j ber-mac3: monoclonal antibody that defines human...

J Clin Pathol 1991;44:936-945

Ber-MAC3: new monoclonal antibody that defineshuman monocyte/macrophage differentiationantigen

E Backe, R Schwarting, J Gerdes, M Ernst, H Stein

AbstractA new monoclonal antibody Ber-MAC3is reported. It recognises a formolsensitive epitope of a not yet clusteredmonocyte/macrophage specific 140kilodalton glycoprotein that is expressedon the cell surface and in the cytoplasm.In 30 cases of acute and chronicleukaemia, Ber-MAC3 staining wasrestricted to 15 myeloid leukaemias ofM4 and M5 types. The tumour cells oftwo cases of true histiocytic malignan-cies were Ber-MAC3 positive, whereasthose of all 280 malignancies of lym-phocytic origin were negative. The latterincluded 52 cases of Hodgkin's diseaseand 41 cases of Ki-l positive anaplasticlarge cell lymphomas which had pre-viously been classified as true histiocyticlymphomas.Ber-MAC3 therefore seems to be of

considerable value for selectiveidentification of monocytes and macro-phages at a certain stage of differentia-tion and seems to be suitable fordiagnosing myelomonocytic or mono-cytic leukaemia and neoplasms of truehistiocytic origin.

Institut furPathologie,Klinikum Steglitz,Berlin, GermanyE BackeR SchwartingJ GerdesH SteinForschungsinstitutBorstel,Berlin, GermanyM ErnstCorrespondence to:Dr Harald Stein,Institut fuir Pathologie,Klinikum Steglitz der FreienUniversitit Berlin,Hindenburgdamm 30,1000 Berlin 45, GermanyAccepted for publication22 May 1991

Considerable morphological and functionaldiversity is seen in cells of the mononuclearphagocyte system (MPS), which includesblood monocytes, resident macrophages ofvarious tissues, starry sky macrophages(equivalent of tingible body histiocytes), andepithelioid type macrophages and multinu-cleated giant cell macrophages of Langhans',foreign body, or Touton type. Because of thisgreat heterogeneity, a clear distinctionbetween the different MPS cells and cells ofother systems is often impossible, especiallywhen they have undergone malignant trans-formation. Several monoclonal antibodieshave been developed which recognise certainsubsets of the MPS.1-25 But a correlationbetween the various functions of these cells-for example, antigen presentation, cytotoxicactivity, secretion of factors regulating cellularfunctions, secretion of enzymes and oxygenfree radicals-and different stages of differen-tiation by defining subpopulations is not yetpossible as it is for B and T cells.A new monoclonal antibody, Ber-MAC3,

defines a distinct stage ofdifferentiation/activa-tion of monocytes/macrophages. Ber-MAC3is unreactive with normal and neoplastic cellsof non-myeloid lineages, and, in myeloid

leukaemia, itsM4/M5 type.

reactivity is restricted to the

MethodsSuspended mononuclear splenocytes (1 x 107)containing monocytes, macrophages, and Bcells, were used for a weekly immunisation ofbalb/c mice over a period of six weeks. Thespleen was obtained fresh from a normal donorafter a traumatic rupture.

Somatic cell hybridisation was performedthree days after the last immunisation accord-ing to the protocol of Oi and Herzenberg.26Supernatants from wells exhibiting hybridgrowth were tested by immunoenzymaticstaining with the alkaline phosphatase anti-alkaline phosphatase (APAAP) method27 onfrozen sections of human tonsil and lymphnode from a patient with Hodgkin's disease.The immunoglobulin subclass of the mono-

clonal antibody was determined by doubleimmunodiffusion using subclass specific rabbitanti-mouse reagents (Serotec, Wiesbaden,Germany).

All cells were cultured in RPMI medium(Biochrom, Berlin, Germany) containing 10%fetal calf serum (FCS) (Gibco, Berlin), 4 mMglutamine 10 U/ml penicillin, and 01 mg/mlstreptomycin (Boehringer, Mannheim, Ger-many). Peripheral blood lymphocytes wereprepared from anticoagulated blood by Ficoll-Hypaque density centrifugation. T cells wereobtained by E-rosette formation using sheepred blood cells treated with 2-aminoethyliso-thiouronium-bromide-hydrobromide (AET).Monocytes were isolated from the non-roset-ting cells by adherence to tissue culture Petridishes. B cells were obtained by carefulremoval of non-adherent cells from the non E-rosette forming cells taken from the tonsils ofchildren after tonsillectomy. T cells werestimulated in normal RPMI medium contain-ing 1% (v/v) phytohaemagglutinin (PHA) or10 Mg/ml Concanavalin A (ConA) for threedays. B cells were stimulated in normal RPMImedium containing 2 x I0-' (v/v) Staphylococ-cus aureus (Calbiochem, Giessen, Germany)cells/ml medium and in medium containing1% (v/v) Pokeweed mitogen. HL-60, U-937,and THP-128 cell lines were stimulated with0-tetradecanyol phorbol diester (TPA) (Sigma,Munich, Germany) at a concentration of100 ng/ml, 10 U/ml, or 100 IU/ml IFN-G(Amersham, Braunschweig, Germany) forthree, seven, and 14 days. Monocytes werecultured for three days in normal medium orwere stimulated either with 10 jug/ml

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Ber-MAC3: new monoclonal antibody that defines human monocyte/macrophage differentiation antigen

lipopolysaccharide Escherichia coli B05:55(Sigma) or 10 IU/ml IFN-G (Amersham), ormonocytes were primed with 10 IU IFN-G/mlfor two and a half hours, to which 10 ng/mllipopolysaccharide were added. For giant cellformation, monocytes were cultured for 14days in medium containing 10% human serumwhich was changed every third day. The effectof Ber-MAC3 on the formation of giant cellswas investigated by incubating monocytes inmedium containing Ber-MAC3 ascites (1 in100, 1 in 1000). Control experiments wereperformed in medium without immuno-globulin and in medium containing anirrelevant non-binding antibody (Ki-27).29

All normal, inflammatory and neoplastic tis-sues, and leukaemic cells orginated from theDepartment ofPathology or the Department ofHaematology, Steglitz Medical Centre, Berlin,FRG. Normal tissues were chosen at randomfrom three different subjects. All malignantnon-Hodgkin's lymphomas (NHL) were clas-sified according to the updated Kiel classifica-tion, using both morphological and immuno-phenotypical criteria.' 3 The cases ofHodgkin's disease were subtyped according tothe Rye classification.32 The myeloid leu-kaemias were typed according to the French-American-British classification. The sectionsfrom all normal tissues were stained at leastthree times.

Control antibodies used for immunohisto-logical staining and biochemical experimentswere anti-CDla NA1/34 (Dakopatts), epithe-lial cell specific Lu5 (Boehringer, Mannheim,Germany), FDC specific R4/23,33 anti-CD30Ber-H2,3 anti-CD 1c S-HCL3,1 monocyte/macrophage specific Ki-M6,22 and prolifera-tion-associated Ki-67.35 As a negative controlantibody, the non-binding variant (W6/32HK)of a class I monoclonal antibody was used.

IMMUNOHISTOLOGY

Biopsy specimens of human tissue were snap-frozen in liquid nitrogen and were stored at- 80°C. Frozen sections of 8 pm were preparedwith a 2800 Frigocut-cryostat (Reichert-Jung,Heidelberg, Germany) and fixed for 30 minutesin acetone and 30 minutes in chloroform. TheAPAAP staining technique was performed as

described by Cordell et al.21

IMMUNOFLUORESCENCEThe cells were fixed by resuspending the cells in70% cold ethanol on ice for 20 minutes beforeincubation with the first antibody. To dis-criminate between cytoplasmic and surfacelabelling non-fixed cells were also used. Cells (1x 106) were incubated with 100 ph of mono-clonal antibody at a saturated concentration at40C for 30 minutes. The cells were washedtwice and subsequently incubated with fluores-cence goat F(ab)2 anti-mouse IgG (Dakopatts),washed twice again, and then analysed on anEPICS 752 flow cytometer (Coulter, USA),equipped with a 5WR-gun laser at an excitationwavelength of 488 nm.

BIOCHEMICAL CHARACTERISATION OF THETARGET ANTIGENViable human alveolar macrophages were

tritiated by the borohydride method36 and lysedwith phosphate buffered saline (PBS) contain-ing 1% Triton X-100 and 2 mM phenyl-methyl-culfonylfluoride (PMSF) (Sigma). Forinternal labelling 6 x I07 cells of the DHL- I3cell line were washed three times with methion-ine-free medium (Gibco) without FCS, thensuspended in medium without methionine,containing 10% FCS (dialysed against meth-ionine-free RPMI 1640) at a concentration of1 x 106 cells/ml and incubated for 30 minutesat 37°C and 5% carbondioxide. Then 1 mCi35S-methionine (NEN-Dupont, Dreieich,Germany) was added and the cells were incu-bated for 20 hours. Cells were harvested,washed three times with medium containingmethionine (Gibco), and the cell pellet waslysed in PBS containing 0-02% NaN3 (Merck,Darmstadt, Germany), 1% NP40 (LKB,Bromma, Sweden), and 1 mM PMSF (Sigma)for 2 hours at 4°C. The antigen wasprecipitated from the cell lysate as well as fromcell culture supernatant. Immunoprecipitationwas performed according to Schwarting et al.1The molecular weight of the target antigen wasdetermined by running the precipitates fromalveolar macrophages on a 5%-20% poly-acrylamide gradient gel and by running theDHL-1 cell precipitates on a 10% polyacryl-amide gel.

FUNCTIONAL ANALYSISInvestigation of the chemoluminescence res-ponse was performed using the modifiedmethod of Kato.38 Cells for detection ofchemoluminescence were suspended inchemoluminescence medium (DMEM con-taining 50 mM of HEPES, pH 7-4, withoutNaHCO3 and phenol red (Boehringer)).Luminol (Boehringer) was dissolved in PBS (2mg/ml), supplemented with 8 pl triethylamin/ml (Kato). Zymosan particles (Sigma) weresuspended at a concentration of 50 mg/ml.Latex beads (1 1 pm diameter) were adjustedto 6 x 109 particles/ml chemoluminescencemedium. A 0-1 mM solution ofFMLP (Sigma)was used. Sheep erythrocytes optimally sensit-ised with rabbit IgG anti-E antibodies wereobtained from Cordis, Miami, USA. The con-centration of red cells was 108/ml.Chemoluminescence was measured in a six-channel luminescence measuring device (LB9505, Berthold, Wildbad, Germany) at 37°C.Cell suspensions (300 p1) were mixed with I lp1of luminol solution. Phagocytosis was startedafter two minutes of chemoluminescence back-ground recording by adding 10 p1 of phago-cytosis stimulus. The data continuously ob-tained were displayed and recorded on line(Apple II computer).The chemoluminescence response was

measured with untreated monocytes as controland with monocytes incubated with Ber-MAC3 ascites (diluted 1 in 100, 1 in 200, and 1in 500), Fab fragments, and purified immuno-globulin (1-150 Mg/ml). Purified immuno-globulins were obtained by using the Bio-RadAffi-Gel Protein A MAPS-II Kit (Bio-Rad,Richmond, California, USA).For preparation of Fab fragments, purified

immunoglobulins were diluted at a concentra-

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separated from the Fc fragments by purificationwith the Bio-Rad Affi-Gels Protein A MAPS-II Kit.An irrelevant monoclonal antibody belong-

ing to the same subclass-that is, Ki-6735-wasused as a control.

ResultsPRODUCTION OF BER-MAC3Screening of about 500 supematants fromhybridomas generated with a mixture of spleencells containing monocytes, macrophages, andB cells showed several antibodies with a macro-

phage tissue binding pattern. The antibodywith the most selective and strongest reactivitywith tissue macrophages was selected forfurther characterisation. This antibody, desig-nated Ber-MAC3, proved to be of IgGI sub-class and ineffective in routinely processedparaffin wax sections.

IMMUNOHISTOLOGICAL REACTIVITY OFBER-MAC3 IN NORMAL AND DISEASED TISSUEImmunostaining offrozen sections from a largerange of normal human tissues withBer-MAC3 showed that it reacted stronglywith most or all macrophages in all tissuesinvestigated (tables 1 and 2; figs lA-C) withthe following exceptions: most starry skymacrophages of lymphoid follicles (fig 2G); allepithelioid type macrophages, and all multi-

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Ber-MAC3: new monoclonal antibody that defines human monocyte/macrophage differentiation antigen

Table I Reactivity ofBer-MAC3 in normal tissuesdetermined by APAAP immunostaining offrozen sections

Type of tissue/cells Ber-MAC3 reactivity

Spleen:Macrophages of the red pulpMacrophages of the white pulpSinus lining cellsB and T cells

Lymph node and tonsil:Interfollicular macrophagesStarry sky macrophagesSinus histiocytesInterdigitating cellsFollicular dendritic cellsB and T cells

Thymus:Cortical and medullary macrophages +Cortical and medullary epithelialcellsThymic nurse cellsCortical and medullary lymphocytes-

Bone:OsteoblastsOsteoclasts +

Bone marrow:

Granulo-monocytesErythrocytesMegakaryocytesMacrophages

Blood:All types of granulocytesMonocytesT lymphocytesLectin-stimulated T cellsB lymphocytesLectin-stimulated B cellsThrombocytesErythrocytes

Skin:Corium macrophagesLangerhans' cellsEpidermal keratinocytes

Macrophages of other tissues:Liver (von Kupffer cells)ProstateBladderKidneyAdrenal glandAlveolar macrophagesHeartCardiumStomachPancreasOesophagusColonJejunumTesticlesBrainPlacenta

+

+

+

++t

*occasional exceptions.tmicroglia cells.

nucleated giant cell macrophages of Langhans,foreign body, osteoclastic, or Touton type (figs2E, 2G). Follicular dendritic cells (FDC) andinterdigitating cells (IDC) also remainednegative. The non-reactivity of Ber-MAC3with IDC was shown on normal lymph nodesand lymph nodes displaying hyperplastic IDC(dermatophathic lymphadenopathy). Thepresence of IDC in these lymph node sampleswas confirmed byNA 1/34 (CD I a) which recog-nises IDC and Langerhans' cells (figs 1D, IE).Comparative stainings of adjacent sections ofnormal skin and oral mucosa with Ber-MAC3and Nal/34 also showed that Ber-MAC3 didnot react with Langerhans' cells.

REACTIVITY OF BER-MAC3 WITH NORMALHEMATOPOIETIC CELLSReactivity with haematopoietic cells was deter-mined by APAAP immunostaining of smearedperipheral blood and bone marrow cellpreparations, as well as by flow cytometryanalysis of suspended peripheral blood cell

preparations. Comparable results wereobtained. Megakaryocytes, erythrocytes, andgranulocytic and monocytic cells, as well aspurified T and B cells were unreactive with theBer-MAC3 (table 1). T cells stimulated withConA and PHA and B cells stimulated withStaphylococcus aureus and Pokeweed mitogenwere also unreactive. Only a small percentage(less than 5%) of fresh monocytes were weaklypositive with Ber-MAC3. Following isolationof monocytes by Ficoll gradient centrifugationand two hours of adherence on plastic Petridishes, a varying percentage (5-30%) of mon-ocytes was weakly reactive in a granular patternwith Ber-MAC3 (fig 2A). Staining intensityand the percentage of stained monocytessteadily increased up to day 3 of culture, when94% of monocytes were strongly positive in agranular pattern with Ber-MAC3 (fig 2B). Theincrease in antigen density and percentage ofBer-MAC3 positive cells were found whenmonocytes were cultured in normal medium, aswell as in medium containing 10% humanserum, LPS, or IFN-G, or when IFN-Gprimed monocytes were stimulated with LPS.Extended stimulation of monocytes (14 days)in the presence ofhuman serum resulted in theformation of large cells with a macrophage-likeappearance (abundant cytoplasm) as well asmultinucleated giant cells. The macrophage-like cells showed a strong but diffuse cytoplas-mic staining (fig 2C); giant cell macrophageswere Ber-MAC3 negative. The percentage ofBer-MAC3 positive cultured monocytes deter-mined by flow cytometry was the same forviable non-fixed and ethanol-fixed cells, butfluorescence intensity was stronger for fixedcells (table 3). This indicates localisation of theantigen in the cytoplasm as well as on themembrane.

REACTIVITY OF BER-MAC3 WITH SOLID ANDLEUKAEMIC NEOPLASMSBer-MAC3 did not bind with neoplastic cellsof any of the 106 various solid, non-lymphomatous lesions, or tumours (table 4).Reactive and residual mononuclear macro-phages were Ber-MAC3 positive; multinu-cleated macrophages were Ber-MAC3 negative(table 4).Immunolabellingofmany different lymphoid

neoplasms (table 5) showed a positive reactionof the tumour cells with Ber-MAC3 in onlytwo cases of true histiocytic malignancies,

Table 2 Reactivity ofBer-MAC3 with various types ofmacrophages in diseased tissue

Type of lesion/cells Ber-MAC3reactivity

Sinus histiocytosis cells +Macrophages around and in necrotic lesions +Foreign body granulomas*:Mononucleated macrophages +Multinucleated macrophagesTouton's giant cells

Small epithelioid cellgranulomas in toxoplasmosis:Macrophages around the granulomas +Epithelioid type macrophages

Large epithelioid cell granulomas:Macrophage around the granulomas +Epithelioid type macrophagesLanghans giant cells

*Induced by foreign body inclusions; ruptured epidermal cystsor cholesterin crystals.

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Backi, Schwarting, Gerdes, Ernst, Stein

Figure 2 Ber-MAC3staining of (A) freshlyprepared humanmonocytes, (B) monocytescultured three days,(C) macrophages andgiant cells differentiatedfrom monocytes after 14days of culture in thepresence ofhuman serum.(D) and (E)Immunostainings ofadjacent sections of the softtissue sarcoma showed thatthe S-HCL3 positive(D) multinucleatedmacrophages wereunreactive withBer-MAC3 (E).(F) S-HCL3 staining of alymph node affected byPiringer's lymphadenitisand (G) Ber-MAC3staining of adjacent sectionshows that Ber-MAC3reacts with neitherepithelioid typemacrophages (arrow) norwith starry skymacrophages.Immunostaining ofadjacent sections of alymph node affected byHodgkin's disease with(H) Ber-H2 (CD30) and(I) Ber-MAC3 showsthat Ber-MAC3 reactswith macrophages and notwith Ber-H2 positiveHodgkin and Sternberg-Reed cells (arrowed).

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941Ber-MAC3: new monoclonal antibody that defines human monocyte/macrophage differentiation antigen

Table 3 Expression of the Ber-MAC3 on cultured human monocytes*

DayO Day I Day 2 Day 3 Day 14

Viabk cells:Percentage of positive cellst 7 40 55 72 ND$Mean channel of fluorescence intensity ND ND 48 125 ND

Fixed cells:Percentage of positive cells< 7 37 55 78 NDMean channel of fluorescence intensity ND ND 125 137 NDPercentage of positive celstt 5/0-30tl 39 81 94 451

*In percentage of the analysed cells.Monocytes were cultured in normal medium without additives.tDetermined by flow cytometry.$ND = not determined.ttDetermined by APAAP-staining; the percentage of monocytes stained with mAb Ber-MAC3 was determined by comparison ofexpression ofmAb Ki-M6, which recognises all resting monocytes.$$<5% on fresh blood monocytes; 0-30% on monocytes following Ficoll centrifugation and adherence 2 h on Petri dishes.¶Mononuclear cells positive; multinucleated giant cells negative.

Table 4 Reactivity ofBer-MAC3 with non-lymphomatous lesions or neoplasms

ReactiveReactive/residual multinucleated Diseasedl

Type of lesion/neoplasm No of cases macrophages macrophages neoplastic cells

Histocytosis X 5 + - -(Langerhans' cell histiocytosis)

Malignant fibrous histiocytoma 7 +Osteosarcoma 3 + - _Rhabdo/leiomyosarcoma 15 + *Carcinoma 76 + *

*Absent.

whereas the neoplastic cells of all types of the Ml, M2, or M3 group according to the FAB280 malignant lymphomas were negative. The classification showed reactivity with Ber-lymphomas studied included 52 cases of MAC3. The number of Ber-MAC3 positiveHodgkin's disease and 41 cases ofKi-I positive cells found in M4, M4/M5, and M5 leukaemiasanaplastic large cell lymphomas erroneously varied between 0% and 100%. All cases ofregarded as true histiocytic lymphoma. All or myeloid leukaemia showing more than 30%nearly all reactive mononuclear macrophages positive cells belonged to the monocytic M5present in these tumours were strongly reactive type.with Ber-MAC3, except those present in theneoplastic follicles of follicular lymphomas. REACTIVITY OF BER-MAC3 WITH ESTABLISHEDEpithelioid type macrophages occurring in HUMAN CELL LINESlymphoepithelioid (Lennert's) lymphoma and Ber-MAC3 reacts with only 1% of cells of theHodgkin's disease were Ber-MAC3-negative, HL-60 cell line, a myeloid leukaemia cell lineas were their counterparts in reactive lesions. consisting of granulocyte precursor cells (tableThe reactivity of Ber-MAC3 on myeloid 7); the percentage of positive cells did not

leukaemias was determined by fluorescence change after stimulation with TPA. Ber-staining ofbone marrow and in some cases with MAC3 did not react with the myeloid/eryth-the APAAP method. Ber-MAC3 reacted with roid leukaemia cell lines KG-1 and K562.only 5% of cells in four of the 10 MI Although stimulation of the myeloid sarcomaleukaemias investigated (table 6), and none of cell line U937 and the monocytic leukaemia cellthe other myeloid leukaemias belonging to the line THP-1 with TPA and IFN-G for 14 days

Table S Reactivity ofBer-MAC3 with true histiocytic malignancies and malignant lymphomas

Reactive/residualType of lymphoma No of cases macrophages Tumour cells

True histiocytic malignancy 2 all + +Low grade B-NHL:

Diffuse 45 all + -

Follicular 32 IF*-* EFt + -

Hairy cell leukaemia 10 all + -

High grade B-NHL:Burkitt's lymphoma 3 all + -

Non-anaplastic large cell lymphoma 38 all + -

Ki-1 positive B-ALC lymphoma 9 all + -

T-NHL:Lymphoblastic (precursor T) 3 all + -

Cutaneous T cell lymphoma 28 all + -

Pleomorphic T cell lymphoma 18 all + -

AILD-type T cell lymphoma 5 all + -

Lymphoepithelioid T cell (Lennert's) lymphoma 5 cm$ +; epm$$- -

Ki-1 positive T-ALC lymphoma 22 all + -

Ki-1 positive O-ALC lymphoma 10 all + -

Hodgkin's disease 52 cm$ +; epmft- -

*IF = intrafollicular macrophages identified with CD11c, CD14, and CD68.tEF = extrafollicular macrophages.$cm = common tissue macrophages.ttepm = epithelioid type macrophages.


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Backe, Schwarting, Gerdes, Ernst, Stein

Table 6 Expression ofBer-MAC3 on myeloidleukaemias

Reactivity:FAB No ofclassification cases Positive cases Positive cells %

Ml 10 0* < 5M2 4 0M3 1 0 -

M4 6 3 <103 <30

M5 9 3 < 2012 >60

*Fewer than 5% positive leukaemic cells were found in fourcases ofMI type.Reactivity was determined by flow cytometry of bone marrowcells or by APAAP staining of cytospin preparations.

resulted in the formation of cells similar tomacrophages in appearance and multinu-cleated giant cells, the cells were unreactivewith Ber-MAC3. DHL-1,37 which is referredto as a histiocytic lymphoma cell line, wasstrongly positive for Ber-MAC3. No T cell-derived cell lines, lymphoblastoid lines,Hodgkin's disease-derived lines,3843 or carcin-oma lines" showed any reactivity with Ber-MAC3.

BIOCHEMICAL CHARACTERISATION OF THETARGET ANTIGENSodium dodecyl polyacrylamide gel electro-phoresis (SDS-PAGE) of immunoprecipitatesobtained with Ber-MAC3 from lysates of 3H-borohydride surface labelled alveolar macro-phages and lysates from internally labelled (35S-methionine) DHL-1 cell line cells both showeda single glycopolypeptide chain of 140 kilo-daltons under reducing conditions and 110kilodaltons under non-reducing conditions.The 140 band and the 110 band could also beprecipitated from the supernatant ofthe DHL-1 cell line, indicating that the Ber-MAC3molecule is released by this cell line (figs 3A-B).

FUNCTIONAL ANALYSISInitial investigations using varying concentra-tions of Ber-MAC3 ascites suggested that theBer-MAC3 antibody has an inhibitory effect onthe chemoluminescence response of mono-cytes. Further experiments with purified Ber-MAC3 antibody and Fab fragments, however,did not show any significant inhibitory effect onthe generation of oxygen radicals. Thisindicates that the reduction of thechemoluminescence response observed withthe crude ascites was not caused by Ber-MAC3antibody, but by other constituents present inthe ascites. The culturing of monocytes in thepresence of Ber-MAC3 did not prevent theirdifferentiation into macrophage-like stronglyBer-MAC3 positive cells, and afterwards intoBer-MAC3 negative multinucleated giant celltype macrophages.

DiscussionThe most important features ofBer-MAC3 areas follows:1 It recognises a single glycopolypeptide chainof 140 kilodaltons which is located in the cellmembrane, and which probably containsintramolecular disulfide bridges as its molecularweight is 30 000 less SDS-PAGE under non-reducing conditions.

2 It is specific for monocytes/macrophages anddoes not cross-react with any other cell type.3 Presence of the Ber-MAC3 reactive antigenis related to a certain stage of monocyte/macro-phage differentiation.

According to the molecular weight and bind-ing pattern to cell lines and a wide range oftissues, Ber-MAC3 seems to be different fromall reported macrophage antibodies. Ber-MAC3 resembled other monocyte/macro-phage-specific antibodies in its strong bindingto macrophages, but differed from these in itsmolecular weight and in its non-reactivity withgranulocytes or monocytes, most starry skymacrophages, epithelioid type macrophagesand multinucleated macrophages, lymphoidsubpopulations, TPA-treated cells of theHL60, THP1, and U937 cell lines, folliculardendritic cells and interdigitating reticulumcells. The only monoclonal antibody thatshows a tissue binding similar to that seen withBer-MAC3 is Ki-M8. The latter, however,differs from Ber-MAC3 in precipitating anantigen of lower molecular weight (30 and 32kilodaltons), by its reactivity with the TPA-stimulated U937 and HL60 cell line and giantcells, as well as its inhibitory effect on thegeneration of reactive oxidants.'

Table 7 Reactivity ofBer-MAC3 with establishedhuman cell lines

Cell line Positive cells %

Myeloid leukaemia cell lines:KG-I 0K-562 0HL-60 1Hl-60 IFN-G stimulated* 1HL-60 TPA stimulated* 1

Monocytic leukaemia cell line:THP-1 < 1THP-1-IFN-G stimulated < ITHP-1 TPA stimulated* < 1THP-1 LPS stimulated < 1

Myeloid sarcoma cell linet:U-937 0U-937 IFN-G stimulated 0U-937 TPA stimulated* 0U-937 stimulated 0

Histiocytic cell line:DHL-1 100

Hairy cell leukaemia cell line:JOK-1 0

Hodgkin's disease derived lines:L428 0L540 0L591 0Co. 0KMH2 0Ho. 0

Lymphoblastoid cell lines:CESS 0B958 0BJA-B 0

Burkitt's lymphoma derived cell line:DAUDI 0RAJI 0

T cell-derived cell lines:MOLT4 0HPB-ALL 0HUT 102 0

Carcinoma cell lines:AA431 (vulva) 0MDA-MB231 (breast) 0MCF-7 (breast) 0ALAB (breast) 0COLO 357 (pancreas) 0ASPC-1 (pancreas) 0

All cell lines were tested with the APAAP method.*Expression of the p150/95 (CD Ilc) complex detected by S-HCL3 was used as a control for the effect of TPA stimulation.tErroneously classified as histiocytic cell line.

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Ber-MA C3: new monoclonal antibody that defines human monocyte/macrophage differentiation antigen

Figure 3AImmunoprecipitation ofBer-MAC3 antigen from3H borohydride-labelledalveolar macrophages.Immunoprecipitates wereanalysed by SDS-PAGEusing a 5%-20%polyacrylamide gradientgel under reducing andnon-reducing conditions.Ber-MAC3 precipitateslanes 2 and 4; precipitateswuith an irrelevantmonoclonal antibody of thesame subclass, lanes I and3.

Figure 3BImmunoprecipitation ofBer-MAC3 antigen from-5S-methionine labelledDHL-1 cell line.Molecular weight wasdetermined by running a10% polyacrylamide gelunder reducing and non-reducing conditions,Ber-MAC3 precipitatefrom culture supernatantlanes 2, 6, from cell lysatelanes 4, 8. Controlprecipitates with anirrelevant monoclonalantibody of the samesubclass lanes 1, 3, 5 and7.

Red KD Non-red

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to 4-

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1 2 3 4 5 6 7 8

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14

Ber-MAC3 was submitted to the 4th Inter-national Workshop and Conference onLeucocyte Differentiation Antigens in Vienna,1989, and compared with clustered and newanti-macrophage antibodies by many differentlaboratories. The result of the workshop andconference studies was that Ber-MAC3 couldnot be assigned to any of the established or newantibody clusters. This supports the view thatBer-MAC3 recognises a new, previouslyundefined macrophage-associated molecule.The most striking characteristic of Ber-

MAC3 is its selective reactivity with monocytesand macrophages of a certain stage of differen-tiation. It stains weakly only a minority of non-activated (fresh) blood monocytes, but stainsvery intensely monocytes stimulated by LPS,IFN-G, and/or plastic adherence, as well astissue macrophages. If the in vitro activatedmonocytes are induced by prolonged culturingto differentiate further into multinucleatedgiant cell macrophages they become unreactivewith Ber-MAC3. The same loss ofBer-MAC3reactivity seems to take place in in vivo macro-phage differentiation, as multinucleated macro-phages of foreign body type, osteoclastic type,Langhans' type, etc., were not labelled by thismonoclonal antibody. The differentiation oftissue macrophages into starry sky macro-phages and epithelioid macrophages alsoproved to be associated with the loss of Ber-MAC3 reactivity. Interestingly, the loss ofBer-MAC3 reactivity of macrophages was alsoseen in certain malignant tumours if a differen-tiation into starry sky-like macrophages, suchas follicular lymphoma, into epithelioid typemacrophages (Lennert's lymphoma, certaincases of Hodgkin's disease), into osteoclastictype or other multinucleated giant cells, such asosteosarcoma, malignant fibrous histiocytoma,etc., occurs. These observations are well in linewith the concept that monocytes undergo adifferentiation step when they emigrate fromthe blood vessels and settle in the tissue asresident tissue macrophages, and that thesemacrophages can further transform into starry

" 4-

I-

sky type, epithelioid type, or various multi-nucleated type macrophages at appropriatesignals. The differentiation step related toemigration is associated with a steep increase ofBer-MAC3 expression. The differentiationstage defined by transformation into epithelioidtype or multinucleated type macrophages isaccompanied by the loss of the Ber-MAC3molecule. In principle, this sequence of eventsis in keeping with other macrophage activationstudies which led to the concept that monocyte/macrophage differentiation takes place instages.45 46

It is conceivable that for the transformationof monocytes into epithelioid type multi-nucleated macrophages, the expression of theBer-MAC3 molecule or the differentiationstage associated with the strong expression ofBer-MAC3 molecule is required. If the formeris true the Ber-MAC3 antigen may represent orbe a part (receptor) of the signal transductionwhich induces the transformation intoepithelioid type macrophages or fusion of sin-gle macrophages to multinucleated macro-phages. This is incompatible, however, withthe findings on the stimulated U937 and THP-1 cell line. Stimulation of these cell lines withTPA and IFN-G leads to the expression ofmany monocyte/macrophage associatedmolecules such as CD 11 c, non-specific esterase,etc,47-49 and, finally, to the formation of multi-nucleated giant cells but not to the appearanceof the Ber-MAC3 molecule. Furthermore, theBer-MAC3 antibody did not inhibit the invitro transformation of monocytes into multi-nucleated macrophages.A further remarkable feature of Ber-

MAC3 is its non-reactivity with cells ofgranulopoiesis, erythropoiesis, and mega-karyopoiesis, but strong reactivity with bonemarrow macrophages. This makes it a suitabletool for isolating bone marrow macrophages bypositive selection or for monitoring such anattempt by negative selection. Other macro-phage monoclonal antibodies are less suitablefor this purpose, as they either recognise

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immature or mature bone marrow cells otherthan macrophages, or they react with a cyto-plasmic but not with a surface constituent.The non-reactivity of Ber-MAC3 with acute

leukaemias of lymphoblastic and non-lym-phoblastic subtype-that is, Ml, M2, or M3subtypes (n = 30), according to the FABclassification, confirms the absence of the Ber-MAC3 antigen in lymphoid and granulopoieticprecursor cells. All cases of acute leukaemiawith more than 30% Ber-MAC3 reactive cellsbelonged to the M5 type, indicating that Ber-MAC3 keeps its specificity even after malignanttransformation. The selective reactivity of Ber-MAC3 with monocytes/macrophages is furthersupported by the observation that Ber-MAC3did not stain any malignant cells in 218 cases ofNHL, 76 carcinomas, various sarcomas (tables4 and 5) and permanent cell lines derived fromneoplastic or vitally transformed epithelialcells, B or T cells, or other cell types (table 7).The tumour cells of Hodgkin's disease have

been regarded as deriving from macro-phages,5°51 but the consistent non-reactivity ofthese tumour cells with Ber-MAC3 corre-sponds with the non-reactivity of other macro-phage reagents-for example, CDl1 c, CD 14,CD68, and lysozyme-with Stemnberg-Reedcells, and thus provides further evidenceagainst the monocyte/macrophage nature ofthese cells. Likewise, Ki-1 positive anaplasticlarge cell lymphomas, which formerly had beenregarded as true malignant histiocytic lym-phomas,30 were negative with Ber-MAC3. Theabsence of Ber-MAC3 antigen from thetumour cells fits in with the absence of othermacrophage antigens, or the presence of T cellor B cell antigens, as well as T cell receptor orimmunoglobulin gene rearrangement in thesetumours, and thus confirms the non-histiocyticorigin of Ki-1 positive anaplastic large celllymphomas.52"3The only two solid neoplasms with which

Ber-MAC3 gave a strongly positive reactionwere the two cases of true histiocytic malignan-cies. This selective reactivity of Ber-MAC3with histiocytic tumours in conjunction with itsstrong binding to the histiocytic lymphoma lineDHL-1 shows the value of Ber-MAC3 foridentifying neoplasms oftrue histiocytic origin.

In summary, Ber-MAC3 seems to distin-guish between activated and non-activatedmonocytes, as well as between common tissuemacrophages on the one hand, and starry skymacrophages, epithelioid type, and multinu-cleated type macrophages on the other hand.Ber-MAC3 is a suitable reagent for the diag-nosis of myelomonocytic and monocytic leuk-aemias (M4/M5 subtype), as well as for thedistinction of non-histiocytic neoplasms fromthose of true histiocytic origin. Thus Ber-MAC3 may be of interest for studies aimed atelucidating the regulation of differentiation ofnormal and neoplastic cells of the mononuclearphagocyte system and for the correlation ofmonocyte/macrophage functions with differen-tiation.

AddendumSequential immunoprecipitation and parallel

labelling studies with the monoclonalantibodies Ber-MAC3 and Ki-M8, which wereperformed after this article had been acceptedfor publication showed that Ki-M8, unlike thereported data,' recognises the same 140 kilodal-ton molecular target and has all the othercharacteristics of Ber-MAC3. Cross-blockingexperiments have shown that Ber-MAC3 andKi-M8 react with different epitopes on the samemolecule.

This work was supported by the Deutsche Krebshilfe e. V. Thepublication contains parts of the doctoral thesis of Eva Backe.

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