Induction of aggregation in porcine lymphoid

cells by antibodies to CD46

Rosario Bullidoa, Jose PeÂrez de la Lastrab,1, Fernando AlmazaÂnc,2,Angel Ezquerraa, Diego Llanesb, Fernando Alonsoa,

Javier DomõÂngueza,*

aCentro de InvestigacioÂn en Sanidad Animal INIA, Valdeolmos, 28130, Madrid, SpainbDpto. de GeneÂtica. Facultad de Veterinaria, Universidad de CoÂrdoba, Cordoba, Spain

cCentro de BiologõÂa Molecular Severo Ochoa, Universidad AutoÂnoma de Madrid, Cantoblanco, Madrid, Spain

Received 17 May 1999; received in revised form 21 October 1999; accepted 21 October 1999

Abstract

CD46 is a major transmembrane glycoprotein that belongs to the regulator of complement

activation (RCA) family. Recently, mAbs to human CD46 were shown to suppress IL-12

production. Here, we describe that mAbs against different porcine CD46 epitopes induced a marked

adhesion of normal lymphocytes. Addition of low amounts of antibody to freshly isolated

lymphocytes or thymocytes resulted in the clustering of the cells. Cross-linking of CD46 molecules

seems essential since Fab fragments failed to induce aggregation. This aggregation was dependent

on active cell metabolism and on the presence of divalent cations and required a functional

cytoskeleton. It was not inhibited by antibodies to CD18, CD29, CD2, CD11a and CD11b.

Staurosporine, an inhibitor of protein kinases, partially blocked the aggregation. This finding is

indicative of a role of protein kinases in the transduction of the signal generated by CD46

engagement. # 2000 Elsevier Science B.V. All rights reserved.

Keywords: Aggregation; CD46; Membrane cofactor protein (MCP); Monoclonal antibody; Pig

Veterinary Immunology and Immunopathology

73 (2000) 73±81

* Corresponding author. Tel.: �34-91-620-2300; fax: �34-91-6202247.

E-mail address: juncal@inia.es (J. DomõÂnguez).1 Present address: Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff,

Wales, UK.2 Present address: Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

0165-2427/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.

PII: S 0 1 6 5 - 2 4 2 7 ( 9 9 ) 0 0 1 5 4 - 3

1. Introduction

CD46 or membrane cofactor protein (MCP) is a type I membrane glycoprotein which

serves as an inhibitor of complement activation on host cells. It binds to membrane-bound

C3b and C4b and allows C3b and 4b to be degraded by factor I, a plasma serine protease,

protecting host cells from self-damage by spontaneous complement activation. On SDS-

PAGE, CD46 appears as a broad heterogeneous doublet with an upper band of 59±68 kDa

and a lower band of 51±58 kDa. This structural heterogeneity is the result of the existence

of different isoforms that arise by alternative splicing of a single gene, although post-

translational modifications, especially glycosylation, also contribute to it (Lublin and

Atkinson, 1989). CD46 is widely distributed, being found on cells of epithelial,

endothelial and mesenchymal origin. All human peripheral blood cells, except

erythrocytes, express CD46; however, it has also been identified on certain primate

erythrocytes (Seya et al., 1988; McNearney et al., 1989, Nickells and Atkinson, 1990).

Apart from its cofactor activity for factor I and that serves as a cellular receptor for

measles virus and Streptococcus pyogenes (Naniche et al., 1993; Okada et al., 1995) , not

much is known about the functions of CD46. Other complement regulatory proteins,

which share some structural features with CD46, such as CR2 or CD59, have been

involved in signal transduction inside the cell. Most recently, Karp et al. (1996) have

shown that cross-linking of CD46 with mAbs led to a marked suppression of IL-12

production by monocytes. Recently, the pig analogue of CD46 has been characterized by

using a panel of mAbs (van den Berg et al., 1997; PeÂrez de la Lastra et al., 1999). Herein,

we show that engagement of CD46 by these anti-CD46 mAbs triggered cell adhesion by a

protein kinase-dependent pathway. This CD46-induced aggregation is temperature- and

energy-dependent and requires for its occurrence a functional cytoskeleton and the

presence of divalent cations.

2. Material and methods

2.1. Cells and reagents

Peripheral blood mononuclear cells (PBMC) were obtained from the EDTA-treated

venous blood of normal pigs by dextran sedimentation, followed by centrifugation on

Percoll gradient. Granulocytes were recovered from the bottom after lysis of residual

erythrocytes by hypotonic treatment (Bullido et al., 1996). Thymus cell suspensions were

prepared by teasing the tissue through a stainless steel sieve. Cells were resuspended at a

density of 106/ml in RPMI 1640 medium containing 10% fetal calf serum (FCS), 2 mML-

glutamine, 5 � 10ÿ5 M 2-mercaptoethanol and 30 mg/ml gentamicin (complete medium).

Staurosporine, genistein, cytochalasin B and sodium azide were from Sigma (St. Louis,

MO). EDTA and EGTA were from Merck (Germany).

2.2. Monoclonal antibodies

The anti-CD46 mAbs 1C5 (IgG2a), 2C11 (IgG1) and 6D8/8 (IgG1) were derived from

two independent fusions of X63-Ag.8.653 myeloma cells with spleen cells from Balb/c

74 R. Bullido et al. / Veterinary Immunology and Immunopathology 73 (2000) 73±81

mice immunized with PBMC and with 2 day-Con A blasts, respectively, using standard

procedures (Kohler and Milstein, 1975). The anti-CD46 mAb 4C8 has already been

described (van den Berg et al., 1997).

The obtention and characterization of mAbs BL1H8 (anti-CD11a) and BL3F1

(CD11b) will be described elsewhere. MAbs TS2/16 (anti-CD29) and Lia 3/2 (anti-

CD18) were kindly provided by Dr. SaÂnchez-Madrid (Universidad AutoÂnoma de Madrid).

Anti-porcine CD2 (MSA4) (Hammerberg and Schurig, 1986) was kindly provided by Dr.

J. Lunney (Beltsville, USDA).

Fab fragments were prepared by papain digestion (Goding, 1983) and purified by

passage over protein A-Sepharose to remove undigested IgG. The purity of the Fab

fragments was assessed by SDS-PAGE, followed by staining with Coomassie blue.

2.3. Flow cytofluorometry

Cells (5 � 105) were incubated on ice with 50 ml of hybridoma supernatants for

30 min. After two washes in PBS containing 0.1% BSA and 0.1% sodium azide

(fluorescence buffer, FB), cells were incubated for 30 min at 48C with 50 ml of FITC-

conjugated rabbit F(ab0)2 anti-mouse Ig (Dako, Denmark) diluted 1/40 in FB. Cells were

washed thrice in FB and fixed in 0.3% paraformaldehyde prior to analysis in an

FACSCAN (Becton Dickinson, USA).

2.4. Aggregation assays

Cells (3 � 105/well) were incubated in flat-bottom 96-well microtiter plates (Nunc,

Denmark). MAb and inhibitors were added in duplicate, in a final volume of 200 ml of the

complete medium, and plates incubated at 378C in a 5% CO2 atmosphere. Aggregation

was then determined at different periods of time by direct visualization of the plate with

an inverted microscope and scored on a semi-quantitative scale from 0 to 5�: 0, no

aggregation; 1�, 10±20 cells per aggregate; 2� , 20±40 cells per aggregate; 3�, 40±60

cells per aggregate, 4�, 60±80 cells per aggregate; 5�, >80 cells per aggregate.

Evaluation of aggregation was done by two independent observers on at least five

randomly chosen fields. All mAbs and inhibitors were tested at least three times.

3. Results

3.1. Anti-CD46 mAbs induce aggregation

In a routine screening on the effect of a panel of mAbs on the proliferative response of

PBMC to mitogens, we found that three mAbs (6D8/8, 1C5 and 2C11) induced cell

aggregation. These Mabs reacted with a 50±60 kDa molecule broadly expressed on

leukocytes, erythrocytes, platelets (Fig. 1), and PK-15 and 38 A1D cell lines, which has

recently been identified as the pig analogue of CD46 (PeÂrez de la Lastra et al., 1999).

We next tested the ability of these mAbs to aggregate other cell types, such as

granulocytes or thymocytes. Like PBMC, both cell types were aggregated; however, the

aggregation was most intense and could be detected earlier in thymocytes. Thereafter,

R. Bullido et al. / Veterinary Immunology and Immunopathology 73 (2000) 73±81 75

most of the experiments were carried out with thymocytes. Aggregation was detected

after 4±5 h, reached a plateau after 20 h and persisted 72 h later. No aggregation was

observed with an isotype matched control antibody to porcine CD45. The aggregation

could be induced by very low concentrations of these mAbs, 40±60% of cells were

aggregated at a concentration of 15 ng/ml. MAb 4C8 that binds to a different epitope of

CD46 (PeÂrez de la Lastra et al., 1999), was also able to induce aggregation.

3.2. Physiological requirements of anti-CD46 induced aggregation

The requirements for cell aggregation triggered by CD46 mAbs are summarized in

Table 1. The process was temperature-dependent and required both, an active metabolism

and cytoskeleton integrity. No aggregation occurred at 48C, but cells rapidly aggregated

within 3±5 h, upon warming of the cultures at 378C. Pretreatment of cells with

cytochalasin B or sodium azide completely blocked the aggregation (Fig. 2). These

Fig. 1. Flow cytometric analysis of porcine peripheral blood lymphocytes (PBMC), granulocytes (PMN),

alveolar macrophages, platelets and erythrocytes stained by mAb 6D8/8. The open histogram corresponds to the

negative control staining using an irrelevant mAb of IgG1 isotype. One representative experiment out of three is

shown.

Table 1

Physiological requirements for anti-CD46 induced cell aggregationa

Condition Aggregation

Medium (control) 4�EDTA 5 mM 0

EGTA 5 mM 0

Azide 0.2% 0

Cytochalasin B (10 mg/ml) 0

48C 0

48C, then 378C 4�PBS 1±2�PBS� MgCl2 4�

a Thymocytes were plated at 3 � 105/well and treated with the different inhibitors for 15 min prior to

addition of mAb 2C11 (20 ml of hybridoma supernatant per well, final dilution 1/10). Sodium azide was used at

0.2%. MgCl2 was at 10 mM. Aggregation was determined 20 h after the addition of the anti-CD46 mAb and

scored on a semi-quantitative scale from 0 to 5 �: 0, no aggregation; 1�, 10±20 cells per aggregate; 2� , 20±40

cells per aggregate; 3�, 40±60 cells per aggregate, 4�, 60±80 cells per aggregate; 5�, >80 cells per aggregate.

76 R. Bullido et al. / Veterinary Immunology and Immunopathology 73 (2000) 73±81

results indicated that cell aggregation was an active process and not an antibody-mediated

agglutination. It was dependent on divalent cations; when the cells were resuspended in

PBS instead of complete medium no aggregation or a faint aggregation was observed, but

it was fully restored by addition of Mg2�. Pretreatment of cells with divalent cation

chelators EDTA or EGTA also abrogated the aggregation. Fab fragments, generated from

mAb 4C8 by papain digestion, were unable to induce aggregation, even at 15 mg /ml, a

concentration >1000 times the necessary for aggregation with the intact immunoglobulin

(Fig. 3). However, the aggregation effect could be recovered by adding a polyclonal

rabbit anti-mouse Ig serum to Fab fragments. Finally, we investigated the effect of two

protein kinase inhibitors, genistein and staurosporine, on the aggregation induced by

CD46 mAbs. Genistein, which selectively inhibits tyrosine kinases, had no effect on

thymocyte aggregation induced by anti-CD46 mAbs, whereas staurosporine, which

inhibits several classes of protein kinases Ð particularly protein kinase C Ð caused a

Fig. 2. Effect of metabolic inhibitors on CD46-induced aggregation. Thymocytes were incubated with A,

medium; B, staurosporine (0.5 mM); C, EDTA (5 mM); and D, sodium azide (0.2%), prior to the addition of

mAb 2C11. Photographs were taken at 20 h (Original magnification, 200 �).

R. Bullido et al. / Veterinary Immunology and Immunopathology 73 (2000) 73±81 77

drastic though not total inhibition of thymocyte aggregation and a complete inhibition of

PBL aggregation (Table 2, Fig. 2). These results are indicative of a role of protein kinases

in the transduction of the signal generated by CD46 engagement.

3.3. Inhibition of CD46-induced aggregation with mAbs

In order to investigate the potential contribution of different adhesion molecules in

mediating the CD46-induced aggregation, we carried out inhibition studies with a panel

Fig. 3. Requirement of divalent antibodies for CD46-induced aggregation. Thymocytes were cultured for 20 h

in the presence of different amounts of anti-CD46 mAb 4C8 (^), F(ab) fragments of 4C8 (*), F(ab) fragments

of 4C8 plus rabbit antibodies to mouse Ig (5 mg/ml) (~), and rabbit antibodies to mouse Ig alone (&).

Aggregation was quantified as described in Section 2. One representative experiment out of two is shown.

Table 2

Effect of inhibition of intracellular signalling pathways on CD46-mediated cell aggregationa

Staurosporine Genistein

� ÿ � ÿThymocytes 2� 4� 4� 4�PBL 0 2� NDb NDb

a Cells were treated, or not, with 0.5 mM staurosporine or 10 mM genistein immediately prior to addition of

anti-CD46 mAb 4C8 (1.5 mg/ml). Aggregation was quantified 20 h later as described in Section 2.b Not done.

Table 3

Effect of different mAbs on the lymphocyte aggregation induced by anti-CD46 mAbsa

Inhibitory mAb Stimulatory mAb Aggregation

Anti-CD11a (BL1H8) 2C11 3±4 �Anti-CD18 (LIA3/2) 2C11 3 �Anti-CD29 (TS2/16) 2C11 3±4 �Anti-CD2 (MSA4) 2C11 3 �Anti-CD11b (BL3F1) 2C11 3 �Anti-CD45 (2A5) 2C11 3±4 �Control medium 2C11 3±4 �

a Thymocytes were pretreated for 15 min with several mAbs (20 ml of hybridoma supernatant), and then, cell

aggregation was induced by adding mAb 2C11 (20 ml of hybridoma supernatant). Similar results were obtained

with anti-CD46 mAbs 6D8 and 4C8. Aggregation was quantified 20 h later as described in Section 2.

78 R. Bullido et al. / Veterinary Immunology and Immunopathology 73 (2000) 73±81

of mAbs to CD2, CD11a, CD11b, CD18 and CD29. Cells were pretreated for 15 min with

the inhibitory mAb, before the addition of the anti-CD46 mAb. None of the mAbs tested

was able to block the aggregation process (Table 3).

4. Discussion

In this report, we describe the induction of homotypic cell aggregation by mAb to

CD46. This effect was observed with all the anti-CD46 mAbs tested, recognizing at least

three different epitopes and belonging to different IgG subclasses (PeÂrez de la Lastra et

al., 1999). This aggregation requires bivalent antibodies and involves an active process, as

it does not occur at 48C or after treatment of cells with metabolic inhibitors such as

sodium azide. Moreover, an intact cytoskeleton is required as demonstrated by the

inhibitory effect of cytochalasin B.

Antibodies to a large variety of cell surface molecules, including the TCR/CD3

complex (Dustin and Springer, 1989), CD2 (van Kooyk et al., 1989), sIg (Dang and Rock,

1991), CD14 (Lauener et al., 1990), CD18 (Keizer et al., 1988), CD19 (Smith et al.,

1991), CD20 (Kansas and Tedder, 1991), CD29 (Campanero et al., 1992), CD39, CD40

(Kansas and Tedder, 1991), CD43 (Cyster and Williams, 1992), CD44 (Koopman et al.,

1990), CD49 (Campanero et al., 1992; Wuthrich, 1992) and MHC class II antigens

(Mourad et al., 1990) have been found to induce leukocyte aggregation. For many of

these molecules, a signalling function has been demonstrated. Similarly, CD46 might act

as a signalling molecule that leads to cell activation and, hence, not being directly

involved in the adhesion process. In this regard, the inhibition found when cells were

treated with staurosporine points to a role of the protein kinases in the aggregation

phenomena, probably by participating in a signalling process. Although this inhibition

was only partial, and the assay used for assessment of aggregation does not allow an

accurate quantification, it was clear and reproducible. It is interesting to note that the

different isoforms of CD46 have been found to be associated with two different

cytoplasmic tails, whose roles in intracellular signalling are at present unknown

(Liszewski et al., 1991). On the other hand, anti-CD46 mAbs may cross-link cells

overcoming the inherent repulsion between them and facilitating interactions between

other cell surface molecules which lead to aggregation, as has been proposed for anti-

CD43 antibodies (Cyster and Williams, 1992).

In most cases, aggregation triggered by signal-transducing molecules have been shown

to involve b1 and b2 integrins. In our studies, it was not possible to identify which

molecules are mediating the aggregation. The failure of available mAbs to abrogate cell

aggregation may depend on the epitopes recognized by these mAbs being different from

those mediating the clustering, although the anti-CD2 and anti-CD29 mAbs used in this

study were able to block rosette formation by T cells and the aggregation of human B

lymphoblastoid cells induced by anti-VLA-a4 mAbs, respectively (Hammerberg and

Schurig, 1986; Campanero et al., 1992). Another possible explanation is that several of

these molecules are involved and that blocking one of them each time is not sufficient to

reduce aggregation. However, no inhibition was either observed when cells were pre-

treated with different combinations of these mAbs (data not shown). A third possibility is

that different molecules from those tested mediate the CD46-induced aggregation.

R. Bullido et al. / Veterinary Immunology and Immunopathology 73 (2000) 73±81 79

The physiological meaning for CD46-induced aggregation is at present unknown.

However, it is possible to speculate that it may contribute to the recruitment of leukocytes

at sites of inflammation. In this regard, it would be interesting to test whether interaction

of CD46 with C3b or C4b fragments bound to the surface of leukocytes as a result of

complement activation leads to aggregation of these cells

Acknowledgements

This work was supported by grants BIO97/402-C02 and 95-0298-OP.

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