Res. Immunol.

0 INSTITUT PASTEUR/ELSEVI&

Paris 1992

1992,

143, 39-41

Monoclonal antibody AMH152 reacts with human monocytes in culture and with inflammatory macrophages A. Martin

(*), R. Audran

cl), B. Collet

(I), G. Lancien

c2) and L. Toujas

(*I

(‘) Laboratoire (21Laboratoire

d’lmmunologie-Immunothkrapie, Centre RPgional de Lutte contre le Cancer, 35033 Rennes Cedex, and d’Anatomie et de Cytologie Pathologiques, Centre Hospitalier, 35033 Renne? Cedex (France)

SUMMARY Monoclonal antibodies (mAb1 raised against human peritoneal macrophages were selected for their non-reactivity with freshly sampled blood cells. One of these mAb, AMH 152, initially non-reactive, bound to monocytes after 18 h of culture, a property which was not shared by an unrelated antibody of the same isotype (IgGl I. The induction of the expression of the antigen detected by AMH 152 on monocytes in culture was not influenced by the addition of serum or by the substrate used, plastic that favoured adhesion or teflon bags. Overnight incubation at 4OC in adhesion conditions did not enable antigen expression. A 1 -h treatment with phorbol myristate acetate or fo!myl-methionylleucyl-phenylalanine did not increase AMH152 binding. Culturing monocytes with cycloheximide tended to inhibit antigen expression. These observations suggested that antigen expression represents an active phenomenon, requiring protein synthesis. The antigen recognized by mAb AMH 152 could be visualized on sections of formalin-fixed and paraffin-embedded tissues. Macrophages of healthy lymphoid organs and tissues that expressed CD68 antigen failed to bind AMH152. In contrast, chronic inflammatory lesions, like those of sarcoidosis, tuberculosis and cat scratch disease, contained epithelioid and multinucleated giant cells that reacted with AMH 152. In serous exudates of cancer metastases, 1 O-40 % of macrophages were also stained. The antigenic material was essentially present at the cell periphery. Thus, mAb AMHI 52 recognized a surface antigen, detectable on paraffin-embedded tissue sections, and which accompanied differentiation of monocytes into inflammatory cells. The expression of this antigen on monocytes in culture suggests that these cells underwent an activation process, even when maintained for some hours in teflon bags and in a serum-free medium. Key-words: Antigen AMH152, Differentiation.

expression,

inflammation,

Introduction Mononuclear phagocytes constitute a heterogenous cell population distributed between three main compartments : bone marrow, in which precur-

Monocyte,

Macrophage;

mAb

sor cells divide and mature into monocytes ; blood, where monocytes circulate transitionally; and peripheral tissues, where they migrate and differenGate into functionally active macrophages. In vitro models have been found suitable to investigate the

Submitted September15, 1991, acceptedOctober 10, 1991.

A. MARTIN

40

differentiation of monocytes into macrophages (Johnson et al., 1977; Kaplan, 1983). Recently, cultures on hydrophobic substances like teflon, which minimize ceil adhesion, have been developed and found to improve viability and allow monocyte differentiation (Andreesen et al., 1983 ; Helinski et al., 1988). The aim of the present work was the production of mAb directed against differentiation markers of monocytes and their investigation in both in vivo and in vitro systems. mAb were raised against cells of human peritoneal cavity and selected for their nonreactivity with freshly collected blood monocytes and for their ability to detect antigens on paraffinembedded tissues. This procedure enabled investigation of macrophage differentiation antigens both in monocyte cultures and on histological sections of tissue samples. One of the mAb produced, AMH152, became reactive with monocytes after some hours of culture and could be detected in vivo in inflamed tissues. Materials

and Methods

Production of mAb against macrophage differenliation antigens Peritoneal exudate cells were sampled from a chylous ascites of a lymphoma patient. Approximately 2 liters of whitish fluid were obtained by puncture and almost 10’ cells were separated by centrifugation. The cells, washed and suspended in foetal calf serum (FCS) supplemented with 15 % dimethylsulphoxide, were frozen and kept in liquid nitrogen. After thawing, 85 070remained viable. These cells were used to immunize BALB/c mice by 3 intraperitoneal injections of 7 x lo6 cells at a IO-day interval, followed 10 days later by intravenous injection of 7 x lo6 cells. The general procedure of mAb production was identical to that described previously (Dazord et al., 1987; Martin et al., 1986). Briefly, spleen cells were fused with SP2/0 plasmacytoma cells and the hybrid culture supernatants were screened for the presence of desired antibody by an indirect radioimmunological assay using immunizing macrophages, B-leukaemic lymphocytes and purified blood monocytes as test cells. Test cells (200,000) were pelleted and incubated with 100 ~1 hybrid culture supernatant for 45 min. After washing and in-

APAAP BSA FCS FMLP FITC

= alkaline phosphatase/anti-alkaline phosphatase. = = = =

bovine serum albumin. foetal calf serum. formyl-methionyl-leucyl-phenylalanine. fluorescein isothiocyanate.

ET AL. cubation with 10’ cpm ‘ZSI-radiolabelled sheep antimouse F(ab)‘, fragment (Amersham, les Ulis, France), the cells were washed 3 times and their radioactivity was measured. Hybrids reacting with immunizing macrophages and not with leukaemic B lymphocytes and monocytes were cloned by the limiting dilution technique and grown extensively for the production of culture supernatants. For each supernatant, antibody isotype and concentration were determined by ELISA (enzyme-linked immunosorbent assay) techniques. Isotype was determined using the mAb-isotyping kit A (Pharmingen). mAb concentration was measured as follows. One-hundred ~1 samples of a I-t*g/ml solution of goat anti-mouse IgGl antiserum (Sigma, La Verpilliere, France) diluted in CO,HNa buffer (pH 9) were deposited in each microplate well (CEB Luxlon, M29 LSC, CML Laboratories, Nemours, France) and incubated overnight. Non-specific adsorption sites were saturated by incubation in 3 070bovine serum albumin (BSA)added PBS (phosphate-buffered saline) for 2 h. Decreasing concentrations of mouse MOPC-3 1C plasmacytoma IgGl (Sigma) were prepared by serial dilution in PBS to which was added 1 % BSA and 0.05 070Tween-20, starting from a 20+g/ml solution. Serially diluted samples of AMH 152 culture supernatants were also made and deposited in the wells. After 2-h incubation, the wells were washed extensively in PBS 0.05 % Tween-20, and a l/400-diluted solution of alkaline phosphatase-labelled goat antimouse antiserum (Sigma) was added. After one hour, a 1 rig/ml solution of p-nitrophenyl phosphate disodium (Sigma) in TRIS 1 M, NaCl 1.5 M buffer, pH 9.8, was added and left overnight. Staining was read at 405 nm in a “Titertek Multiscan” spectrophotometer. The supernatants thus calibrated were tested against a panel of paraffin-embedded tissues including liver, kidney, lung, spleen and intestine. Test cells and tissues Blood mononuclear cells were obtained by centrifugation on a Ficoll-methrizoate cushion (MSL, Eurobio, Paris). Monocytes were purified from this cell population by centrifugation with “SepracellMN” (Sepratech Corporation, Clinisciences, Paris), a medium containing modified silica particles. Bloodderived macrophages were obtained by culturing pu-

mAb PBS PE PMA

= = = =

monoclonal antibody. phosphate-buffered saline. phycoerythrin. phorbol myristate acetate.

MONOCYTES,

INFLAMMATION,

rified monocytes for 6 days in “Chex-all II Teflon” tissue culture bags (J-Bio, les Ulis, France) with RPMI 1640 medium supplemented with the various components indicated by Dumont et al. (1988) and with 5 % non-heat-inactiated autologous serum. Alveolar macrophages were collected by bronchoalveolar lavage. Granulocytes were obtained by centrifuging heparinized blood from healthy donors on a cushion of “Mono-poly” resolving medium (Flow, Les Ulis, France). Malignant B lymphocytes were collected from the peripheral blood of a leukaemic patient and kept frozen in liquid nitrogen. The erythroblastic K562 cell line was maintained in RPMI-1640 (Aqual; AES Laboratories, 35370 Combourg, France) supplemented with 2 mM glutamine, 160 kg/ml gentamycin and 10 % FCS (Anval, 35830 Betton, France). In some experiments, cells were treated with 100 nM phorbol myristate acetate (PMA) (Sigma) or 10 nM (formyl-methionyl-leucylphenylalanine) (FMLP) (Sigma). In some experiments, cultures were achieved in the presence of cycloheximide (Sigma). The various pathological or healthy tissues mentioned in “Results” were sampled during surgical operations, fixed in 10 % formalin, dehydrated in acetone and embedded in paraffin. CeN and tissue reactivity

with antibodies

Cell suspensions in PBS with 0.5 % BSA added were reacted with various mAb in addition to mAb 152 : fluorescein-isothiocyanate (FITC)-labelled anti-CD1 lb (IOM-1 Immunotech, Marseille), phycoerythrin (PE)-labelled anti-CD14 (IOM-2 Immunotech), FITC-labelled anti-CD4 (Immunotech), PE-labelled anti-CD8 (Immunotech) and control IgGl immunoglobulin isolated from MOPC-21 plasmacytoma (Sigma). Immunofluorescence with directly labelled mAb (IOM-I and IOM-2) required a single incubation, as indicated by the manufacturer, followed by 3 washings. Indirect immunofluorescence techniques (AMH152, MOPCZI) included an additional exposure for 45 min with FITC-labelled sheep anti-mouse F(ab)‘, (Silenus Laboratories, Eurobio, Paris) at 1/4~-dilution and 3 washings. Before flow cytometry analysis, cells were post-fixed and stored in 0.3 % form~in-added PBS. C~ofluorimetry was achieved with the technical assistance of Catherine Picot (Centre Regional de Transfusion Sanguine, Rennes) using the H-50 system Ortho fitted with a “Persona 1600 Logabax” computer or a “FACscan” (Becton-Dickinson). Cytograms of the cell populations studied were made on the basis of the right angle and forward angle scatters. This allowed the exclusion of cellular debris by choosing an appropriate electronic threshold and the determination of a domain corresponding to the monocytes present in suspensions of blood mononuclear cells. Histograms were achieved from the populations selected by plot-

Ag EXPRESSIUN

AND

mAb AMHf52

41

ting fluorescence intensity in abcissa against cell number in ordinate. Double fluorescence investigations were also performed. Cell smears and tissue sections were reacted with AMH152 and an anti-CD68 mAb kindly provided by D.A. Mason (Oxford). The reaction was revealed by the alkaline phosphataseianti-alkaline phosphatase (APAAP) technique according to Cordell et al. (1984). Briefly, sections were rehydrated and proteinbinding sites saturated with a BSA-containing buffer. The slides were then exposed successively to the m.Ab studied, rabbit anti-mouse immunoglobulin antibodies (Dako-Sebia, Issy-les-Moulineaux, France) and APAAP complex (Dako). Alkaline phosphatase activity was revealed by incubation with a substrate containing fuchsin, and the sections were counterstained with Harris haematoxylin. Results Prod~cr~on

of mAb AMH152

mAb were prepared by mouse immunization against ascites cells sampled from a patient with a chylous ascites. More than 80 % of these cells displayed round cytoplasmic inclusions stained with osmium tetroxide, extractible with organic solvents and interpreted as fat vacuoles. Immunofluorescence staining showed, upon microscopic examination, that 85 % of the cells reacted with anti-CD1 lb and 83 % with anti-CD14. The ascites cells were thus considered as containing more than 80 070macrophages and their fat vacuoles were interpreted as resulting from the phagocytosis of lipids pathologically discharged from chyliferous in the peritoneal cavity. Cell fusions for production of mAb were repeated twice. Hybrid supernatants reacting against immunizing macrophages and not against leukaemic B lymphocytes were selected with a radioimmunological test. Eleven of them did not bind to freshly sampled blood monocy-tes ; of these, 4 (AMH152, -225, -469 and -488) reacted with paraffin-embedded tissue sections. AMH152 studied here was found to be of the IgGl kappa isotype. It did not react with cells of haematopoietic origin, except for immunizing macrophages, as judged from a radioimmunoassay performed with a panel of such cells (table I). Modification differentialion

of AMHl52 reactivity of blood monocytes

during in vitro

Monocytes purified from peripheral blood by centrifugation on “Sepracell” medium were cultured in teflon bags for various times and allowed to react with AMH 152 (10 pg/ml), with an unrelated IgG1

A. MARTIN

42 Table I. Reactivity

of mAb AMH152

ET AL.

with cells of haematopoietic origin, as studied by immunofluorescence and cell flow analysis. Percent of positive cells

Mean fluorescence intensity

53 2 0 0 0 0 0 0 0 0

38 5 -

Immunizing cells Monocytes, adherent (I) Monocytes, susgnsion (‘I T lymphocytes B-leukaemic lymphocytes Polymorphonuclear cells Bone marrow cells Red blood cells K562 cell line Platelets

(I) Purified by adhesion for 2 h at 37°C. 01 Identified from a Ficoll separation by light scattering properties. (3) Purified from blood mononuclear cells by passage througha glasswoolcolumn.

substantially AMH152 if compared to MOPC21. After 24 h of culture, the percentage of MOPC21-binding cells tended to decline, whereas binding of AMH152 increased strongly. CD14 expression also increased during the course of the culture, but not as much as AMHl5Zrelated antigen. A double-labelling method in which monocytes were identified as CD14+ cells was also used to investigate AMH152 binding. The percentage of CD14+ cells that also bound AMH152 passed from 13 to 99 % after 24 h of culture (fig. 2).

0

7.

4

6

6

DAYSOFMONOCYTECULTURE

Fig. 1. Reactivity of various antibodiesat different times of a monocyte culture. Each point representsthe mean of 3 to 6 determinations.

immunoglobulin (MOPC21 protein) at the same concentration and with anti-CD1 lb and anti-CD14 mAb. Flow cytometry analysis enabled us to identify monocytes by their light scattering properties and to quantify antibody binding. The results are presented in figure 1. Freshly collected monocytes did not bind

The role of various factors in the onset of AMH152 reactivity with Sepracell-purified monocytes was investigated. The presence of serum in the culture was unnecessary. Culturing on plastic vessels or in teflon bags did not modify results (data not shown). A temperature of 37°C was required, and leaving the cells overnight at 4°C enabled them to adhere, but not to express the antigen (table II). A l-h treatment with PMA or FMLP did not influence AMH152 reactivity (not shown). In the experiment presented in table III, blood monocytes were cultured for 24 h in the presence of cycloheximide, an inhibitor of protein synthesis. This treatment strongly reduced CD14 expression, did not substantially affect CD1 1b, but moderately diminished reactivity with AMH152. Tissue reactivity of mAb AMH152 The reactivity of mAb AMH152 with tissue sections was investigated using the APAAP technique. CD68 antigen, present in virtually all macrophages

MONOCYTES,

INFLAMMATION,

Ag EXPRESSION

UNCULTURED

AND

24

43

mAb AMH152

h - CULTURED

Fig. 2. Double immunofluorescence labelling of monocytes by anti-CD14 and AMH152. Comparison between uncultured and 24-h-cultured monocytes. Anti-CD14 was directly conjugated with PE whereas AMH152 was revealed by an indirect immunofluorescence assay using FITC-labelled anti-mouse F(ab)‘,.

(10 mg/ml), anti-CD14 and of the unrelated MOPC21 IgGl protein (10 mg/ml) with monocytes adhered at 4°C or 37°C for 18 h in the absence of serum.

Table II. Reactivity of AMH152

24-h incubation AMHl52 Anti-CD14 MOPC2 1 Antibody

Percent positive cells 24-h incubation

binding

was

measured

by immunofluorescence

and

of culture with cyclohexamide No. culture

0

67 50 13

76 84 89

at 37°C

37 58 9

0 64 0

Table III. Influence

CDllb CD14 AMH152

at 4°C

flow

cytometry.

upon expression of various antigens on monocytes.

Percent antibody-binding cells 24-h culture with cycloheximide @g/ml) 2.5 5 10

(Bielefeldt-Ohmann et al., 1988 ; Davey et al., 1988 ; Kelly et al., 1988), was revealed in parallel and taken as a positive control. As a general rule, healthy tissues did not contain macrophages reacting with AMH152. Kupffer cells and various tissue macrophages from skin, kidney and intestine mucosa, which exhibited the CD68 antigen, were negative with

80 21 75

81 17 36

88 4 37

20 78 5 48

AMH152. In lymph node, spleen and thymus, only very rare and isolated cells among those reacting with anti-CD68 bound AMH152. In lung, macrophages present in the alveolar cavity were AMH152and CD68+. Surprisingly, type II pneumocytes of the aIveolar wall bound AMH152 but did not express CD68 antigen. Chronic and subacute inflammations,

Fig. 3. Staining of giant cells with

AMH152 (a) and anti-CD68 (b). Control without antibody (c).

MONOCYTES,

INFLAMMATION,

particularly sarcoidosis, tuberculosis, cat scratch disease, synovial subacute lesions and a foreign-bodyinduced granuloma were also investigated. The proportion of CD68+ cells binding AMH152 was clearly more elevated than in healthy lymphoid tissues. Many epithelioid cells were circled with a fine line of antibody-stained material. The deposit appeared thicker around multinucleated giant cells (fig. 3a). Certain cells were stained more intensely than others. This did not depend on their morphological aspect, whether it be Langhans or foreign body type. Staining predominated at the periphery of the cells, in contrast to CD68, which was expressed only in the cytoplasm (fig. 3b). Most non-lymphoid tissues were negative, including liver and kidney parenchyma, muscle and connective tissue. In epithelial tissues, however, some glandular formations stained positively, including tracheal and bronchial mucous glands, intestinal cryptic glands, about 10 % of pancreatic exocrine glands, and skin sweat glands. The reactivity of AMH 152 was also tested on various cell smears by the APAAP technique. In peritoneal and pleural metastatic exudates of ovary (2) and breast (2) cancers, a variable proportion (lo-30 070)of cells containing cytoplasmic vacuolar inclusions and thought to be macrophages reacted positively (data not shown). The microscopic examination of the immunofluorescence and APAAP-stained cells showed that mAb AMH152 bound essentially to the surface membrane, although some antigen could also be detected in the cytoplasm.

Discussion mAb directed against human macrophage differentiation antigens have been raised by immunizing mice against monocytes exposed to interferon gamma and corticoids (Morganelli and McGuyre, 1988) or cultured for several days and transformed into macrophages (Andreesen et al., 1988; Zwadlo et al., 1985). Some mAb were also derived from U937 or HL60 cell lines which differentiated into macrophage-like cells under the effect of various activators, in particular phorbol esters (Zuckermann et al., 1987). Tissue macrophages, with the exception of alveolar macrophages and breast milk macrophages (Kelly, 1988; Biondi el al., 1984), have rarely been taken as immunogens. mAb prepared against macrophages from peritoneal cavity, which are very common in mouse, do not yet seem to have been prepared in man. In the work presented here, advantage was taken of a pathological situation in which a large number of macrophages in an almost pure population were present in the peritoneal cavity.

Ag EXPRESSION

AND

mAb AMH152

45

AMH152, one of the mAb produced by mouse immunization against these cells, reacted both in vitro with monocytes cultured for some hours and in vivo on paraffin tissue sections. In vitro experiments showed that the binding of antibody AMH152 was not related to binding via Fc receptors, since a nonrelated IgGl used at the same concentration as AMH152 did not bind to cultured monocytes. Several mAb have been reported to increase their reactivity with monocytes during culture (Hogg ef al., 1985). As shown by Andreesen et al. (1990), antiCD 16, anti-CD5 1, max-1 and max-3 were reactive after several days of culture, whereas anti-CD71, max-26 and max-11 did not depend on maturation into macrophage and were expressed after adhesion. Anti-M03e mAb also increased its binding after a 24-h culture (Todd et al., 1987) and, unlike AMH152, upon stimulation by PMA and lipopolysaccharide. Like AMH152, simple adhesion at 4°C did not modify M03e expression. Rapid expression of an antigen on the surface membrane may correspond to the transfer to the cell surface of material stored in the cytoplasm. Miller et al. (1986, 1987) showed that adhesion proteins Mac-l and p150,95 were induced to pass within a few minutes from an intracellular vesicular compartment to the cell surface following treatment with FMLP, platelet-derived growth factor, tumour-necrosis factor, C5a and leukotriene B4. Midoux et al. (1989) also reported an antigen recognized by mAb 5B5, initially present in the cytoplasm of macrophage and expressed on the surface membrane after adhesion at the very site of cell attachment to plastic. These findings prompted us to investigate the onset mechanism of the expression of AMH152-related antigen in the course of culture. The observation that simple cell attachment to plastic was not sufficient to induce antigen expression, and the diminished binding of AMH152 under the effect of cycloheximide showed that the expression of the antigen was an active process, requiring protein synthesis. The failure of PMA and FMLP to provoke immediate antigen expression and the effect of cycloheximide, which diminished AMH152 but not anti-CD1 1b binding, might constitute features different from certain leukocyte cell adhesion molecules. Intercellular adhesion and adhesion to culture substrate (Patarroyo et al., 1988) have been shown to be inhibited by mAb directed against leukocyte cell adhesion molecules. Antibody AMH 152 did not alter monocyte adhesion and spreading. Studies on the histological localization of AMH152-reacting cells showed that such cells were selectively present in inflammatory lesions. The inflammation process is known to generate new categories of cells, like macrophage exudate cells, epithelioid cells and multinucleated giant cells (Van Furth, 1980). mAb AMH152 reacted remarkably with these three cell types, which suggests that it may

A. MARTIN be considered as a marker accompanying differentiation of monocytes into inflammatory cells. The relationship between in vitro and in vivo observations presented here merits certain remarks. Detection of the antigen on cultured monocytes contrasted with its presence on histological sections only in well differentiated inflammatory cells. Experimental investigations in mice have indeed shown that monocyte differentiation into epithelioid cells or giant cells required at least 4 days (Papadimitriou and Spector, 1971; Mariano and Spector, 1974), and therefore some monocytes could be expected to react positively in the inflammatory foci examined. It seems conceivable that the antigen detected by immunofluorescence on intact cells was partly destroyed by fixation and embedding techniques or that it was expressed amounts too low on monocytes in to be detectable by the immunohistological method used. The amount of antigen could increase during the maturation of inflammatory cells and become progressively detectable. Particularly thick layers of stained material were seen at the periphery of giant cells. The presence, in cultures of monocytes, of a marker of differentiation into inflammatory cells suggests that, even in optimal conditions using serumfree medium and teflon bags as substrate, monocyte activation occurs after a short period of culture. Acknowledgements

Thisworkwassupported by theAssociation pourla Recherche surle Cancer.

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Ag EXPRESSION

AND

mAb AMH152

41

Van Furth, R., Diesselhof-den-Dulk, M.M.C. & Mattie, H. (1973), Quantitative study on the production and kinetics of mononuclear phagocytes during an acute inflammatory reaction. J. exp. Med., 138, 13141330. Zuckerman, S.H., Tang, J., Gitter, B.D. & Scheetz, M.E. (1987), 7C3, a marker for the differentiation of human macrophage cell lines. J. Leuk. Biol., 42, 491-497. Zwadlo, G., Brocker, E.B., Von Bassewitz, D.B., Feige, U. & Sorg, C. (1985), A monoclonal antibody to a differentiation antigen present on mature human macrophages and absent from monocytes. J. Immunol., 134, 1487-1492.