the presence of 10 mM EDTA, both being read after 20 min. The effect of 13 monoclonal antibodies on monocyte aggregation in presence of phorbol ester (%P(Bu), aggr.) was tested. The cells were incubated with antibody (either IgG or Fab fragments) at 20 wg/ml for 20 min at room temperature and for 10 more min at 37°C before being shaken and treated with phorbol ester. U’nbound antibodies were also present during P(Bu), treatment. The effect of the antibodies (inhibition) was expressedas %P(Bu)* aggr. in control sample - %P(Bu)* aggr. in presence of antibody x loo %P(Bu)* aggr. in control sample Monocyte ,aggregationin the absence or presence of phorbol ester was 45 and 73%, respectively. To measure monocyte adhesion to plastic surfaces, cell suspension aliquots of 0.3 ml ( lo5 cells) in 96-well flat-bottom tissue culture plates (Nunc, Kamstrup, Denmark) were centrifuged (5 min at 400g) in the absence or presence of P(Bu), (60 nM) and then incub,ated at 37°C for 20 min. After pipeting each well 10 times, the percentage of adherent cells was measured indirectly by counting the number of cells present in a determined area of the hemocytometer and substracting this value from the corresponding total number of cells in the presence of 10 mM EDTA. The effect of monoclonal antibodies on monocyte adhesion to plastic in presence of phorbol ester was measured as for monocyte aggregation. Monocyte adhesion to plastic surfaceswas in the absenceand presenceof phorbol ester 57 and 70%, respectively. To measure binding of monocytes to blood lymphocytes, a slight modification of the procedure for monocyte (aggregationwas used. Monocytes were labeled with the vital dye carboxyfluorescein diacetate (CFD; Molecular Probes, Inc, Junction City, OR) as previously described ( 19). After washing, labeled monocytes and unlabeled blood lymphocytes were mixed at 1:10 ratio, respectively, to obtain a cell suspension of approximately 10 X lo6 ml. When required, 20 pg of Fab fragments from antibody 60.3 was added simultaneously to the cell mixture. Incubation with the antibody, shaking, and Pi treatment were as described for cell aggregation. Thereafter, the percentage of monocytes binding lymphocytes was determined in a fluorescence microscope by counting the number of fluorescent cells which bound one or more unlabeled cells. The blood lymphocyte population was obtained from the mononuclear cells nonadherent to plastic after additional passagethrough a nylon-wool column (39) and it contained more than 90% T cells. Indirect immunoj7uorescence and morphological studies. Cells were stained with the antibodies by indirect immunofluorescence by using a fluorescein-conjugated F(ab);! fraction made from rabbit immunoglobulin to mouse immunoglobulins (Dakopatts, Copenhagen, Denmark) asthe second-stepreagent. Incubation with the antibodies was done at 4°C. After staining, the cells were treated with glycerol and observed in ,a Leitz fluorescence microscope (Schott, FRG). Morphological changes were followed by light microscopy at a 320X. Photomicrographs were taken with a Wild micrloscope camera (Heerbrugg, Switzerland), using Pan Plus X Kodak films.

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PATARROYO ET AL. TABLE 2 Effect of Various Monoclonal Antibodies (IgG) to Cell-Surface Antigens on Adhesion of Monocytes to Each Other and To Plastic in the Presenceof Phorbol Ester

Monoclonal antibody”

% Inhibition of monocyte aggregationh

% Inhibition of monocyte adherence to plastic’

60.3 Anti-LFA- 1 (IOT- 16) LB-2 60. I Anti-Leu-MS Anti-C3bR W6/32 Anti-HLA-DR Fl O-44-2 T29/33 TA- 1 OKM5 OKM 1 Anti-LFA- 1 + 60.1 + anti-Leu-MS

91 35 22 10 IO 6 5 0 -I -II -15 -17 -22 71

61 11 15 25 NT 3 -3 -1 12 I NT NT NT 67

’ Twenty pg/ml of IgG; mean of at least four experiments. b Effect of antibody on monocyte aggregation after 20 min of treatment with 60 nkt P(Buh at 37°C and 100 rpm. Monocyte aggregation was 73%. ’ Effect of antibody on monocyte adherence to plastic after 20 min of treatment with 60 nM P(Bu), at 37°C. Monocyte adherence was 70%.

Cell-surface labeling, immunoprecipitation, and polyacrylamide gel electrophoresix Monocytes were surface-labeled with 3H after periodate oxidation (40). Immunoprecipitation was performed as described previously by using rabbit antimouse Ig (Dakopatts) in the second step (41). Polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate was done according to Laemmli (42) using 8% acrylamide gels. The 3H-labeled gels were treated for fluorography according to Bonner and Laskey (43). 14C-labeledstandard proteins were obtained from the Radiochemical Centre (Amersham, UK). RESULTS Eflect of Monoclonal Antibodies (IgG) to Cell-Surface Antigens on Adhesion ofMonocytes To Each Other and To Plastic in the Presenceof Phorbol Ester Thirteen monoclonal antibodies were tested on aggregation of monocytes after 20 min of treatment with P(Bu)z. At this time, 73% of the cells were aggregated. All antibodies reacted with 80% or more of the monocytes as shown in Table 1. Only antibody 60.3 inhibited in more than 90% the stimulated cell aggregates,followed by anti-LFA- 1, LB-2, 60.1, and anti-Leu-MS antibodies which exerted a much less inhibitory effect (Table 2). The other antibodies did not significantly inhibit cell aggregation or, in some cases,enhanced intercellular binding. At the same point, monocyte adhesion to plastic in the presence of phorbol ester was 70%. In comparison to cell aggregation, stimulated adhesion to plastic was less sensitive to inhibition by

283

ADHESION MOLECULES OF MONOCYTES TABLE 3 Effect of Fab Fragments from Antibody 60.3 on Adhesion of Monocytes to Each Other and To Plastic in the Absence or Presenceof Phorbol Ester” W3u)2

Type of adhesion Cell aggregation

Adhesion to plastic

treatment*

Antibody 60.3’

% Adherent cells

Absent Present Absent Present Absent Present Absent Present

45 4 73 5 57 28 70 39

% Inhibition of adhesion 90 97 53 53

’ Mean of at least five experiments. * 60 nM for 20 min at 37°C. c Cells were incubated with 20 pg/ml of Fab fragments.

antibody 610.3(Table 2). Low inhibition was exerted by anti-LFA- 1, LB-2, and 60.1 antibodies. When compared to antibody 60.3 alone, combination of anti-LFA-1, 60.1, and a.nti-Leu-MS antibodies was similarly effective on monocyte adherence to plastic but lessinhibitory on monocyte aggregation (Table 2). Eflect ofFab Fragmentsfrom Antibody60.3 on Adhesion ofMonocytes to Each Other, to a Plastic Surface, and To Blood Lymphocytes in the Absence (Basal) or Presence (Stimulated) of Phorbol Ester Monocytes are known to aggregate and to adhere to plastic surfaces “spontaneously.” Under the present experimental conditions approximately half of the monocytes exerted a basal adhesion to each other (45%) or to plastic surfaces (57%). After phorbol esltertreatment monocyte adhesion increased to 73% and 70%, respectively (Table 3). To avoid any possible artifact introduced by the Fc portion or the ability to crosslink cell-surface antigens, Fab fragments from antibody 60.3 were prepared. The monovalent fragments also inhibited most efficiently monocyte aggregation in the presence of phorbol ester in a dose-dependent manner (Fig. 1). Fifty percent inhibition was obtained with approximately 0.8 pg/ml. Both basal and stimulated monocyte adhesion to each other and to plastic were inhibited by the antibody fragments (Table 3). ,4s observed with the intact antibody, monocyte adhesion to an inert surface was 1e:sssensitive than monocyte aggregation. The Fab fragments also inhibited flattening of the cells on the plastic surface (Fig. 2). Adhesion between purified monocytes and lymphocytes was analyzed by labeling one population and inducing cell conjugation with Pi. Phorbol ester treatment increased conjugate formation from 2 to :29%(Table 4). Basal cell conjugation was minimal but slightly higher in allogeneic ‘combination. After 20 min treatment with P(Bu), , approximately 30% of monocytes, bound autologous or allogeneic lymphocytes, suggesting no major histocompatibility complex restriction in the process. Fab fragments inhibited cell conjugation almost completely (Table 4, Fig. 3).

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PATARROYO ET AL.

mAb

Fab

concentration

(pa/ml)

FIG. 1. Effect of different concentrations of Fab fragments from antibody 60.3 on aggregation of mono-

cytes in presence of phorbol ester. Cell aggregation in absenceof the antibody was 73%.

FIG. 2. Light micrographs ofthe effect of Fab fragments from antibody 60.3 on aggregation ofmonocytes in the absence or presence of phorbol ester. In a and c cells were not treated with P(Bu)*. In b and d cells were treated with 60 nM P(Bu), for 20 min at 37°C. In c and d cells were preincubated with 20 pg/ml of Fab fragments. Cells were photographed in an inverted microscope at 320X. All samples were continuously rotated at 100 rpm. Bar = 20 pm.

285

ADHESION MOLECULES OF MONOCYTES TABLE 4 Effect of Antibody 60.3 on Adhesion of Monocytes to Autologous and Allogeneic Lymphocytes in the Absence or Presenceof Phorbol Ester” Cell donor combination Autologous

Allogeneic

Wuh treatmentb

Antibody 60.3

% Adherent cellsd

Absent Present Absent Present Absent Present Absent Present

2
% Inhibition of adhesion

93

93

a Means of three experiments. ’ 60 t&f at 37°C for 20 min. ’ Cells were incubated with 20 pg/ml of Fab fragments. d Percentage of fluorescent cells binding one or more unlabeled cells.

Immunoprecipitation with Monoclonal Antibodies Monocytes were surface-labeled with 3H after periodate treatment. Immunoprecipitation with antibody 60.3 revealed, under reducing conditions, three major sialylated polypeptides with apparent molecular weights of 90,000, 130,000, and 160,000 (Fig. 4A). The broad GP160 band also contained a sialoglycopolypeptide with an apparent molecular weight of 155,000, which is associated to GP90 and which is precipitated by antibodies OKMl and 60.1 (Figs. 4D and 4K, respectively). Both anti-LFA- 1 (Figs. 4B and 4H) and TA- 1 (Fig. 4C) antibodies precipitated two sialo-

FIG. 3. Light micrographs of the effect of Fab fragments from antibody 60.3 on adhesion of monocytes (indicated by arrows) to autologous blood lymphocytes. In a and c cells were not treated with P(Bu)* In b and d cells were treated with 60 nA4 P(Bu)* for 20 min at 37°C. In c and d cells were preincubated with 20 pg/ml of Fab fragments. The ratio of monocytes to lymphocytes was 1:10. Cells were photographed in a conventional microscope at 250X. To differentiate the two populations, monocytes were prelabeled with fluorescein. All samples were continuously rotated at 100 rpm. Bar = 20 pm.

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FIG. 4. A fluorogram of a polyacrylamide slab gel of immune precipitates obtained from surface-labeled monocytes. A, pattern obtained with antibody 60.3; B, with anti-LFA- 1 antibody; C, with antibody TA- 1; D, with antibody OKM 1; E, with anti-Leu-MS antibody; F, with mouse preimmune serum; G, with antibody 60.3; H, with anti-LFA-1 antibody; I, with anti-Leu-MS antibody; J, with antibody 60.3; K, with antibody 60.1. The lanes A-F were exposed for 1 week; G-I for 1 day; J-K were from a separate experiment. GP160 = glycoprotein with an apparent molecular weight of 160,000,etc.

glycopolypeptides with apparent molecular weights of 160,000 and 90,000, while anti-Leu-MS antibody precipitated sialoglycopeptides with apparent molecular weights of 130,000 and 90,000 (Figs. 4E and 41). Preclearing of the cell lysate with antibody 60.3 resulted in no precipitate by any of the previous antibodies (data not shown). Although antibody LB-2 is known to precipitate a single glycoprotein with an apparent molecular weight of 84,000 from activated B cells (24) immunoprecipitation from monocytes was unsuccessful. DISCUSSION The phorbol ester-induced leukocyte aggregation represents a unique model to analyze leukocyte adhesion since these small compounds bypass early steps and molecules mediating cell recognition and activation (44). Antibodies which identify adhesion-mediating molecules should not inhibit other cellular responsesinduced by phorbol esters such as enhanced mobility of membrane proteins and degranulation ( 17, 18). Since these compounds active protein kinase C they may induce adhesion by changing the conformation of adhesion molecules or associated structures by phosphorylation. Moreover, leukocyte adhesion may be enhanced by increasing surface expression of adhesion molecules, a phenomenon known to occur in monocytes as measured by flow cytometry and antibody 60.3 (45). Half of the monocytes displayed adherence in the absence of phorbol esters. Although stimulation of the cells during the purification procedure cannot be excluded, a large basal adherence has also been observed in monocytes isolated by countercurrent centrifugal elutration (45) suggestingthat these cells have an appreciable intrinsic adhesiveness.Adhesion of monocytes to T lymphocytes appears to be required for proliferation of the latter cells since antibody 60.3, shown to block monocyte/ lymphocyte adhesion, inhibits IL-2 production (20). Binding between mononuclear phagocytes and other cells may constitute a signal or allow interaction between cellsurface ligands and receptors. This physical contact is known to mediate tumor cell lysis (4) killing of intracellular parasites (46) and modulation of bone-marrow cell proliferation (3). As in granulocytes (3 1, 16) antibody 60.3 may well inhibit phagocy-

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tosis of unopsonized large particles and migration in monocytes, functions known to require cell-surface interaction with the particle and the substrate. GP90, either alone or associatedto GP 160, GP 155, or GP 130, mediates leukocyte adhesion I(17- 19, 2 1, 22). Monoclonal antibodies to each glycoprotein and to different epitopes allow an immunological mapping of these structures. Antibody 60.3 reacting with GP90 (CD 18) inhibits most efficiently adhesion in T cells, granulocytes, B cells, and monocytes, while anti-LFA- 1 antibody (GP 160, CD I 1a) inhibits mainly lymphocyte adhesion, has a partial inhibitory effect on monocytes, and has a minimal effect on granulocytes. In contrast, antibody 60.1 (GP155, CD1 lb) blocks almost completely granulocyte adhesion, minimally blocks monocyte adhesion, and has hardly any effect on lymphocyte adhesion. Antibodies TA- 1 and OKM 1 appear to recognize “irrelevant” epitopes on GP 160 and GP 155, respectively, since they do not inhibit adhesion in any type of leukocyte (17-19, 2 1, 22, 47, this paper). Recently, anti-Leu-MS antibody (GP 130, CD1 1c) was reported to inhibit the conjugate formation between certain cytotoxic T-cell clones, which expresslarge amounts of the antigen, and t,argetcells, indicating that the antibody detects an epitope relevant to leukocyte adhesion (48). Thus, GP90 is the structure common to adhesion in all types of leukocytes. The combination of anti-LFA- 1, 60.1, and anti-Leu-MS antibodies was less effective than antibody 60.3 alone in inhibiting the monocyte aggregation. This phenomenon may be due to some passive bridging between cell-surface antigens and Fc receptors mediated by certain subclassesof IgG, since the antibody combination and antibody 60.3 alone were similarly effective on monocyte adherence to plastic. Since GP84 is not upregulated on monocytes after the short treatment with phorbol ester (data not shown), this passive bridging may also explain why antibody LB-2 inhibits monocyte aggregation, but no adherence to plastic, slightly better than antibody 60.1, which labels more intensively the cells. However, a possible specificity of the adhesion molecules,should also be considered. Recently, Mentzer et al. (7) reported an almost complete inhibition of interferony-induced aggregation of human monocytes by anti-LFA-1 antibody TS1/22 (CD1 la) and a smaller inhibitory effect by anti-0 chain antibody TS1/18 (CD1 8). The inversed effect of their antibodies on monocyte adhesion is intriguing and deservesfurther investigation. Previously, intact antibody 60.3 was reported to abrogate monocyte adhesion to endothelial cells and inert surfaces(45). However, direct blocking of adhlesion-mediating molecules could not be concluded since possible mechanisms induced by IgG, such as capping, shedding, or internalization of the antigen, could not be excluded. Thus, the use of monovalent Fab fragments clarifies this issue. In addition to adhesion of the monocyte, adhesion of T and B cells and granulocytes to vascular endothelial cells is inhibited by the Fab fragments (52). Only partial inhibition of monocyte adherence to plastic was observed with both the intact immunoglobulin and Fab fragments from antibody 60.3. This finding suggests that some of the adhesion occurs through a CD 1g-independent mechanism. Moreover., the absence of monocyte spreading on plastic in the presence of the antibody indicates the participation of GP90 and substrate adhesion in this morphological change. Parallel immunoprecipitations with the various antibodies and preclearing studies indicate and confirm the association of GP90 (CD1 8) with either GP160 (CD1 la), GP155 (CD1 lb), or GP130 (CD1 lc) (25, 47, 49). The weak reactivity of antibody LB-2 with monocytes may explain the failure to obtain any immunoprecipi-

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tate. However, lower sensitivity of the molecule to the isotope-labeling may also be involved. This antibody reacts strongly with lectin-activated blood mononuclear cells and Epstein-Barr virus-immortalized normal B cells and precipitates from the latter cells a single glycoprotein, GP84, distinct from the GP90-GP160 complex (24). Although partial, the inhibitory effect of antibody LB-2 on monocyte aggregation in the presence of phorbol ester indicates participation of the corresponding molecule in monocyte adhesion. Participation of GP90 in monocyte adhesion is further indicated by analysis of cells from patients with a congenital leukocyte adhesion deficiency. Monocyte adhesiondependent functions such as lectin-induced lymphocyte proliferation and monocyte adherence to plastic are defective in these patients, and antibodies 60.3, OKM 1, and anti-LFA-1 fail to react with their leukocytes, including monocytes (45, 50). Moreover, the primary defect appears to reside in GP90 (5 1). Interestingly, lymphocytes from these patients can be bound by normal monocytes (data not shown). Since this processis also inhibited by antibody 60.3 the surface expression of CD 18 on only one leukocyte population is enough for the intercellular adhesion. Abnormal adhesion patterns of mononuclear leukocytes may contribute to vascular and inflammatory disorders as well as to malignancies of these cells. ACKNOWLEDGMENTS We thank Dr. H. Wigzell for revising the manuscript and Dr. J. W. Fabre for providing monoclonal antibody FIO-44-2, as well as Ms. I. Lindfors for excellent assistancein the typing of this manuscript. This project was supported by the Karolinska Institute, the Swedish Cancer Society, the Academy of Finland, and the Sigrid Juselius Stiftelse.

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