Scatid. J. Immunol. 22, 6] 1-017.

Distinct Effects of /Interferon on HumanB-Lymphocyte Precursor Cell Lines

H. KllL BLOMHOFF. C. DAVIES. E. RUUD. S. FUNDERUD& T. GODALLaboratory of Itnmunology. Dcpartmctit of Pathology and Department of Biophysics. NorskHydro's Institute for Cancer Research. The Norwegian Radium Hospital, and The NorwegianCancer Society. Oslo. Norwav

Kiil BlomholT. H.. Davies. C . Ruud. F. . Funderud. S. & Godal. T. Distinet Ffleets of yInterferon on Human B-Lymphoeytc Preeurs(»r Cell Lines. Scand. J. Imnuinol. 22, dl I-(S17.

The effect of y inierferon (IFN-y) on proliferation and antigenic eharaeteristics of eell linesbelonging to the B-cell progenitor compartment was studied. We ohservcd a selective efl'ect ofreeonibinant IFN-i'hut noi IFN-rron prolileralion of ihc B-precursorcell lines Reh and KM.l,Onday 4. after addition of 4(M)///ml IFN-;.'ihe pH|thymidine uptake in these eells was reducedto W^'f and 45''V respeetively. while no effect of IFN-j' was evident on the proliteraiion of ihemore mature B-cell lines Raji. Ramos. BS5. and Daudi. On the other hand, both Reh andRamos showed induction ot major histocompatibility complex (MHC) elass I antig^en expres-sion in response to 400/^''ml IFN-;'. In contrast to 12-0-tetradecanoyl-phorbol-l.^-acetiite(TPA). IFN-;'did not induce increased MHC elass U aniigen expression on Reh cells. Takentogether, our results indicate thai IFN-;' liiltils distinct lunctions al different levels in ihedevelopment of B cells.

Heidi Kul Blomhoff. Laboratory of Immunology. The Norwegian Radium Hospital. Mon-tebcllo. Oslo .i, Morwav

Interferons (IFN) are a family of proteins withdistinctive biological properties. In addition tothe antiviral activity for which they were namedand classified [11]. IFN are now known to inhibitcell growth [7. 9]. modify cell structtirc anddifferentiation [3. 7]. and to have immuno-regulatory activities [10, 20]. IFN-y has beenfound to increase expression of both class I andclass 11 histocompiitibillty antigetis in murineand human monocytes. as well as murine celllines of the myeloid lineage [2. 23. 26]. whileonly class I antigens have been indueed by IFN-(rtreatment of the same cells [12]. In murine lym-phoid cell lines, elevated levels of both class Iand class II antigens have been found uponIFN-j'treatment [26]. while in human lymphoideell lines only class I antigens have been induced[241.

The purpose of our study was to explore theeffect of recombinant IFN-j' on human B-pre-cursor cell lines, with respect to both inhibition

of eell growth and differentiation, and to com-pare the results with studies on cells at moremature stages in the B-eeil development. In ourexperiments we have concentrated on the effectof IFN-J'. sinee in several studies IFN-j'scems tohave stronger immunoregulatory and growth-inhibitory potential than IFN-fl- or/9 [ 12. 18. 22].Our study shows that the effeet of IFN-j' onB-precursorcell lines differs both from the effeeton other haematopoietic ceil lines such asHL-6(). and the more mature B cell lines B85.Ramos. Raji. and Daudi.

M A T E R I A L S AND M E T H O D S

Cell lines. The origins and ihe phcnotypic expres-sion of ihe various human lymphoid cell lines studiedare summarized in Table I. The Reh. KMIl. and EH^cell lines were kindly provided by M. F. Greaves.Imperial Cancer Researeh Fund Laboratories. Lon-don. UK: while the Ramos. Daudi. and Raji cell lines

611

(il2 //. Kid Blomhoff et al.

TAHI I- 1. Properues of lyniphtiid eeil lines

Cell line Origin EBV*

RehKM3BS5RamosRajiDaudi

e-ALLHe-ALLBALL

BL||BLBL

B! HH2S

• EBV. presence of Epstein-Barr virus genome.tC// . cytoplasniic immunoglobulin.+ sIgM. suriace nieinbraiie imnuinoglobulin.SHUi arid HI 12 are monoclonal antibodies specific Tor B cells [21|. ABl is a monoclonal antibody which also

delects H-cel! precursors [15].II ALL. acute lymphatic leukaemia.II BL. Burkitt's lymphoma.

were kindly supplietl by Ci. Klein. KarolinskaInstitute. Stockholm. Sweden. HL-Wtisii promyelocy-tic leukaemia ccW line provided by Ci. Gaudernaek.Rikshospiuilct. Oslo. Norway. The cell lines weregrown as suspension culture in RPMI 1641) medium,supplemented wiih \U''r heat-inaetivaicd letal calfserum (Gibco) anti 100///ml penicillin at 37^C in ahumidilied atmosphere o( '5 ' / CO..

A.sses.sment of eell f^rovvth. The effeet of IFN-/ oncell growth was assessed by two different methods,[-'H|thymidine incorporalion and cell counting. In the['H|thymidine incorporalion assay, the cells weregrown in %-well mierotitre pkites (Costar). at0.15x10'' cells/ml ;is an initial density. The experi-ments were run in triplieate. ami pulsed with 5x10'BL| |/ncf/i\7-'H]lhymidine (Radioehemitral Centre.Amersham. Fngland) for 2\ h al M'-C before beingharvested on a cell hitrvester (l i i iak. Skatron) asdescribed elsewhere [(i|. Initial experimcnis showedthat the grouth-inhibitory effect ol IFN-;'on Reh eellsinereased with mcreasing eell density. However, initialcell densities abtive 2x10' cells/ml eould nol beassessed, because ol'eeli cieaih at the lime of harvest.Thus an initial eell density of 1.5x1(1^ cells/ml wasused in all subsequenl experiments.

Cell counting was earried out on cells incubated at0.15x10" cells/ml in fi-well plaies (Cosiar). The eellswere counted in a Coulter Counter (C'ouller Fleetro-nies. England), and the numher ol viable cells wasmeasured by the trypan blue exelusion test [4|.

Reai-ents. IFN-;/ was a recombinani produet fromCienentee. kindly provided by O. R. Adolf at Eriist-Boeringer-Inslitui t'tir Arzencimiitellorschung.Vienna. Austria, with a specific activity of 12x|()"/t/mg (bioiissay: inhibition of CPE virus and EMCvirus on A544 human lung eareinoma eells). Therecombinani IFN-(r wtis a produet of InrcrlcronLaboratory. Hjoerrini;, Denmark. Both IFN werestored diluted in RPMI.'l(r'rFCSiil-70"C\ l2-a-tetra-decanoyl-phorbol-LVacetiite ( IPA) was purchiisedirom Consolldiited Midland Corporation. Brewsler.N.Y. The monocl(inal antibodies (MoAb) were AB-.^and E2-14. AB-3. developed in our lahoratory [15|,reacts with a monomorpliic LIUSN II epitope. as shown

by immunoprecipitation and L celK Iransfecled withelass M genes. The E2-14 aniibody. also developed inour laboralory. has been shown to reaet with /i-2mierogloliuiin. by ELISA and by the reactiviiy pat-tern, on LI large number of eell lines (e.g. negative onDaucli cells, which are kn(iwn lo lack surfaee /i-2mieroglobulin).

MoAh staining liininuiu/fluoreseenee). Changes inthe expression of MHC antigens and lineage-specificantigens were assessed by MoAb staining of untreatedeells. or eells treiited with IFN-)'or TPA. The stainingof cell siirfiices with mouse MoAb w;is eviilualed afterindirect antibody fluorescence labelling by the use offlowcylometrv. One to two x Id" cells were ineubatedwith monoclonal antibodies for 45 min at 4^C undersaturating conditions. After 2 washes, ihe eelts wereincubaied lor another 45 min with FITC-labelledsheep anti-mouse Ig (131. •"'"J linally washed 3 limes.

Flow eyiometry. The antigen expression wasmeiisured with a Coulter EPICS V flow cyiometer.rhe4SS[im iirgon laser line was used to excite fluores-cein liuorescenee. whicb wiis detected in ihe speetralinierval from 515 to 5h(l nni. The forward angle light-scatter signal discriminaled between live and deadeells. 1 he fluorescenee histograms were gated on thelighi-scaller signal to avoid unspecific liuoresceneefriim dead cells and debris, and lluoreseenee fromaggregates of cells.

RESULTS

Cell firowth

Logarithmicaliy growing cells were exposed tovarying concentrations of IFN-y for 4 and 6days respectively, and cell proliferation wasmeastired as |'H|tbymitliiie inc(>rporation (Fig.I). IFN-y markedly reduced cell proliferation inthe B-precursor cell lines Reh and KM3 in adose-dependent manner (Fig. lA. B). while noreduction in [^Hjthymidine incorporation wasevident in the more differentiated B-cell lines

100

50 -

Inierferon on llunum B-precursor Cell Lines 613

B C

.E 100

50

0

- I

40 400 4000 40 400 4000 40 400 4000

40 400 4000 40400 4000 40 400 4000U/ml IFN-y

FIG. L Dose response ol" IFN-;'on eell lines measured by|'H|thyrnidine ineorporation. Cell lines at 0.15x10"eells/ml were expt)sed to different eoneentrations ofIFN-y (or 4 (0 ) and 6 (U) days respeetively. "I'he resultsare presented as radioaetiviiy ineorporaiion, in percent oteontrol. The vertical hars indicate SEM: A=Reh.B=KM3. r -HL-W) . D = R a j i , F - B 8 5 . F -Ramos .

BS5. Raji. and Ramos. On day fi. after additionof IFN-)'. the proliferation was reduced byapproximately 60''; and 75'"; in Reh and KM.̂cells respectively. The viability of the H-N-v-treatcd cells on day 4 was >95''^(. and on day 6>9()%. of that of the control. The effect of IFN-;'on the mature B-cell line Daudi has heen foundby others to be marginal |H. 14|. and in otir hands4U0 ///ml IFN->' reduced the proliferation byonly about 5%. However, at a similar concentra-tion. IFN-(/ inhibited the proliferation by 611'';.On the other hand. IFN-fj- had no substantialeffeet on the B-cell precursors, since 4(10///mlIFN-(f reduced the proliferation in (Itese cells byapproximately 1(1'r tinly.

In the HI.-6() cell line, which belongs to ano-ther haematopoietic eell lineage. IFN-y has beenfound to strongly induce differentiation j 12. 2^\.We therefore also examined the effect of IFN-yon the proliferation of these cells. As shown inPig. IC, IFN-x reduced the cell growth ofthese

eells in a dose-dependent manner. The prolifera-tion was reduced by 6?^? with 4()()///ml IFN-y.

The dose-dependent effeet of II-'N-y on Iheinhibition of cell growth was further confirmedby direct cell counting of the continuouslyII'N-y-exposed cell lines. Again. Reh and KM3showed reducetl proliferation, while Ramos wasunaffected (Fig. 2). Fig. 2 also shows the kineticsof IFN-y-induced inhibition of cell growth. Theeffect on cell number was not apparent until thethird day. after addition of IFN-y. independentof the concentration of IFN. WhenPH I thymidine incorporation was measured,again the inhibititin of proliferation appeareil on

Cell differentiation. Since precursors of otherhaematopoietic lineages have been shown todifferentiate in response to IFN-y [2. i2| , wewanted to examine whether IFN-y also affecteddifferentiation of the B-precursor cell line Reh.As shown in Fig. 4. IFN-y increased t)nly the

614 H. Kiil Blomhoff ei al.

Days in cultureFIG. 2. Effeet of IFN-yon cell growth. Cells wore grown indifferent coneentrations of IFN-;'. and the number ofviable eells was eounted at different times alter addition oflFN-3'. ( • ) eontrol; ( • ) 4(l///ml IFN-y; (O) 40(1///mlIFN-y; (D). 4(M)()///ml IFN-y. A = Reh. B=KM3 andC= Ramos. Vcrtieal hars indieale the range of the countedvalues.

expression of class I MHC antigens on Reh cells,under conditions by which IFN-y induced class IIantigens on HL-60 cells. On the other hand, asreported by others [25]. TPA increased e.xpres-sion of class II MHC antigens on Reh cells. Anincreased class I antigen expression by IFN-ywas also found on Ramos cells, although nogrowth inhibition was observed on this cell line.

DISCUSSION

The results presented in this paper describe aselective effect of IFN-y on cell hncs of B-pre-cursor origin, with respect to inhibition of pro-liferation. Both the B-cell precursors and themature B cells respond, however, to IFN-y byenhanced expression of class 1 but not of class IIMHC antigens.

In order to assess growth inhibition on B-eell

precursors, we decided to use cell lines, sincehuman B-preeursor cells are not available insufficient quantities to allow such studies to bemade. Moreover, direct effects are difficult todistinguish from indirect effects mediated by Tcells, macrophages. or other cells, unless purecell cultures, i.e. cell lines, are used.

Among various substances tested, we foundthat reeombinant IFN-y had strong growth-inhibitory properties on the B-precursor celllines tested, i.e. Reh and KM3. These cell lineshave been found to differentiate along the B-eelllineage in response to TPA |I9| . Further, bothReh and KM3 are recognized by the B celllineage-specific monoclonal antibody ABl [15].,which indicates that these cell lines belong to theB-cel! compartment.

IFN-y reduced proliferation in both Reh andKM3 eells. measured by |'H]thymidine incor-

y Interferon on Human B-precttrsor Cell Lines 615

100

50 -

0 1 2 3 4 5Days after oddition of IFN-y

FIG. 3. The kinetics of IFN-y induced inhibition of cellproliferation. Cells (Reh) at (1.15x lO''cells/ml were grnwnin the presence of 4(H)///ml IFN-j'. The cells were harvested;tt different times after addition of IFN-y. ['H|thymidinewas added 21 h before harves^ting. Vertical hars indieatedthe SEM.

C:Romos D:H L- 60

t ''':\

Loganfimit fluorescence

FRi.4. Differentiation of cells in response to IFN-y or TPA. Reh(A.B). Ramos (C). and HL-6n (D) wereexposed to 50(1///ml IFN-y or III " M TPA for 3 days. As control, the cells were incuh;ited in RPMI. U)%FCS. On day? the cells were stained with E2-14 fordeterminationofelass I antigens I A.C). and stained withAB-3 fordclerminiuion of class II antigens (B.D). =eontrol; = IFN-j';A A A A - l PA.

616 H. Kiil Blomhoff et al.

poration. Since IFN-y has been shown tn inter-fere with the uptake of nucleic acid precursorsinto the soluble pool [5], we confirmed ourresults by direct counting of the IFN-j'-treatedcells. The inhibitory effect was not due to celldeath, since the viability was always greater than90% of that of the untreated cells. The inhibitionof proliferation was dose-dependent, and theeffect appeared between 48 and 72 h after addi-tion of IFN-J'. When given at the same doses asIFN y, IFN-rt did not reduce the proliferationsubstantially in Reh and KM.^.

IFN-(T and/j have been shown to reduce pro-liferation in mature B-cell lines | l . y | . but we didnot find any effect of IFN-y on mature B-celllines. The lack ofeffect of IFN-j'on proliferationof B-cell lines is in agreentent with the findings ofHannigan et al. [H]. and underlines the distinc-tive biological effects of IFN-y as compared toIFN-rt on different parts of the B-cel! lineage.The difference in response between B-cell pre-cursors and surface immunoglobulin-positive Bcells is particularly interesting when comparingReh and KM3 with BS5. since all three cell linesare derived from acute lymphoblasticleukaemias. Adams ei ill. fl] suggested thatdifferent sensitivities to the antiproliferativeeffeets of IFN might be eaused by the effect onthe expression of the Epstein-Barr virus (EBV)genome in the cells. However, this cannotexplain our results, since of the cell lines testedin our study, only Raji and Daudi are known toexpress the EBV genome |16|. We can ;ilsoexclude the possibility of influence trom con-taminating bacterial lipopolysaccharides (LPS)in the recombinant IFN-y preparation, since thefinal amount of LPS in the diluted cultures is<lt.(JI pg/ml. and therefore negligible.

Reduced proliferation appears to be associ-ated with differentiation and/or maturation. Wetherefore studied the effect of IFN-y on a num-ber of surface markers. We found a clearincrease in the expression of MHC class I anti-gens. This change was found on Reh as well as onB-cel! lines, suggesting that it is not related to thegrowth-inhibitory effeet. On the other hand, wedid not detect any increase of MIIC class 11antigen expression by IFN-y oti Reh. TPAhowever, as found by others [25|. increasedMHC class II antigens, showing that Reh alsohas the potential for increased class II antigenexpression. Moreover, under similar conditions.

IFN-y increased expression of MHC class II anti-gens on the promyelocytic cell line HL-6(). IFN-ywas found to induce only class I histocom-patibility antigens in murine pre-B-cell lines aswell 127).

The physiological significance of IFN-y-induced expression of class I antigens in cellsbelonging to the B-cell precursor compartmentremains unclear. A eharacteristic feature of thiscompartment is the large turnover of cells |17].Thus, it is possible that increased expression ofclass I molecules facilitates growth-control andkilling by suppressor and/or cytotoxic T ceils.By the same mechanisms, increases in class Iantigens may perhaps have an additional effecton neoplastic processes where B-precursor ceilsare involved.

Taken together, our results indieate thatIFN-y exerts distinctive effects on ceils ofdifTerent haematopoietic lineages and stages ofdevelopment. First of all. there seems to be adistinction between immunogiobulin- negativeand surface inmiunogiobulin-positive B ceiiswith respeet to II-'N-y-induced inhibition of cellproliferation; the former being highly sensitiveto this effect, and the iatter insensitive.Secondly, the regulation of IFN-y-inducedeffects on proliferation and expression of MHCclass I antigens seems different, since mature Bcells, which are insensitive to reduced prolifera-tion, still respond by increases in MFIC class Iexpression. Thirdly, IFN-y appears lo inducetlifferent types of MHC-associated antigens indifferent haematopoietic cell lines, since inHL-60, in which proliferation was also inhibited,high levels of class II MHC antigens wereexpressed in response to IFN-y. The controlexperiments carried out with TPA also indicatedthat the mechanism of action of TPA and IFN-ywere different in cell lines of progenitor origin.

These differences in effeet could be due todifferent amounts or affinities of receptors, ordifferences in the mediator substances associ-ated with signal transmission of IFN-y. Finally,changes in the programming of the genome asso-ciated with maturation and differentiation maybring different responses to the same mediatorsubstances.

A C K N O W L E D G M E N T S

We thank Monica Laukus for excellent technicalassistance. Kare Fundiniisrud for his assistance

y Inierferon on Human B-precursor Cel! Lines 617

with the flow cytometer analysis, and Iva Hirschfor preparing the manuscript carefully. Thiswork was supported by The Norwegian CancerSociety.

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Received 28 January 1985Received in revised form 15 April 1985

40