Monoclonal antibody 2F411 recognizes the α chain of a porcine β2 integrin involved in adhesion and complement mediated phagocytosis

JOURNAL OF IMMUNOLOGICAL METHODS ELSEVIER

Journal of Immunological Methods 195 ( 1996) 12% 134

Monoclonal antibody 2F4/11 recognizes the ct! chain of a porcine & integrin involved in adhesion and complement mediated phagocytosis R. Bullido, F. Alonso, M. G6mez de1 Moral, A. Ezquerra, B. Alvarez, E. Ortuf~o, J. Dominguez * Cenrro de Inuestigaci6n en Sanidad Animal. INIA, Valdeolmos 28130, Madrid. Spain Received 30 October 1995; revised 21 December 1995; accepted 3 May 1996

Abstract The characterization of a new mAb, named 2F4/11, specific for porcine myelomonocytic cells is described. This mAb immunoprecipitates a non-covalently linked heterodimer of 1.55000/95 000, which is expressed by granulocytes, monocytes and tissue macrophages but not by lymphocytes, erythrocytes or platelets. Immunoblot analysis localizes the 2F4/11 epitope on the largest subunit of the heterodimer. Mab 2F4/11 is able to block phagocytosis of complement-opsonized zymosan particles by PMN granulocytes and alveolar macrophages, as well as adherence to plastic surfaces of PMA-activated PMN. Together, these results suggest that mAb 2F4/11 recognizes the CD1 lb or (Y chain of the porcine complement type 3

receptor (CR3). Keywords: Monoclonal

antibody:

Granulocyte;

Monocyte;

Macrophage:

1. Introduction Few monoclonal antibodies (mAb) are currently available for the characterization of porcine myelomonocytic cells (Lunney, 1993; Haverson et al., 1994). At present, only three clusters of differentiation (CD14, CD16 and SWC3) have been defined for porcine myelomonocytic cells, represented respectively by mAbs against human CD14 that crossreact with pig (Jacobsen et al., 1993; ZieglerHeitbrock et al., 19941, mAb G7 (CD161 (Halloran et al., 1994) and mAb 74-22-15 (SWC3) (Lunney, 1993). Recently, Haverson et al. (1994) have re-

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author. Fax: (34) 1 6202247.

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CD1 lb; Mac-i;

Integrin: (Pig)

ported the characterization of four mAbs against porcine myeloid cells, but the lack of molecular data on the antigens recognized by these mAbs precludes a definitive classification. Mac-l or complement type 3 receptor (CR3), a member of the & integrin family, has been considered to be one of the most reliable markers for myeloid cells. This antigen is present on blood monocytes, macrophages and granulocytes, NK cells being the only non-myeloid cells expressing it. The Mac-l molecule is composed of two non-covalently associated polypeptides, an CY subunit (CD1 lb) of IV& 165 000 and a p subunit (CD18) of IV, 9.5 000 that is shared with the other members of the family, LFA-1 (CD1 la) and LeuM5 (CD 1 lc) (SBnchezMadrid et al., 1983; Larson and Springer, 1990), and

0 1996 Elsevier Science B.V. All rights reserved.

qi k-h>a novel member that has recently been reported in dogs (Danilenko et al.. 1995). CDl I b/CD1 8 serves as a multifunctional receptor for several different ligands. It binds to a fragment of the third component of complement (iC3b) and mediates adherence and phagocytosis of iC3b-coated particles by granulocytes and monocytes (Belier et al., 1982; Wright et al., 1983; Arnaout et al., 1983). It also mediates some interactions of myeloid cells, being involved in phorbol ester-induced PMN homotypic adhesion and chemotaxis (Anderson et al., 1986), and contributes to PMN and monocyte adherence to endothelium and subsequent PMN extravasation to the sites of inflammation (Arnaout et al.. 1988). Interaction of CD 11b/CD 18 with coagulation factors, fibrinogen and factor X, and with ICAM-I has also been reported (Wright et al., 1988; Altieri et al., 1988; Lo et al., 1989; Diamond and Springer, 1993). Here we describe the generation and characterization of a mAb. named 2F4/ 1 1. which is specific for the (Y chain of a porcine & integrin selectively expressed on myelomonocytic cells. This mAb acts as a potent inhibitor of the phagocytosis of opsonized particles and adherence of activated PMN to plastic, two functions in which CD1 1b/CD18 plays a major role.

2. Materials

and methods

2.1. Animals

and cells

thawed 24 h before assays, rapidly washed in RPM1 1640 medium containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 PM 2-mercaptoethanol and 30 pg/ml gentamicin (complete medium) and distributed in a 6-well plate at 5 X lo6 cells/well. After being incubated for 6 h at 37°C non-adherent cells were removed and macrophages were detached from the plastic by washing with Hanks’ buffer containing 10 mM EDTA. 2.2. Mvnvclvnal

antibody production

MAb 2F4/11 was derived from a fusion of X63Ag.8.653 myeloma cells with spleen cells from BALB/c mice immunized with porcine alveolar macrophages using standard procedures (Kohler and Milstein, 1975). Supernatants were tested for activity against porcine alveolar macrophages and PBMC by flow cytofluorometry (FAcs). Cells from positive wells were cloned at least twice by limiting dilution. Class and subclass of mAbs were determined by ELISA, using rabbit antisera specific for mouse heavy and light chains and a peroxidase-conjugated goat anti-rabbit Ig (Bio-Rad. USA). MAb 2F4/11 was of IgG I isotype. MAb LIA3. an anti-human CD 18 that crossreacts with porcine CDl8, was kindly provided by Dr. F. Sanchez-Madrid (Universidad Autonoma de Madrid. Spain). 2.3. Irnmunoprecipitatiorl

Large White pigs weighing between 30 and 40 kg were used as blood and tissue donors. Peripheral blood mononuclear cells (PBMC) were isolated on Percoll discontinuous gradients, after blood sedimentation in dextran, as has been previously described (Gonzalez et al.. 1990). Granulocytes were recovered from the lower Percoll phase. Residual erythrocytes were lysed by hypotonic treatment. Platelets were obtained from the platelet rich plasma fraction resulting from the centrifugation of 50 ml of normal swine blood for 30 min at 350 X g. Alveolar macrophages were collected by bronchoalveolar lavage as described by Carrascosa et al. (1982), washed in Hanks’ buffer containing 2 mM EDTA and frozen in liquid nitrogen until use. Alveolar macrophages were

analysis

Alveolar macrophages (5 X 107) were washed three times in phosphate buffered saline (PBS) and resuspended in 5 ml of PBS. Sulfo-NHS-biotin (Pierce, USA) (0.4 mg/ml, final concentration) was added to the cells and incubated for 15 min at 4°C. After washing three times with PBS, cells were lysed with 1 ml of a lysis buffer consisting of 10 mM Tris-HCI, 150 mM NaCl, 1 mM EDTA. 1% NP40. pH 7.4. I mg/ml bovine serum albumin (BSA) and 1 mM phenylmethylsulphonylfluoride. The lysate was precleared twice with 50 ~1 of a 25% (v/v) suspension of protein G-Sepharose (Pharmacia, Sweden) in lysis buffer, and then incubated with the different mAbs. 0.3 ml of hybridoma supernatant was added to 0.1 ml of lysate and incubated for 2 h at room temperature. Then, 40 ~1 of 25% (v/v)

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suspension of protein G-Sepharose were added and incubated for 1 h with gentle mixing. Beads were washed three times with lysis buffer, boiled in electrophoresis sample buffer (0.062 M Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.7 M 2-mercaptoethanol, 0.00 1% bromophenol blue) and the supematants run on a 7.5% SDS-PAGE and transferred to nitrocellulose. Filters were incubated with streptavidinperoxidase (Pierce, USA) and the bands visualized with the ECL detection assay following the recommendations of the manufacturer (Amersham, UK).

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on nitrocellulose were blocked with PBS-2% powdered milk. Thereafter, strips were incubated with hybridoma supematants for 1 h at room temperature, followed by a 1 h incubation with a peroxidaselabelled rabbit anti-mouse Ig (Dako, Denmark). Peroxidase activity was visualized using the ECL detection assay (Amersham, UK). In some experiments, alveolar macrophage lysates were subjected to immunoprecipitation with mAb LIA3. Bound proteins were resolved by 7.5% SDSPAGE and analysed by immunoblotting with mAb 2F4/ 11 as described above.

2.4. Western blotting 2.5. Flow cytojluorometry Alveolar macrophages (5 X 107) were washed twice with PBS and solubilized in 0.5 ml of lysis buffer for 1 h at 4°C. After centrifugation at 12 000 X g for 30 min, the supematant was mixed with the sample buffer and run on a 7.5% SDS-PAGE gel under reducing conditions. The separated proteins were transferred to nitrocellulose. Free binding sites

Cells (5 X 105) were incubated on ice with hybridoma supematant for 45 min. After two washes in PBS containing 0.1% BSA and 0.1% sodium azide (fluorescence buffer, FBI, cells were incubated for 45 min at 4°C with FITC-conjugated rabbit F(ab’>, anti-mouse Ig (Dako, Denmark) diluted l/50 in FB.

A MO

7n;

RBC

n7

Platelets

B

0

Fluorescence

intensity

FSC Fig. 1. A: flow cytometric analysis of porcine peripheral blood mononuclear cells (MO), erythrocytes (RBC), and platelets stained by mAb 2F4/11. The grey profile conjugate alone. B: comparison of the reactivity of mAb 2F4/11 with peripheral according their forward (FSC) and side scatter (SSC) profiles. Representative results

(PBMC), granulocytes (PMN), alveolar macrophages corresponds to background staining with fluorescent blood lymphocytes (Rl) and monocytes (R2), gated of three independent experiments are shown.

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Fig. 2. Cryostat sections of porcine intimately associated with cortical zone (arrows) in the spleen with paracortex of a mesenteric lymph lymphoid sheath.

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tissues stained with mAb 2F4/ I 1 by the immunoperoxidase method. A: positive cells in thymic cortex thymocytes (magnification X 250). B: thymic medulla (magnification X 2.50). C: staining of marginal few scattered cells stained in the red pulp (RP) (magnification X 100). D: large positive cells in the node (magnification X250). Sections were counterstained with methylene blue. PALS, periarteriolar

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Cells were washed three times in FB and fixed in 0.3% parafotmaldehyde prior to analysis in a FACSCAN (Becton Dickinson, USA). 2.6. Immunohistochemical

studies

Porcine tissues were snap frozen in isopentane/liquid nitrogen and stored at -70°C. 6 pm cryosections were fixed in cold acetone for 10 min and then washed with PBS. The sections were stained by an indirect immunoperoxidase technique as previously described (Minguez et al., 1988), and counterstained with methylene blue. 2.7. Phagocytosis

assays

Zymosan particles were incubated in fresh normal pig serum for 1 h at 37°C then washed extensively, and resuspended in complete medium. Freshly isolated granulocytes or alveolar macrophages were incubated with mAb preparations for 30 min at 4°C and thereafter exposed to opsonized zymosan particles for 1 h at 37°C. Cells were then fixed and stained with toluidine blue and examined with a light microscope (Leitz Ortolux, Germany). The results were expressed as a percentage of cells containing two or more particles for granulocytes or five or more for alveolar macrophages. Hybridoma supernatants were used as the source of mAbs. Phagocytosis by granulocytes was also assessed by monitoring the changes in side scatter (SSC) and forward scatter (FSC) parameters of these cells after ingestion of zymosan particles in a flow cytometer.

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10% Giemsa solution for 10 min, plates were washed in tap water, dried, and the retained dye solubilized in methanol. Stain was quantified by measuring absorbance at 460 nm in an automatic plate reader (Dynatech Lab, USA).

3. Results 3.1. Cell and tissue distribution The hybridoma secreting mAb 2F4/ 11 was originally selected for its strong reactivity with alveolar macrophages, which were the cells used as immunogen. Further FACS analyses showed that the antigen recognized by mAb 2F4/ 11 was restricted to myeloid cells, while there was no reactivity on lymphocytes, platelets and erythrocytes (Fig. 1). Immunohistochemical analyses of lymphoid organs confirmed the restricted reactivity of this mAb with monocyte/macrophages, but not with lymphocytes (Fig. 2). In the spleen, 2F4/11 labelled large cells of irregular shape located in the outer zone of the periarteriolar lymphoid sheath (PALS) and in the

a

b

c

a 205-

204s 121_82-O-

m

121x

2.8. Adhesion

assays

Adhesion assays were performed as described by Rosen and Gordon (1987) with some modifications. Briefly, freshly isolated porcine granulocytes were suspended in complete medium and plated at a density of 5 X lo5 cells/well in 96-well tissue culturetreated plastic plates (Nunc, Denmark). After preincubation at 4°C for 30 min with hybridoma supernatants, cells were stimulated with phorbol myristate acetate (PMA) (Sigma Chemical Co., USA) (final concentration 10 ng/ml) for 1 h at 37°C. Then, plates were washed three times in PBS and adherent cells were fixed with methanol. After staining with

86Fig. 3. Molecular characterization of the antigen recognized by mAb 2F4/11. A: analysis by immunoprecipitation. Alveolar macrophages were surface labelled with biotin, solubilized in detergent and immunoprecipitated with antibodies and protein G-Sepharose. Immunoprecipitates were subjected to SDS-7.5% PAGE under reducing conditions followed by blotting with streptavidin-peroxidase. Lane a, 2F4/11; lane b. LIA3 (antiCD18); lane c, irrelevant control mAb. B: analysis by immunoblotting. A lysate from alveolar macrophages was subjected to SDS-7.5% PAGE under reducing conditions, transferred to a nitrocellulose membrane and immunochemically stained with mAb 2F4/11 followed by rabbit anti-mouse Ig peroxidase and ECL. The position of the molecular weight markers is given on the left side of each panel.

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marginal zone with a few cells scattered throughout the red pulp. In lymph nodes positive cells were located predominantly within the subcapsular sinus and medullar cords and some in the interfollicular areas of paracortex. In the thymus positive cells were observed predominantly in the medulla. However, a few positive cells that appeared to be associated with the neighbouring thymocytes were also found in the cortex. In the gut a few rounded positive cells were scattered in the lamina propria. Neither Kupffer cells in the liver nor mesangial or interstitial macrophages in the kidney were stained (data not shown). 3.2. Biochemical

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kDa, both under reducing and non-reducing conditions. These comigrated on the same gel with the lowest bands identified by LIA3, a cross-reactive anti-human p2 mAb (Fig. 3A). On immunoblotting, the 2F4/ 11 epitope was located on the 155 kDa band (Fig. 3B). The identity of these molecules was further confirmed by the ability of mAb 2F4/11 to recognize the band of 155 kDa on immunoblots of immunoprecipitates with the anti-p, mAb (data not shown). 3.3. IZfect of mAb 2F4/ trdhesiorl to plastic

I I on phagocytosis

and

characterization The cellular distribution and biochemical properties of the 2F4/11 antigen strongly resemble those integrins, particularly the of the p1 family CD1 lb/CD18 and CD1 Ic/CDl8 moieties. Therefore, we next examined the effect of mAb 2F4/11 on the phagocytosis of complement-opsonized part-

The molecular weight of the antigen recognized by mAb 2F4/ 11 was estimated by immunoprecipitation from biotin-labelled alveolar macrophage lysates and immunoblotting (Fig. 3). This mAb immunoprecipitated a heterodimer with chains of 155 and 95

ii?

Methods

8

Alveolar macrophages 1

Polymorphomuclear

p100,

cells 7

150,

25l74 --t

150;

24175 ---I

I

0

Fig. 4. A and B: inhibitory effect of 2F4/11 mAb on the phagocytoais of opaonized zymosan particles by alveolar macrophages (A) and PMN (B). MAb 3C3. which recognizes an isoform of CD45 not expressed by granulocytes and macrophages, was used as a negative control. C: analysis by flow cytometry of phagocytosis of zymosan particles by PMN: 1. PMN alone: 2. 3 and 4. PMN plus zymosan particles preincubated in medium alone. 2F4 and 3C3 hybridoma supernatants respectively. Numbers indicate the percentage of cells within the histogram regions defined by horizontal bars.

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cles, and on the adhesion of granulocytes (PMN) to two functions mediated through the plastic, CD1 lb/CD18 molecule. Phagocytosis of complement-treated zymosan particles by PMN and/or alveolar macrophages was strongly inhibited when these cells were preincubated with 2F4/11 hybridoma supematant, in comparison with cells preincubated with an irrelevant control mAb or untreated cells (Fig. 4A and B). Phagocytosis of non-complement-treated zymosan particles was negligible under our assay conditions (data not shown). Results from examination of the samples by light microscopy were confirmed by flow cytometric analysis. Ingestion of zymosan particles resulted in an increase of SSC and FSC parameters of the cells, which were more evident for SSC. As can be seen in Fig. 4C, after incubation of PMN with zymosan particles, untreated cells or cells preincubated with the irrelevant mAb showed an increase of the SSC parameter, % cell adherence

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whereas most of the cells treated with mAb 2F4/11 remained unmodified. Phorbol esters induce, within minutes of treatment, aggregation of monocytes and granulocytes, as well as adhesion of these cells to artificial substrates such as plastic (Patarroyo et al., 1990). Freshly isolated PMN were distributed in a 96-well tissue culture plate and preincubated with mAb 2F4/ 11 or an irrelevant mAb supematant for 30 min at 4°C prior to warming to 37°C and treatment with PMA. While PMN preincubated in medium alone or with the irrelevant mAb supematant become flat and well spread, cells preincubated with mAb 2F4/ 11 showed a rounded morphology, appearing to adhere loosely to plastic, and were washed away (Fig. 5). Although mAb 2F4/ 11 strongly reduced the adhesion to plastic of PMA-treated PMN, these cells showed an increased aggregation, forming clusters of larger size than those observed in wells incubated with medium alone or irrelevant mAb. No effect of the mAbs was observed on resting PMN.

,I-:s-“B

4. Discussion Medium

2F4

3c3

PMA+2F4

I

Fig. 5. Effect of mAb 2F4/ 11on the adherence of PMA-activated granulocytes to plastic. Data are expressed as % of the binding of PMA-treated cells preincubated with medium alone. Results are representative of two independent experiments and show the means of triplicates.

In spite of the efforts of several laboratories to produce mAbs which specifically recognize porcine monocyte/macrophage surface antigens, very few have been fully characterized. Furthermore, the number of mAbs against monocyte/macrophage antigens of other species that crossreact with pig is still low. In the present study we have characterized a mouse monoclonal antibody that recognizes the (Y chain of a porcine & integrin selectively expressed on myelomonocytic cells. The porcine & integrin family appears to be similar to that described in humans. Using a cross-reactive anti-human CD18 mAb and two-dimensional gel electrophoresis, Hildreth et al. (1989) were able to distinguish four polypeptide bands that correspond in molecular weight to the human CD 1 la, CD1 lb, CD 1 lc and CD1 8 molecules. However, only two (Y subunit bands were detected by SDS-PAGE. Similarly, in the present study, only two bands corresponding to the (Y subunits, nearly alike in intensity, were resolved by SDS-PAGE when alveolar macrophage lysates were immunoprecipitated with a cross-reactive anti-human CD1 8 mAb. The failure to

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detect a third LYsubunit band might result from its low abundance in the lysate or its co-migration with one of the other bands. Although the 1X/95 kDa mol. wt. could be consistent with that of CD1 Ic, from the SDS-PAGE analysis it is not possible to determine whether the cx band recognized by mAb 2F4/ 11 corresponds to either CDI 1b or CD1 Ic. However, the cellular distribution and functional properties of the molecule recognized by 2F4/11 strongly resemble those of human CD1 1b. Similarly, the 2F4/1 1 antigen is highly expressed on granulocytes and monocytes, while CDllc is weakly expressed on these cells (Miller et al., 1986; Freyer et al., 1988; Myones et al., 1988). In addition, mAb 2F4/11 causes a drastic reduction in the phagocytosis of complement opsonized zymosan particles by granulocytes. This function is primarily mediated in humans by CD1 1b. whereas CD 1 lc does not appear to contribute significantly to the binding and phagocytic ingestion of C3-opsonized particles by granulocytes (Myones et al., 1988; Anderson et al.. 1986). Immunohistochemical analyses of lymphoid tissues confirm the restricted distribution of this antigen on cells of the myelomonocytic lineage. Although different staining patterns have been obtained with various mAbs to human or rat CD1 lb (Knowles et al., 1984; Robinson et al., 1986; Damoiseaux, 199 l), probably reflecting differing accessibilities of distinct epitopes, the tissue distribution observed with mAb 2F4/11 is comparable to that described in other species with anti-CD1 1b mAbs (Flotte et al., 1983; Splitter and Morrison, 1991; Gupta et al., 1993). It is of special interest to note the association found between cells stained by 2F4/11 in thymus and cortical thymocytes. The formation of rosettes between phagocytic cells of the thymic reticulum and cortical thymocytes has been reported by several authors and shown to involve CD1 lb/CD18 molecules (Kyewski et al., 1982; El Rouby et al., 1985). On the other hand, mAb 2F4/11 does not stain other tissue macrophage populations, including red pulp stromal macrophages in the spleen. Kupffer cells in the liver, or kidney interstitial macrophages. that are stained by other porcine macrophage-specific mAbs, i.e., 74-22-15 (Dominguez, unpublished observation). This is consistent with the observation of Gordon et al. (1988) that CD1 1b/CD18 is present

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on recently recruited monocytes/macrophages but becomes undetectable on ‘fixed’ or tissue embedded macrophages. probably as the result of selective down-modulation by environmental signals or masking by interaction with its ligands, since it is rapidly up-regulated once macrophages are isolated from tissues and maintained in culture (Gordon et al., 1992). Data on CD 11b/CD 18 expression by pulmonary alveolar macrophages are controversial. While some authors have reported that in man and rat 25-35% of these cells show a strong expression of this receptor (Knowles et al., 1984; Robinson et al., 1986), others have found this receptor to be barely detectable (Freyer et al., 1988). In our study, as has been described for sheep (Gupta et al., 1993), the majority of alveolar macrophages was strongly labelled by mAb 2F4/11. These results can be explained by either differences in the expression of the CD1 lb molecule between species or differences in the state of activation of these cells. In this regard, alveolar macrophages have been considered to be in a more activated state than other tissue macrophages because they are continuously exposed to exogenous material inhaled into the alveoli. However, Freyer et al. (1988) did not find any increase in the expression of CD 1 I b/CD18 by alveolar macrophages when these cells were exposed to activating agents. Furthermore. due to the functional redundancy of the leukointegrins (Larson and Springer. 1990). it is possible that a particular function might be primarily performed by different molecules in different animal species. Integrins participate both in cell to cell and cell to extracellular matrix interactions and are also involved in signalling across the cell membrane. In the mouse and human, CD1 1b/CD18 mediates adherence of PMN and monocytes to vascular endothelium. prior to diapedesis (Amaout et al., 19881, contributes to phorbol ester-induced PMN aggregation (Anderson et al., 1986; Patarroyo et al., 19901, is involved in phagocytosis of complement-coated particles (Carlos and Harlan, 1990) and in the adhesion of myelomonocytic cells to plastic (Patarroyo et al., 1985; Rosen and Gordon, 1987). Multiple functional domains have been defined in the CD 1 I b/CD 18 molecule (Dana et al., 1986; Graham and Brown. 1991). MAb 2F4/ 11 is able to inhibit

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adhesion of PMA-activated PMN to plastic, in addition to inhibiting the phagocytosis of opsonized zymosan particles by PMN and alveolar macrophages. Interestingly, it also increases the size of the aggregates induced by PMA between these cells. This result suggests that aggregation is mediated by a domain of CD1 1b different from the one involved in phagocytosis or plastic adherence. However, this increase in aggregation may also be secondary to the reduced adhesiveness to plastic that facilitates cell to cell surface interactions mediated by other molecules. It might also be the result of divalent binding of the antibody to different cells, which seems unlikely as 2F4/ 11 does not induce aggregation of non-activated PMN. However, PMA, like other inflammatory stimuli, is known to increase surface expression of CD 11b/CD 18 in PMN (Lo et al., 1989; Vedder and Harlan, 1988), that may influence the outcome of the experiment. In fact, stimulation of PMN with PMA for 20 min resulted in a twofold increase in the surface density of the 2F4/11 antigen, whereas expression of other antigens remained unaltered (data not shown). In summary, we have produced and characterized a mAb to the (Y chain of a porcine & integrin selectively expressed on myelomonocytic cells, whose functional properties strongly resemble those of CD1 1b. This mAb may be a useful reagent for the isolation and characterization of porcine macrophage subpopulations and for the investigation of their heterogeneity and differentiation, as well as for the evaluation of the role of this molecule in different inflammatory and infectious diseases. Acknowledgements We thank Drs. Agustin Zapata. Francisco Sanchez Madrid and Nieves Domenech for critical reading of the manuscript. This work was supported by INIA grant SC93- 155 and EC Project AIR3-CT93-1332. R. Bullido and M. Gomez de1 Moral are recipients of INIA and CICYT Ph.D. fellowships respectively. References Altieri. D.C., Morrissey, J.H. and Edgington, T.S. (1988) Adhesive receptor Mac-l coordinates the activation of factor X on

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stimulated cells of monocytic and myeloid differentiation: an alternative initiation of the coagulation protease cascade. Proc. Natl. Acad. Sci. USA 85, 7462. Anderson, D.C., Miller, L.J., Schmalsteig. F.C., Rothlein, R. and Springer, T.A. (1986) Contributions of the Mac-l glycoprotein family to adherence-dependent granulocyte functions: structure-function assessments employing subunit-specific monoclonal antibodies. J. Immunol. 137, 15. Amaout, M., Todd, R., Dana. N.. Melamed, J.. Schlossman. S. and Colten, H. (1983) Inhibition of phagocytosis of complement C3 or immunoglobulin G-coated particles and of C3bi binding by monoclonal antibodies to a monocyte-granulocyte membrane glycoprotein (mol). J. Clin. Invest. 72, 171. Arnaout. M.A.. Lanier. L. and Faller. D.V. (1988) Relative contribution of the leukocyte molecules Mol, LFA-1, ~150, 95 (LeuM5) in adhesion of granulocytes and monocytes to vascular endothelium is tissue- and stimulus-specific. _I. Cell. Physiol. 137, 305. Beller, D., Springer, T. and Schreiber, R. (1982) Anti-Mac-l selectively inhibits the mouse and the human type three complement receptor. J. Exp. Med. 156. 1000. Carlos, T.M. and Harlan. J.M. (19901 Membrane proteins involved in phagocyte adherence to endothelium. Immunol. Rev. 114, 5. Carrascosa. A.L., Santaren. J. and Viiiuela. E. (1982) Production and titration of African swine fever virus in porcine alveolar macrophages. J. Virol. Methods 3, 303. Damoiseaux, J. (1991) Macrophage heterogeneity in the rat. Ph.D. Thesis, Free University, Amsterdam. Dana, N.. Stryrt, B.. Griffin. J.D., Todd, R.F., Klempner, M.S. and Arnaout. M.A. (1986) Two functional domains in the phagocyte membrane glycoprotein Mol identified with monoclonal antibodies. J. Immunol. 137, 3259. Danilenko, D.M., Rossito, P.V., Van der Vieren, M., Le Trong, H., McDonough, S.P., Affolter. V.K. and Moore, P.F. (1995) A novel canine leukointegrin, q, &, is expressed by specific macrophage subpopulations in tissue and minor CD8+ lymphocyte subpopulation in peripheral blood. J. Immunol. 155, 35. Diamond. M.S. and Springer, T.A. (1993) A subpopulation of Mac- 1 (CD1 1b/CD 18) molecules mediates neutrophil adhesion to ICAM- and fibrinogen. J. Cell Biol. 120, 545. El Rouby, S.. Praz, F.. Halbwachs-Mercarelli, L. and Papiemik, M. (1985) Thymic reticulum in mice. IV. The rosette formation between phagocytic cells of the thymic reticulum and cortical type thymocytes is mediated by complement receptor type three. J. Immunol. 134, 3625. Flotte, T.J., Springer, T.A. and Thorbecke, G.J. (1983) Dendritic cell and macrophage staining by monoclonal antibodies in tissue sections and epidetmal sheets. Am. J. Pathol. 111, 112. Freyer, D., Morganroth, M.. Rogers, C., Arnaout, M. and Todd, R. (1988) Modulation of surface CD1 l/CD18 glycoproteins (Mel, LFA-1, ~150.95) by human mononuclear phagocytes. Cell Immunol. Immunopathol. 46. 272. Gonzalez, S.. Mendoza. C., SBnchez-Vizcaino. J.M. and Alonso, F. (1990) Inhibitory effect of African swine fever virus on

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