[CANCER RESEARCH 48, 82-88, January 1. 1988]

Effect of Endogenous and Exogenous Interferons on the Differentiation ofHuman Monocyte Cell Line U9371

Ugo Testa, Didier Ferbus,2 Marco Gabbianelli, Brunella Pascucci, Giovanni Boccoli, Fawzia Louache, and

Ming Nguy ThangINSERM, Unité245 et Centre National de la Recherche Scientifique, VA.C. ¡13,Centre INSERM, HôpitalSt-Antoine, 184, rue du Faubourg St-Antoine, 75571 ParisCedex 12, France ¡D.F., M. N. T.]; INSERM, Unité91, HôpitalHenri Mondar, SI, avenue du Maréchalde Lattre de Tassigny, 94010 Creteil, France [F. L.J; andInstituto Superiore di Sanità,Laboratorio di Ematologia, Viale Regina Elena, 299-00161 Roma, Italy [U. T., M. G., B. P., G. B.J

ABSTRACT

The effect of interferons (IFNs) on the differentiation of hematopoieticcells was examined with the human monocyte cell line U937. Thedifferentiation of U937 was induced by hydroxyvitamin I), and wasevaluated through the study of specific markers.

The induction of the U937 differentiation was associated with aproduction of IFN and with a marked increase in (2'5') oligoadenylate

synthetase. Addition of anti-IFN-a//3 antibodies inhibited the enhancement of (2'5') oligoadenylate synthetase and reduced the inhibitory effect

of hydroxyvitantin I), on cell growth. Nevertheless, neutralization ofendogenous IFN excreted during U937 cell maturation did not modifythe expression of the differentiation markers examined. Exogenous natural IFN-a, IFN-/Õ,or recombinant (r) 11-N-7, when added to the culturemedium, did not promote a "global" U937 differentiation. Most of the

differentiation markers, except for reduction of nitroblue-tetrazolium,were not induced by IFN-a or -ß.However, ribN-T was able to induce

the appearance of several monocytic membrane markers at an extentcomparable or slightly inferior to that elicited by hydroxyvitamin D.i.Different effects on the expression of HLA antigens were obtained withthese IFNs: IFN-a or -ßenhanced mainly class I HLA antigen expression, whereas rIFN-7 increased selectively the expression of class IIHLA IK 'I but not HLA DR antigens. In contrast, phytohemagglutinin-

leukocyte conditioned medium elicited a marked and selective enhancement of the expression of HLA-DR antigens. This induction of HLAIMI antigens by rIFN-f was not observed in two other leukemic celllines (III ,6(1and HEL). The present study shows that IFN-a or -ßmay

participate in the antiproliferative effect occurring during cellular differentiation, while IFN-7 may be involved in the induction of the expression

of specific monocytic markers involved in cellular immunoregulation.

INTRODUCTION

In the past few years, evidence has accumulated regardingthe possible involvement of IFN3 in cellular regulation, includ

ing growth and differentiation [reviewed by Taylor et al. (i)].Recently, endogenous IFN production (2) has been detectedduring cell differentiation and the induction of hematopoieticcell differentiation associated with a marked enhancement ofIFN-induced 2-5 A synthetase has been reported (3-6). Thus,it has been suggested that interferon plays a general role ingrowth and differentiation via 2-5 A synthetase induction.

The hematopoietic system represents a unique model forinvestigation of the biochemical events associated with cellular

Received 3/17/87; revised 9/14/87; accepted 9/30/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This research was supported in part by grants from INSERM (U.245, U.91),

Centre National de la Recherche Scientifique (U.A.C. 113), Fondation pour laRecherche Médicale,and Associazione Italiana per la Ricerca sul Cancro.

2To whom requests for reprints should be addressed, at U.245 INSERM,Centre INSERM, Hôpital St-Antoine, 184. rue du Faubourg St-Antoine,75571 Paris Cedex 12, France.

3The abbreviations used are: IFN, interferon; 2-5 A synthetase, (2'5') oligoadenylate synthetase; TPA, 12-O-tetradecanoylphorbol-13-acetate; hydroxy-vit D3,la,25 dihydroxyvitamin D3; NBT, nitroblue tetrazolium; r!FN->, recombinant -yinterferon; FMLP, formyl-methionyl-leucyl-phenylalanine; PHA-LCM, phyto-hemagglutinin-stimulated leukocyte conditioned medium; poly(I)-poly(C), co-polymer of polyinosinic and polycytidylic acid; III ,60. human promyelocytic cellline; TCA, trichloroacetic acid.

differentiation. The dtta concerning the possible role of interferon and 2-5 A synthetase in the process of hematopoietic celldifferentiation afford evidence about different actions of thevarious IFNs on different aspects of cellular differentiation (7-9). Thus, some studies have shown a role of endogenous IFNin inducing growth arrest, a phenomenon constantly coupledwith cellular differentiation/maturation (10); other studies,however, provide evidence in favor of a role of IFN-/32 as apromoter of growth and differentiation of B lymphocytes: infact, IFN-02 is capable of inducing synthesis of immunoglobulinby Epstein-Barr virus-transformed human B-cells (11, 12).Some studies suggest that IFN-7 (13-14) or (2'5') oligoaden-

ylates (15) might help to induce differentiation of the humanpromonocytic U937 cell line derived from pleural fluid of apatient with diffuse histiocytic lymphoma (16). hydroxy-vit D3,a supposedly physiological inducer, induced complete differentiation of the U937 cells in mature monocytes (17). We havepreviously shown that this induction of U937 differentiationwas associated with a production of interferon in the medium(10 units/ml) and a marked increase in 2-5 A synthetase, whichwas essentially dependent on an endogenous production ofinterferon since the enzyme activity was inhibited by anti-IFNantibodies ( 18). These results were in agreement with a previousstudy by Yarden et al. (19) showing that the induction of 2-5 Asynthetase observed in U937 cells induced to differentiate byphorbol esters mainly results from the autogenous productionof IFN-0.

We decided to reinvestigate these conflicting results by studying the effect of endogenous and exogenous IFN-a, -ß,and -7on the expressions of 12 differentiation markers and the induction of the major histocompatibility complex during the differentiation of U937 cells.

MATERIALS AND METHODS

Cells and Cell Culture. The human histiocytic lymphoma cell lineU937 (16) was cultured in RPMI 1640 medium supplemented with10% heat-inactivated fetal calf serum (Boehringer, Mannheim, Germany). All cells were inoculated at 1 x 10* cells/ml and grew exponen

tially for 3-4 days. The inducer hydroxy-vit D, was dissolved in absolute

ethanol and added to the cultures at a final concentration of 500 ini.hydroxy-vit D3 reduced the rate of cell proliferation. When the U937cells were induced with hydroxy-vit D3 on the fourth day of culture, thecells were diluted 2-fold with fresh medium and fresh hydroxy-vit D3

was added in the culture medium to maintain the initial concentration.Preliminary dose-response experiments showed that hydroxy-vit D3 at

the doses used is not cytotoxic (cell viability > 95% until the sixth dayof culture).

Assay of Properties of Differentiated Cells. To evaluate the differentiation of U937 cells, several tests were performed. Cells with C3 andFc receptors were detected according to the method of Lotem and Sachs(20) by measuring rosette formation with sheep erythrocytes coatedwith rabbit anti-sheep erythrocyte antibody and mouse complement orIgG.

Reduction of NBT was assayed using a kit from Sigma (St. Louis,MO) according to the conditions recommended by the manufacturer.

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The percentage of cells containing intracellular blue-black formazandeposits was determined.

o-Naphthyl acetate esterase activity was examined in cytocentrifugepreparations using a Sigma kit. Binding of monoclonal antibodies tothe cell surface antigens was determined by both indirect immunofluo-rescence and measuring the binding of a second [125I]-labeledantimouseantibody. The monoclonal antibody MO2 was obtained from Coultron-ics France. OKM1 and OKM5 were obtained from Ortho Lab., MAS072 from Sera-Lab (Cambridge, United Kingdom), TEC MGI, NK1,and M1 from Technogenetics (Milano, Italy), and Leu M3 from Becton-Dickinson (USA). MO2 was a monoclonal antibody, entirely restrictedto the monocyte macrophage series. It recognizes a true monocyteantigen because it was resynthesized after protease treatment of mono-cytes (21). OKM1, TEC Ml, and MAS 072 were distinct antigens,specific for monocyte-macrophages (22, 23). OKM5 monoclonal antibody recognizes an antigen present both on mature monocytes andplatelets, TEC MG1 monoclonal antibody specifically reacts with anantigen present on the surface of both granulocytic and monocytic cells.Monoclonal antibodies which specifically recognize HLA classes I andII antigens were obtained from Technogenetics (Milano, Italy). Theseantibodies were TEC HLA A, B, C; TEC HLA-DR; TEC HLA DC1;TEC anti ft-microglobulin, whose specificity was previously reported(24-^26). The secondary antibodies [goat anti-mouse ¡mmunoglobulinsF(ab')2 fragment] conjugated with fluorescein isothiocyanate were pur

chased from Cappel Laboratories, Inc., West Chester, PA.For indirect fluorescence the following procedure was used. The cells

were first washed 3x in Hanks' saline solution and then incubated 60min at 4 ( with 100 ul of an optimal dilution (in Hanks' saline solution

containing I mg/ml bovine serum albumin of each monoclonal antibody. The cells, washed at 4"( ' in Hanks' saline solution, were incubatedfor 60 min at 4°Cwith fluorescein isothiocyanate-labeled F(ab')z frag

ments of immunoadsorbent-purified sheep antibodies against mouseIgGs. After 3 additional washes, the cells were mounted on slides in50% glycerol in phosphate-buffered saline.

The proportion of stained cells was scored in incident light with aLeitz standard universal fluorescent microscope equipped with a set offilters for narrow fluorescence. For the quantitative assay of monoclonalantibodies binding to the cells, the cells were first incubated as forindirect immunofluorescence. After 3 washes with cold Hanks' salinesolution, cells were incubated for 60 min at 4°Cwith 0.2 ml of '"I-labeled I (;ib'): sheep anti-mouse immunoglobulins appropriately di

luted (50 ng/ml). At the end of the incubation, the cells were layeredon a cushion of dibutyl phthalate oil (density, 1.025) and then centri-fuged at 10,000 x g for 2 min. The supernatant and most of the oilwere removed by aspiration, and the radioactivity in the cell pellet wasmeasured in a gamma counter. In order to measure the "nonspecific"binding of I25l-labeled F(ab')2 sheep anti-mouse ¡mmunoglobulins to

U937 cells, monoclonal antibodies were replaced by mouse myelomaprotein TEPC 183 (IgM-K). Lysosome was assayed (urbimetrically(27) in cells lysed with 0.5% Triton X-100 (Sigma) using egg-whitelysozyme (Sigma) as standard. Results are expressed in units permicrogram of egg lysozyme.

Generation of Superoxide was measured by the ability of this aniónto reduce ferricytochrome C (28). The stimulus for generation wasprovided by TPA. One ml of Hanks' balanced salt solution (pH 7.40)containing 5 x IO6 cells, 0.12 mM ferricytochrome C (Sigma) and,

where appropriate, 100 ng freshly diluted TPA with or without 60 ngSuperoxide dismutase (Sigma), was incubated for 60 min at 37°C.The

reaction was stopped by submerging the tubes in an ice bath. Cells werepelleted by centrifugation at 4°Cand 500 x g for 5 min and the

absorbance for the supernatant was then determined at 550 nm in aGilford 240 spectrophotometer. The quantity of Superoxide generatedwas expressed as the A/60 min/5 x 10* cells which is inhibited by 60

/IRSuperoxide dismutase.Assay for FMLP Receptors. Two million U937 cells were incubated

in 15 nM [3H]FMLP (New England Nuclear, Boston, MA) in a total

volume of 0.2 ml in the presence or absence of 10 MMunlabeled FMLP(Sigma). After 3 h at 4"( ', the cell suspensions were rapidly filtered

onto glass fiber discs (Whatman GF/C; Whatman, Inc., Clifton, NJ)and washed with 30 ml of 4*C phosphate-buffered saline. Radioactivity

on the discs was measured by liquid scintillation spectroscopy. Specificbinding was defined as the total amount of labeled FMLP bound minusthe amount bound in the presence of an excess of unlabeled cold FMLP.Specific binding measured as cpm was converted to sites per cell.

Assay of 2-5 A Synthetase Activity. The procedure used has alreadybeen described (29). Briefly, cells were lysed in a buffer containing 20mM Tris-HCl, pH 8.0, 5 mM magnesium acetate, 10% (v/v) glycerol,and 0.5% (v/v) of NP40. The lysate was centrifuged at 10,000 x g for10 min. The supernatant was added to a suspension of poly(I)-poly(C)agarose in the same buffer. After 10 min of enzyme binding to thepoly(I) •poly(C) ligand, the supernatant was discarded by centrifugation.The poly(I)-poly(C) agarose beads were washed twice with the samebuffer.

To the pellet of poly(I) •poly(C) agarose, 10 /¿Iof an incubationmixture were added to make final concentrations, in 25 ¿il,of 3 mM[a-"P]ATP (0.15 ¿¿Ci/incubation),20 mM magnesium acetate, 1 mM

dithiothreitol, 0.1 mg/ml bovine serum albumin, 0.1 mg/ml creatinekinase and 6 mM phosphocreatine. The mixture was incubated at 37°C

for 2 h. The reaction products were analyzed by thin-layer chromatog-raphy on polyethyleneimine-cellulose; 3 n\ of each mixture were spottedand the chromatogram developed with 2 M Tris-HCl, pH 8.6.

Spots corresponding to ATP and (2'5') oligoadenylates located by

autoradiography were cut and their radioactivity was determined byCerenkov counting. One unit of 2-5 A synthetase was defined as theamount incorporating 1 nmol AMP residues into 2'5' oligoadenylates/min at 37°C.

Protein concentrations were determined according to the method ofSpector (30).

|"I l| I li\midine Incorporation. DNA synthesis was measured as | '111

thymidine incorporation into acid-precipitable materials. Two nC\ of[mefAy/-3H]thymidine (40 Ci/mmol; New England Nuclear) were addedto U937 cells grown in liquid suspension for 2 h at 37°C.The cells,

washed 3 times in cold phosphate-buffered saline, were lysed by incubation for 10 min at 37"C in 0.1% sodium dodecyl sulfate (Bio-Rad

Laboratories). One hundred n\ 0.1% sodium dodecyl sulfate for 1 x10' cells were added and to this an identical volume of cold 10% TCAwas added. After 10 min at 4°C,the TCA precipitates were collected

on 2.4-cm Whatman GF-C filters and washed 3 times with 5 ml cold10% TCA. The filters were then dried, treated for 10 min at 37°Cwith

0.5 ml NCS solubilizer (Amersham/Searle), and counted in a PPO-POPOP-toluene scintillation mixture.

IFNs and Anti-IFN Antibodies. IFN-o was purchased from InstitutPasteur Production, Rueil Malmaison, Paris, France, IFN-/3 was donated by UPSA Laboratoire, France, and recombinant IFN-7 was agift from Biogen. Their respective specific activities were IO6, IO7,and1.4 x 10' units/mg protein. Anti-IFN-a was kindly provided by Dr. K.Berg, Denmark, and anti-IFN-,8 by Dr. A. Billiau, Belgium. The respective neutralizing titers of these antibodies were 20,000 and 40,000units/ml. Anti-IFN-7 was purchased from Cistron Co. (Eurogenetics,Turin, Italy) and possesses a neutralizing titer corresponding to 10,000units/ml.

Preliminary dose-response experiments showed that IFNs at thedoses used (10-500 units/ml) were noncytotoxic for U937 cells; athigher concentrations (1000 or more units/ml) all the types of IFN (a,ß,or 7) were significantly cytotoxic.

Interferon-7 Assay. IFN-7 was measured by a radioimmunoassay(IMRX; Centocor, Inc., Malvern, PA) based upon the use of polystyrenebeads coated with a mouse monoclonal antibody specific for humanIFN-7, incubated with test supernatants, and subsequently washed, andof a second monoclonal antibody to human IFN-7 labeled with 125Ithatreacts with IFN-7 bound to the beads. The radioactivity bound to thebeads, quantified in a gamma scintillation counter, is proportional tothe concentration of IFN-7 ¡n'he specimen, within the working rangeof the assay. Results were expressed in units calculated using a preparation of recombinant IFN-7 as standard reference.

RESULTSEffect of Anti-a and -fi Interferon Antibodies on the Differen

tiation of U937 Cells Induced by hydroxy-vit D3. Cellular differentiation is characterized by 2 related phenomena: (a) progres-

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Table 1 Effect ofanli-ßInterferon antiserum on differentiation ofU937 cellsDifferentiation was initiated by exposure of U937 cells (1.105 cells/ml) with hydroxy vit D3 (500 nM). Anti-IFN antibodies were added to the culture medium

immediately after the addition of the inducer of differentiation, at a final dilution sufficient to neutralize 200 units/ml of corresponding IFN. On the fourth day ofculture, cells were diluted 2-fold with fresh medium plus antiserum. Differentiation markers were examined on the sixth day, and the 2-5 A synthetase activity wastested in cell extracts as described in "Materials and Methods"; the enzyme activity is expressed in units/mg protein. Results are mean ±SD obtained in 5 separate

experiments.

a-Naphthyl acetate esterase (% positive cells)NBT reduction (% positive cells)Lysozyme Gig/mg of protein)MO2 binding (% positive cells)MAS O72 binding (% positive cells)Control0.14

1.20.10.1Ami-,!

anti-serum0.2

31.10.10.1la,25(OH)2-vita-

minIh85

952.3

5947la,25(OH)2-vi-

tamin D, andanii-fj antiserum82

902.3

5549

2-5 A synthetase (units/mg) 0.98 ±0.07 0.62 ±0.08 4.5 ±0.15 1.02 ±0.13

sive and coordinate acquisition of a panel of specific markers;(b) progressive loss of cellular growth capacities. Since inter-

feron is produced during differentiation of U937 induced byhydroxy-vit D3 (18), the question raised was on the role thatinterferon may play in inducing the process of cellular differentiation. IFN-7 was not detectable by a very sensitive radio-immunoassay in the culture medium of control as well as ofhydroxy-vit D3 treated U937 cells. Thus, we tested whether theaddition of both hydroxy-vit D3 and IFN antibodies couldmodify the induction of differentiation of U937 cells. In orderto evaluate the differentiation of U937 cells, several differentiation markers were investigated: a-naphthyl acetate esteraseactivity, NBT reduction, lysozyme activity, FMLP receptors,and M02, OKM5, Ml and MG1 membrane antigens recognized by monoclonal antibodies. These markers are expressedlittle or not at all in undifferentiated promonocytic U937 cellsbut are highly expressed in mature monocytes. We have previously shown that hydroxy-vit D3 induced both complete differentiation of U937 cells into mature monocyte and a very markedincrease in 2-5 A synthetase (18). Thus, 2-5 A synthetase(interferon marker) can be considered as a biochemical markerof cell status and differentiation in the monocyte line. Thisenhancement of 2-5 A synthetase activity was dependent onendogenous interferon production because addition of anti-IFN-0 antibodies reduced the enzyme level to that of nonin-duced cells (Table 1). The doses of antibodies used couldneutralize 20 times the interferon level detected in the mediumculture (10 units/ml) of U937 induced to differentiate by hydroxy-vit D3.

Our results clearly showed that the addition of anti-IFNantibodies neutralized all the endogenous IFN produced byhydroxy-vit D3 induced U937 cells. However, the anti-IFN-/Santibodies added contemporaneously with hydroxy-vit D3 wereunable to inhibit the appearance of the monocytic, membrane,or cytoplasmic markers induced by hydroxy-vit D3 (Table 1).Similarly, the contemporaneous addition of hydroxy-vit D3 anda neutralizing anti IFN-7 antibody did not prevent the appearance of monocyte markers induced by hydroxy-vit D3 (data notshown). Thus, endogenous interferons are not necessary for theinduction of most of the differentiation markers examined.

Inhibition of Cell Growth during Differentiation. Induction ofcellular differentiation is usually associated with a loss of theproliferative potential. Thus, hydroxy-vit D3 induced a reduction of cell growth of U937 cells which became particularlyevident after the second day of culture (Fig. \A).

Hydroxy-vit D3 reduced by 55% the rate of cell growth ofU937 cells and the addition contemporaneously of anti-IFN-a/ßantibodies in the culture medium to hydroxy-vit D3 reducedthe inhibitory effect of hydroxy-vit D3 on cell growth. The

12C10

8

c 4K 3LU •»m23 2

mS

A

B

1234

TIME (Day)

Fig. 1. A, effect of ami-«and anti-ßIFN antibodies on the induction of growthinhibition during differentiation. U937 cells were exposed to hydroxy-vit D3 (X),hydroxy-vit D, + anti-a and ami-/} IFN antibodies (0), anti-«and anti-/3 IFNantibodies (•),medium control (O), and assayed for growth inhibition; averagevalue from 5 separate experiments. The difference in growth rate between anti-IFN treated cells and control cells was statistically not significant (/'>().!), whilethe difference between hydroxy-vit D3 or hydroxy-vit D3 + anti-IFN antibodytreated and control cells is statistically significant (/' < 0.01). H, effect ofexogenous IFN-/J and rlFN-y on cell proliferation. U937 cells were grown in theabsence (O, control) or in the presence of rIFN-i (D, 250 units/ml; •.1000 units/ml), and IFN-/3 (A, 10 units/ml; A, 100 units/ml). Results are the mean of 5separate experiments.

difference in the cell number observed at different days ofculture of U937 grown with hydroxy-vit D3 alone or in combination with anti-IFN antibodies was statistically significant (P< 0.01). This result suggests that during U937 differentiation,the endogenous IFNs produced by differentiating U937 cells

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IFNs AND MONOCYTIC DIFFERENTIATION

contribute to the inhibition of cell growth. This hypothesis isin accordance with the well-established fact that interferonshave a marked antiproliferative effect. Thus, addition of as littleas 10 units/ml IFN-/8 induced an inhibition of U937 growthsimilar to that elicited by hydroxy-vit D3, while rIFN-7 exhibited an inhibitory activity only at high doses, as shown in Fig.IB.

The effects of IFNs on ['Hjthymidine incorporation into

U937 cells were also tested (Fig. 2). In control cells a peak of[3H]thymidine was observed 1 day after subculture in freshmedium. This early [3H]thymidine incorporation peak was inhibited by IFN-/3 or rIFN-7 as well as by hydroxy-vit D3;furthermore, no cooperative effect in inhibiting DNA synthesisin U937 cells was observed in the presence of both hydroxy-vitD3 and IFNs.

Effect of Exogenous a, 0, or y Interféronson Induction ofU937 Differentiation. The effect of exogenous interferons onthe induction of U937 differentiation was investigated by incubating U937 cells, respectively, in the presence of IFN-«,-ß,or -y and following the evolution of a series of differentiationmarkers (Table 2).

We observed that most monocytic markers were not expressed under the action of exogenous natural IFNs with theexception of NBT reduction and Fc receptors. Moreover, theeffects of interferons on these 2 markers were different depending on the nature of the IFN used. The expression of "NBTreduction" was higher in U937 cells treated by IFN-a or -ß

than by rIFN-7. The 2-5 A synthetase was strongly enhancedby IFN-a or -ßbut very little by IFN-7. The expression of Fc

_ 80

70

S 60

Op

et2atO

so

40

aiZo

¡30

- 20

10

DAY OF CULTUREFig. 2. Effect of hydroxy-vit D3, exogenous IFN-/J, and rIFN-i on [3H]-

thymidine incorporation into U937 cells. U937 cells were grown in the absenceor presence of hydroxy-vit D3 or different concentrations of exogenous IFN-0and rIFN-ï: control (O); IFN-/3 (A, 1000 units/ml); hydroxy-vit D3 (x, 250 ng/ml); hydroxy-vit D3 + IFN-0 100 units/ml (C); hydroxy-vit D3 + rIFN-T, 250units/ml (i-!). [3H]thymidine incorporation was measured at different days ofculture. Control doses of IFN-0 (D, 100 units/ml) and rlFN-^ (A, 250 units/ml)are for the first day only. The results represent the average value obtained in 3separate experiments.

receptors was increased by rIFN-7 only. In addition, rIFN-7was able to induce the appearance of several monocytic markers(mostly membrane markers) in U937 cells at a level comparableto that elicited by hydroxy-vit D3 (Table 2).

In a separate experiment we followed the kinetics of inductionof monocytic membrane markers induced by rIFN-7, hydroxy-vit D3, and both of these compounds added together. Thisexperiment showed that (a) most of membrane monocyticmarkers were fully expressed from the fourth to sixth day ofculture on U937 cells grown under all the above-mentionedconditions and (¿>)the contemporaneous addition of rIFN-7and hydroxy-vit D3 resulted in an additive stimulatory effect inthe induction of all membrane monocytic markers studies (Fig.3).

Table 2 Effect of a, 13,and y interferons on differentiation of U93 7 cellsExponentially growing U937 cells (1.10* cells/ml) were treated with hydroxy-

vit D3 (500 nM), IFN-a (800 lU/ml), -ft (800 lU/ml) or -y (250 lU/ml). On thefourth day of culture, cells were diluted 2-fold with fresh medium, and freshhydroxy-vit D3 or IFN was added to maintain the initial concentration. Differentiation markers were examined on the sixth day and 2-5 A synthetase (Interferonmarker) was assayed in cell extracts as described in 'Materials and Methods."

activity being expressed in units/mg protein. Results are means obtained in 5separate experiments, with SD < 8%.

«,25(OH)2-Control IFN-« IFN-fJ r!FN--y D3 Vitamin

NBT reduction (% positivecells)a-Naphthyl

acetate esterase (% positivecells)Superoxidegeneration(O2-generated-A55o/2.5x

lO'cells/h)LysozymeFc

receptors (%positivecells)MO2

binding (%positivecells)OKM1

binding (% positivecells)TEC

Ml binding (% positivecells)OKM5

binding (% positivecells)TECMG1

(%positivecells)FMLP

receptors(sites/cell(xIO"3)]2-5

A synthetaseactivity(unils/mg)320.040.97-8<1<12<12160.9090<10.031.47-8<1<11.5<12075.590<10.031.47-8<1<12<12385.54540.050.861433645879131.295850.552.3645957925698354.5

OY OF CULTURE

Fig. 3. Kinetics of induction of membrane monocytic markers on U937 cellsgrown in the presence of rIFN-7 and/or hydroxy-vit D3. U937 cells grown in thepresence of either rlFN-f (100 units/ml, left), hydroxy-vit D3(250 ng/ml, middle),or both rIFN-> and hydroxy-vit D3 (right) were analyzed at different days ofculture for the presence of membrane monocytic markers. The expression of thesemarkers was analyzed by means of specific monoclonal antibodies and the bindingof these antibodies on the cells measured using a '"(-labeled F(ab')2 donkey anti-

mouse immunoglobulin as second antibody.

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IFNs AND MONOCYTIC DIFFERENTIATION

Fig. 4. Effect of hydroxy-vit D3, TPA andhydroxy-vil Dj plus r!FN--x on the expressionof ft-microglobulin. HLA-DC. HLA-DR. andHLA-DCI. U937 cells were grown in the presence of hydroxy-vit D, (250 ng/ml). TPA (16nM), or hydroxy-vit D3 (250 ng/ml) -f rIFN--x(100 units/ml) and the expression of classes Iand II I II \ antigens was analyzed by meansof specific monoclonal antibodies. The level ofbinding of these antibodies on U937 cells wasassessed using a '"I-labeled F(ab')2 donkeyanti-mouse immunoglobulin as second antibody. A, hydroxy-vit D3; B, hydroxy-vit D3 +rlFN-y; C, TPA; •,HLA-DC; BL HLA-DR;H, HLA-ABC; D. /¡j-microglobulin.

roo

60

50

40

30

20

o. 10

=: = x

0246

DAY OF CULTURE

Effect of Exogenous a, /9, or y Interferons on Induction ofMajor Histocompatibility Complex during Differentiation ofU937 Cells. Control uninduced U937 cells express class I HLAantigens (100% positive cells), while only a minority of thesecells express class II HLA antigens [25 ±5 (SD) and 10 ±4%of the cells were HLA-DR and HLA-DCI positive, respectively]. However, in previous studies, U937 cells before differentiation have been reported to exhibit only or low expressionof class I HLA antigens (19); there is much variation betweenU937 clones and this may explain this discrepancy. Inductionof the differentiation of U937 cells by hydroxy-vit D3 or byTPA is accompanied, with similar kinetics for both inducers,by a marked enhancement of the expression of HLA-DCIantigens, and by no modification of HLA-ABC antigens expression, as shown in Fig. 4. hydroxy-vit D, induced a slightdecrease of the expression of HLA-DR antigens; however, nochange was observed during TPA induction. The modificationsin the expression of HLA antigens mostly occurred during thepassage from the second to sixth day of culture, which corresponds to the time when monocytic markers appear on U937cells. In contrast, rIFN-7 induced in U937 cells a markedincrease in the level of HLA-DCI antigen expression and anysignificant change in HLA-DR and HLA-ABC antigen expression (Table 3). Moreover, the expression of HLA-DC antigens,as compared to the effect of hydroxy-vit D3 alone, was highlyenhanced by the contemporaneous addition of hydroxy-vit D3and rIFN-7. This treatment modified the kinetics of HLA-DCIantigens induction since maximal HLA-DCI expression wasobserved at days 2-4 of culture, while in cells grown withhydroxy-vit D3 only peak expression of HLA-DCI antigensoccurred at day 6 of culture (Fig. 4). This stimulatory effect ofrIFN-7 on HLA-DC expression seems to be specific for U937cells since control experiments performed on other leukemiccell lines, K562, HEL, and HL-60 showed no HLA-DC antigens induction by rIFN-7 (data not shown). Additional experiments showed that rIFN-7 clearly enhances the expression ofHLA-DC antigen in normal human monocytes (data notshown). The addition of IFN-«or -ßelicited a moderate enhancement of HLA-ABC antigen expression and no significantmodification in the level of expression of both HLA-DR and -

DC1 antigens (Table 3).Interestingly, the addition of PHA-leukocyte condition me

dium, which is known to contain one or more compoundscapable of inducing the differentiation of U937 cells (14),induced a moderate increase in the expression of HLA-ABCantigens and a marked enhancement of HLA-DR antigenexpression associated with only a very moderate stimulation of

Table 3 Effect of a, 0, and y interféronson classes I and li HLA antigenexpression of V93 7 cells

U937 cells were grown 72 h in the absence or presence of IFNs at the dosesindicated. HLA antigen expression was investigated as described in "Materialsand Methods." Results are the mean level observed in 3 separate experiments.

'"I-labeled F(ab')2 donkey anti-mouse ¡mimmoglobulins bound (cpm/10* cells x IO3)of cell

sample

02-micro-HLA-DR HLA-DCI HLA-ABC globulin

ControlIFN-«(500units/ml)1FN-0(500units/ml)rIFN--y(100 units/ml)23.622.923.523.91.441.91.813.649.563.460.954.943.448.949.757

3.0

2.5

O

x 2.0 •M

8 ,.5 i

o.o

1.0

0.5

• CONTROL

D 1% PHA-LCM

HLA-DR HLA-DC HLA-ABC

Fig. 5. Effect of PHA-leukocyte conditioned medium on the expression ofclasses I and II HLA antigens. U937 cells were grown for 3 days in the absence(control) or presence of \% PHA-LCM; HLA antigen expression was evaluatedas described in the legend to Fig. 4.

HLA-DCI antigen expression (Fig. 5). Furthermore, the addition of anti IFN-7 antiserum to PHA-LCM at a concentrationcapable of neutralizing all IFN-7 contained in PHA-LCMprevented the enhancing effect of PHA-LCM on HLA-DCIantigen expression but did not modify the stimulatory actionon HLA-DR antigen expression (data not shown). Taking allthese data into account, it appears clearly that in U937 cells,class II HLA antigens, HLA-DR and HLA-DC, are submittedto differential regulation: HLA-DC antigen expression is modulated by IFN-7, while HLA-DR antigen expression is apparently controlled by a cytokine different from IFN-7. Preliminary attempts to determine the cytokine responsible for HLA-DCI antigen modulation in U937 cells have shown that it isdifferent from tumor necrosis factor, interleukin-1 and -2.

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IFNs AND MONOCYTIC DIFFERENTIATION

DISCUSSION

IFNs exert pleiotropic effects on myelomonocytic cells (31).It was shown that IFN-7 is capable of enhancing the expressionof class II HLA antigens (32), of inducing or enhancing theexpression of the high-affinity Fc receptor for monomeric IgG(33), of inducing myeloid cells from established lines, fromnormal bone marrow, or from patients with chronic or acutemyelogenous leukemia, and of expressing a series of surfacemarkers, enzymes, and functional activities characteristic ofcells differentiating along the monocytic pathway (34). However, evidence concerning this last point is conflicting. Thus,early reports afforded some evidence that IFN-«and -ß(13) orIFN-7 (14, 35, 36) was able to induce the differentiation ofU937 cells to mature monocytes. In the aforementioned studiesthe monocyte-inducing properties of IFNs were evaluated byinvestigation of only I to three monocyte-specific markers. Inthe present study, this question was reevaluated, investigatingthe effect of endogenous and exogenous IFNs on the expressionof 12 differentiation markers in U937 cells. Previous studieshave shown that the production of endogenous interferon (IFN-.-f)as well as the activity of (2'5') oligoadenylate synthetase

markedly increases when murine (Friend erythroleukemia cells)or human leukemic (U937) cell lines are induced to terminalmaturation (2, 7-10, 18, 19). This endogenous IFN is mostlyrepresented by IFN-0 since its effect is neutralized by anti-IFN-ßantiserum, while IFN-7 is undetectable. Attempts have beenmade to suggest that this endogenous IFN could play a role insome cellular and biochemical processes leading to cellulardifferentiation. We showed that immunological neutralizationof endogenous IFN produced by U937 cells induced with hy-droxy-vit D3 did not inhibit the appearance of maturationmarkers of U937. These results strongly suggest that endogenous IFN does not play a major role in the induction of cellulardifferentiation of U937 cells. Our results clearly showed that,contrary to hydroxy-vit D3, exogenous IFNs were unable toinduce a marked enhancement of all these markers. Thus, IFN-»and -D'were capable of inducing only an important enhance

ment of NBT reduction and 2-5 A synthetase activity. Theseresults afford clear evidence that IFN-a and -ßwere unable toinduce the differentiation of U937 cells to mature monocytes.A different conclusion has to be drawn for rIFN-7 since thiscompound, although not being a "global" inducing agent of the

differentiation of U937 cells, is capable of inducing the appearance of several membrane monocytic markers on these cells, ina similar manner with respect to hydroxy-vit D3.

The immunological neutralization of endogenous IFN byanti-IFN-a/|0 antiserum partially reduced the inhibition of cellular proliferation observed in U937 cells induced with hydroxy-vit Dj. On the other hand, exogenous IFN-/3 or rIFN-7 blocked[3H]thymidine incorporation, occurring after subculture of

U937 cells in fresh medium, in accordance with previous observations (37, 38). These results suggest that endogenous IFNmay represent one of the factors which contribute to the loss ofcellular proliferation observed when hematopoietic cells areinduced to terminal differentiation. This conclusion is conceptually in line with recent studies (2, 10, 39, 40), suggesting thatendogenous IFN works in a feed-back loop which limits growthand ensures a negative control of cell growth.

Furthermore, we showed that exogenous IFNs may modulatethe expression of HLA antigens of U937 cells: the effect ofIFN-a and -ßis more pronounced on class I HLA antigens,while the action of rIFN-7 is restricted to class II HLA-DCantigens. These data are apparently at variance with otherresults showing that rIFN-7 enhance HLA-DR antigen expres

sion in U937 cells (41). However, this discrepancy could betentatively related to the heterogeneity of U937 for the expression of HLA-DR antigens (42). Furthermore, our data on themodulation of HLA antigen expression by rIFN-7 in U937cells are of some potential interest since several studies on othercell types, such as fibroblasts (32), vascular endothelium (43),or keratinocytes (44) showed that rIFN-7 markedly enhancesHLA-DR and only moderately enhances HLA-DC antigens.Moreover, there is clear evidence that rIFN-7 modulates theexpression of both HLA-DR and -DC antigens in normalhuman monocytes cultured in vitro (45-47). Thus, modulationof HLA-DC antigens by rIFN-7 may represent a particularfeature of monocytic cells; this phenomenon seems to have aphysiological role since the membrane expression of HLA-DCplays a key role in antigen presentation by monocytes (48).Furthermore, the observation that PHA-leukocyte conditionedmedium is capable of selectively enhancing HLA-DR antigensexpression leads us to the obvious conclusion that one or moresubstances different from IFN-7 contained in PHA-LCM is/are capable of modulating HLA-DR antigens in U937 cells.This conclusion is directly supported by the observation thatthe addition of a neutralizing anti-IFN-7 antibody prevents thestimulatory effect of PHA-LCM on HLA-DC antigen expression but does not modify the enhancement of HLA-DR antigenexpression. This observation is consistent with recent studiesshowing that the expression of la antigens on mouse lymphoidB-cells (49) and human diploid fibroblasts or large bowel ade-nocarcinoma cells (50) was increased by a lymphokine otherthan IFN-7.

In spite of the fact that IFN-7 is not a "global" inducing

agent because there are several monocyte characteristics whichare not inducible by IFN-7 treatment, the effect of this substance on the induction of surface antigens, enzymes, andfunctional activities characteristic of cells differentiating alongthe monocytic pathway may have important biological significance. In line with this statement, it is of some interest tounderline that several macrophage functions such as tumor cellcytolysis, antibody-dependent cell-mediated cytotoxicity, andphagocytic activity are known to increase after rIFN-7 treatment (31). It is tempting to suggest that the induction of somemonocytic membrane components by IFN-7 plays a role in theabove-mentioned activities. The inducing effect of IFN-7 on Fcmonomeric receptor for IgG is in agreement with previousstudies (33,51 ). IFN-7 may also induce IgE receptor expressionon U937 cells (52). Furthermore, recent observations haveshown that tumor necrosis factor exhibits a synergistic effectwith IFN-7 in inducing the monocytic differentiation of eitherU937 or HL-60 cells (53).

Finally, the observation that hydroxy-vit D3 and rIFN-7exhibit a cooperative effect in inducing the synthesis of monocytic membrane markers of U937 cells is in accordance with arecent study performed on the HL 60 cell line (54) suggestingthat these 2 compounds may act through different mechanismsin stimulating the synthesis of monocytic membrane proteinsduring monocytic differentiation.

ACKNOWLEDGMENTS

The authors wish to thank Drs. K. Berg and A. Billiau for anti-«andanti-/3 IFN antibodies, Biogen for rIFN-7, and C. Denolf and V.Martinez for secretarial assistance.

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IFNs AND MONOCYTIC DIFFERENTIATION

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