Interferon-a Restores Normal Adhesion of Chronic Myelogenous LeukemiaHematopoietic Progenitors to Bone Marrow Stroma by Correcting Impaired 381Integrin Receptor FunctionRavi Bhatia, Elizabeth A. Wayner, Philip B. McGlave, and Catherine M. VerfaillieDepartments of Medicine and Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455

Abstract

Treatment of chronic myelogenous leukemia (CML) withinterferon-a frequently results in normalization of periph-eral blood counts and, in up to 20% of patients, reestablish-ment of normal hematopoiesis. Wehypothesize that interfer-on-ar may restore normal adhesive interactions betweenCMLprogenitors and the bone marrow microenvironmentand restore normal growth regulatory effects resulting fromthese progenitor-stroma interactions. Wedemonstrate thattreatment with interferon-a induces a significant, dose-de-pendent increase in the adhesion of primitive long-term cul-ture initiating cells and committed colony-forming cells(CFC) from CMLbone marrow to normal stroma. Adhe-sion of CFCseen after interferon-a treatment could be in-hibited by blocking antibodies directed at the a4, a5, and,B1 integrins and vascular cell adhesion molecule, but notCD44 or intracellular adhesion molecule, suggesting thatinterferon-a induces normalization of progenitor-stroma in-teractions in CML. Because FACS® analysis showed that thelevel of a4, a5, and fl1 integrin expression after interferon-atreatment is unchanged, this suggests that interferon-a may

restore normal f81 integrin function. Normalization of inter-actions between CML progenitors and the bone marrow

microenvironment may then result in the restoration of nor-

mal regulation of CMLprogenitor proliferation, and ex-

plain, at least in part, the therapeutic efficacy of interferon-at in CML. (J. Clin. Invest. 1994. 94:384-391.) Key words:

chronic myelogenous leukemia * hematopoiesis * interferon* integrins - extracellular matrix

Introduction

Chronic myelogenous leukemia (CML), 1 a malignancy arisingfrom the hematopoietic stem cell, is characterized by a vast

Address correspondence to Catherine Verfaillie, MD, Department ofMedicine, Box 480 UMHC,420 Delaware Street SE, Minneapolis, MN55455.

Received for publication 13 December 1993 and in revised form 2March 1994.

1. Abbreviations used in this paper: BM, bone marrow; BMMNC,bonemarrow mononuclear cells; CFC, colony forming cells; CM, conditionedmedium; CML, chronic myelogenous leukemia; LTBMC, long-termbone marrow culture; LTC-IC, long-term culture-initiating cells; MIP,macrophage inflammatory protein.

expansion of hematopoietic cells belonging to the malignantclone resulting from unregulated proliferation of hematopoieticprogenitors ( 1). In addition, greatly increased numbers of circu-lating progenitors are seen as a result of their premature releaseinto the circulation (2). Malignant CMLcells are characterizedcytogenetically by a translocation between chromosomes 9 and22 (t9:22), the Philadelphia chromosome (3), and at the molec-ular level by the bcr/abl fusion oncogene (4).

Unregulated proliferation and abnormal circulation of CMLmalignant progenitors can, at least in part, be explained byabnormal adhesive interactions between the bone marrow mi-croenvironment and CMLprogenitors. Close contact betweennormal progenitors and stroma is thought to be important notonly for localization of hematopoiesis in the bone marrow butalso for regulation of progenitor cell growth. Normal primitivehematopoietic progenitors present in close contact with bonemarrow stroma are usually quiescent (5). This may be the resultof increased availability of growth inhibitory cytokines such astransforming growth factor-3 (TGF-f3) and macrophage in-flammatory protein-la (MIP-la) in proximity with the bonemarrow stroma (6-9). Recent studies from our laboratory sug-gest also that direct contact per se between normal progenitorsand stroma may inhibit progenitor proliferation (10, 11). Nor-mal progenitors adhere to stroma through a variety of cell sur-face adhesion receptors including the a4f31 integrin receptorswhich can bind to VCAMand the CS-I region in the COOH-terminal heparin binding domain of fibronectin and a5,(1 inte-grin receptors which recognize the RGDcontaining cell bindingdomain of fibronectin (12-16). Unlike normal progenitors,CMLprogenitors fail to adhere to normal stromal layers (17,18) and to fibronectin or its proteolytic fragments (18). How-ever, a4,61 and a5,681 integrin receptors are present at normallevels on CMLprogenitors, which suggests that the function ofthese receptors may be impaired in CML( 18). Since a4f31 anda5,61 receptors may transmit proliferation inhibitory signalsfrom the microenvironment to normal hematopoietic progeni-tors, decreased adhesion of CMLprogenitors to the microenvi-ronment through these receptors may underly the observationthat CMLprogenitors in the stromal adherent layer of long-termbone marrow cultures (LTBMC) are continuously proliferating.

Interferon-a has been used successfully in the treatment ofCML. Treatment with interferon-a induces hematologic remis-sions in up to 75% of patients treated. Moreover, interferon-atreatment can result in a gradual, selective suppression of themalignant clone with restoration of normal nonclonal hemato-poiesis (19). The mechanism s) by which interferon-a restoresnormal hematopoiesis in CMLis not clear. It has been demon-strated that interferon pretreatment of normal bone marrowstroma followed by culture of CMLprogenitors in the continu-ing presence of interferon results in enhanced adhesion of pro-genitors to stroma (20). It is, however, not clear if this effectis the result of alterations in the progenitors, the stroma or both.

384 Bhatia, Wayner, McGlave, and Verfaillie

J. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/94/07/0384/08 $2.00Volume 94, July 1994, 384-391

In this regard, interferon-a can restore the deficient expressionof LFA-3 on CMLprogenitor cells suggesting that it may simi-larly enhance expression of other critical adhesion receptorsthat may be deficient on CMLprogenitors (21).

To further elucidate the mechanisms whereby interferon-arestores normal hematopoiesis in CML, we studied the effectof interferon-a treatment of CMLprogenitors on their adhesionto normal BM stroma. Treatment of CML progenitors withinterferon-a resulted in enhanced progenitor adhesion to stromain a dose-dependent fashion. Wedemonstrate that adhesion ofinterferon-a-treated cells to normal stroma involves the a4/31and a5p3I integrin receptors and that this is the result of modula-tion of receptor function rather than an increase in their quantita-tive expression.

Methods

Bone marrow samples14 patients with CMLand 9 normal healthy volunteers were evaluatedafter informed consent was obtained using guidelines approved by theCommittee on the Use of Human Subjects at the University of Minne-sota. All patients were in the chronic phase of CMLand had receivedtreatment with hydroxyurea only. For each patient, treatment was dis-continued at least 4 d before study. Heparinized bone marrow sampleswere obtained by aspiration from the posterior iliac crest. Bone marrowmononuclear cells (BMMNC)were isolated by Ficoll-Hypaque (SigmaDiagnostics, St. Louis, MO)density gradient separation (specific gravity1.077) for 30 min at 370C and 400 g.

Selection of purified progenitor populationsSelection of purified progenitor populations was performed using meth-ods previously described (22, 23). CML or normal BMMNCwereinjected into an elutriator system (Beckman Instruments, Palo Alto,CA) with rotor speed maintained at 1,950 rpm and temperature at 10C(24). After loading, 200 ml of effluent was collected at a flow rate of12 ml/min for CMLbone marrow and 14 ml/min for normal marrow.Fraction 12 or 14 cells were depleted of T lymphocytes and NK cellsby sheep erythrocyte rosetting (25) and depleted further of committedB lymphocytic, myelomonocytic and erythroid cells using immunomag-netic beads (Advanced Magnetics Inc., Cambridge, MA) and mono-clonal antibodies directed at CDl9, CDl lb (Becton Dickinson, Moun-tain View, CA), MY-8 (Coulter Cytometry, Hialeah, FL), and glyco-phorin-A (Amac, Inc., Westbrook, ME). The resultant lineage negativepopulation (lin-) was labeled with anti-CD34-PE and anti-HLA-DR-FITC antibodies and sorted on a FACStar'LUS@ laser flow cytometrysystem equipped with a CONSORT32 computer system. Cells wereselected for low vertical and horizontal light scatter properties and forexpression of CD34 and HLA-DR antigens based on isotype controlstains. In normal individuals primitive progenitors capable of initiatingLTBMC(long-term culture initiating cells, LTC-IC) are concentratedin the Lin-34+DR- (DR-) fraction and more differentiated progenitorscapable of forming colonies in short-term methylcellulose progenitorcultures (colony forming cells, CFCs) in the Lin-34+DR+ fraction(DR+) (22). In CMLpatients, however, CFC and LTC-IC derivedfrom the malignant clone are both present in the DR+ fraction. TheCMLDR- fraction on the other hand contains cells that give rise tocolonies of nonclonal origin during LTBMC(23).

Bone marrow stromal layersBone marrow stromal layers were established in T-75 or T-150 flasksby plating normal bone marrow in LTBMCmedium (IMDM; GibcoLaboratories, Grand Island, NY with 12.5% fetal calf serum [HycloneLaboratories Inc., Logan, UT], 12.5% horse serum [Terry Fox Labora-tories, Vancouver, BC, Canada], 2 mML-glutamine, penicillin [1,000U/ml] and streptomycin [100 U/ml; Gibco] and 10-6 Mhydrocortisone[A-Hydrocort; Abbott Laboratories, North Chicago, IL]). Confluent

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Figure 1. Increased adhesion of CMLbut not normal progenitors tonormal bone marrow stroma after interferon-a treatment. CMLDR+cells (a) and normal DR- and DR+ cells (b) were preincubated withinterferon-a (10,000 U/ml) for 48 h. Cells were washed, panned, orplated on normal stromal layers for 2 h, and then replated in eithermethylcellulose progenitor culture or LTBMCto assay for CFCs andLTC-ICs, respectively. Percent adhesion of progenitors was calculatedby dividing the number of progenitors in cultures initiated with pannedcells by the number of progenitors in cultures initiated with plated cells.Results are expressed as mean±SEM. Black bars represent adhesion inthe absence of interferon, while cross-hatched bars represent adhesionafter interferon exposure. Significance levels: comparison of adhesionof progenitors with and without interferon-a treatment; *P < 0.0001,¶P < 0.05. The number of BFU-E, CFU-GM, CFU-MIX, and LTC-ICin 5,000 CMLDR+ cells was 210.6±25.7, 97.8±12.3, 15.3±1.8 and236.4±53.7, respectively. The number of BFU-E, CFU-GMand CFU-MIX in 5,000 normal DR+ cells was 119.7±20.6, 128.9±15.8 and15.9±2.6, respectively. The number of LTC-IC in 5,000 normal DR-cells was 105.9±15.9.

stromal layers were formed after 4-5 wk of culture and were irradiatedat 1,250 cGy using a Cesium irradiator to eliminate hematopoietic cells.Adherent cells collected after digestion with 0.1% collagenase (Boeh-ringer Mannheim Corp., Indianapolis, IN) were subcultured at a concen-tration of 350,000 stromal cells/well in 24-well plates (Costar Corp.,Cambridge, MA) (22).

Cell adhesion assaysNormal DR- and DR+and CMLDR+progenitor cells were incubatedin bone marrow stroma conditioned medium (CM) containing interfer-on-a2b (Schering Corp., Kenilworth, NJ) at 37°C in a humidified atmo-sphere with 5% CO2. The interferon concentrations used ranged from100 to 10,000 U/ml. Cells were exposed to interferon-a for time periodsranging from 20 min to 48 h. A control population of cells was incubatedunder identical conditions but without interferon-a, After incubation,cells were washed three times to remove excess unbound interferon,

Interferon Restores /31 Integrin Function in Chronic Myelogenous Leukemia 385

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resuspended at 5,000 cells/ml in LTBMCmedia and coincubated withnormal bone marrow stromal layers in 24-well plates for 2 h at 370C.Nonadherent cells were removed by three vigorous washes using warm

IMDM (panning). Tightly adherent progenitor cells were harvestedusing trypsin and plated in short-term methylcellulose assay to evaluatethe percent adherent CFCsand in LTBMCculture to evaluate the percentadherent LTC-ICs (22). For normal progenitors, DR+ cells were usedfor CFCassays and DR- cells for the LTC-IC assays. For CMLpatients,DR+ cells were used in both assays. The percent adherent progenitorswas calculated as: [the number of progenitors adherent to stroma(panned cells) divided by the total input of progenitor cells (platedcells)] x 100. Experiments were carried out simultaneously with normaland CMLprogenitors.

Adhesion inhibition assays

CMLDR+ cells were incubated for 48 h with or without 10,000 U/mlinterferon-a in stroma conditioned medium in a humidified atmospherewith 5%CO2 at 370C. Cells were washed three times and resuspendedin LTBMCmedia at 5,000 cells/well. Interferon treated CMLDR+cells or untreated CMLand normal DR+ cells were incubated with theindicated antibodies for 30 min before the adhesion assays. Alterna-tively, stroma was incubated with antibodies to VCAMor ICAM for30 min before the adhesion assays. Progenitors were then plated incontact with stroma in the presence of the indicated antibodies for 2 hafter which nonadherent cells were removed and adherent cells platedin methylcellulose progenitor culture as described above. Percent adhe-sion of interferon-treated CML progenitors in the presence of theseantibodies was calculated as [adhesion in the presence of antibodydivided by adhesion in the absence of antibody] X 100%.

1 0 1 0

Figure 2. Interferon-a enhances adhesion ofCMLprogenitors to normal bone marrowstroma in a dose-dependent manner. CMLDR+ cells (n = 5) were preincubated withincreasing concentrations of interferon-a for48 h. Cells were washed, panned and platedon normal stromal layers for 2 h, and thenreplated in either methylcellulose progenitorculture or LTBMC. Percent adhesion of (a)CFU-GM, (b) BFU-E, (c) CFU-MIX, and(d) LTC-IC was calculated by dividing thenumber of progenitors in cultures initiatedwith panned cells by the number of progeni-tors in cultures initiated with plated cells. Re-sults are expressed as meantSEM. Compari-son of adhesion of progenitors with and with-out interferon-a treatment; *P < 0.05.

Progenitor cultureLong-term bone marrow culture. Long-term bone marrow cultures were

established by plating or panning 5,000 CMLDR+ cells or normalDR- cells in direct contact with normal stromal layers subcultured in24-well plates. Cultures were maintained for 5 wk in a humidifiedatmosphere, at 370C and 5%CO2. Weekly media changes were carriedout by removing half the cell free supernatant medium and replacing itwith fresh LTBMCmedium. To determine the number of CFCpresentin long-term cultures, cells were harvested by digesting the stromallayers with trypsin and replated in methylcellulose progenitor culture(22).

Short-term methylcellulose progenitor culture. DR+ cells recoveredfrom panning assays or the progeny of cells in LTBMCwere plated inmethylcellulose (final concentration 1.12%) (Fisher Scientific, FairLawn, NJ) with 11DMsupplemented with 30% FCS, antibiotics, 5 x

10-1 M2-mercaptoethanol (Sigma Chemical Co.), 3 IU recombinanterythropoietin (Epoetin; Amgen Biologicals, Thousand Oaks, CA) and4 ng/ml recombinant interleukin 3 (a kind gift from Dr. Wong, GeneticsInstitute, Boston, MA). Cultures were incubated in a humidified atmo-sphere at 37TC and 5%CO2for 14-18 d. The cultures were then assessedfor the presence of CFU-GM, BFU-E, and CFU-MIX colonies as pre-viously described (25).

FACSanalysis of cell surface adhesion receptors on CMLprogenitorsLineage negative CMLcells were incubated with or without interferon-a in stroma-conditioned medium for 48 h, washed, and labeled withantibodies to adhesion receptors in conjunction with antibodies to CD34

386 Bhatia, Wayner, McGlave, and Verfaillie

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as follows: 100,000 lineage negative cells were sequentially incubatedwith primary antibodies to the a4, a5, and fl integrins, FITC conjugatedgoat anti-mouse antibody (Tago Inc., Burlingame, CA), mouse IgG(Sigma, Chemical Co.) to block unoccupied binding sites on the goatanti-mouse antibody and PE-conjugated anti-CD34 antibody (BectonDickinson). Controls were labeled sequentially with control mouse IgG,goat anti-mouse FITC, mouse IgG, and then either IgGI-PE or anti-CD34-PE. Cells with low horizontal and vertical light scatter propertieswere selected and the expression of adhesion receptors on cells coex-pressing CD34 within this population measured using FACSO.

AntibodiesAntibodies against a-4 (P4C2), a-5 (P3DIb), 63-1 (P5D2), ICAM(P4Fl 1) and VCAM(P8Bl ) were developed by Dr. Elizabeth Wayner.Anti-CD44 Hermes 3 was a kind gift from Dr. Eugene Butcher, PaloAlto, CA and antibody 50B4 was a kind gift from Dr. DominiqueLetarte, Toronto, Canada. Mouse IgG was obtained from Sigma Chemi-cal Co.

Figure 3. Effect of duration of preincubation with inter-feron-a on the adhesion of CMLprogenitors to normalstroma. CMLDR+ cells (n = 4) were preincubatedwith interferon-a (10,000 U/ml) for time periods rang-ing from 20 min to 48 h. Cells were washed, pannedand plated on normal stromal layers for 2 h, and thenreplated in methylcellulose progenitor culture. Percentadhesion of (a) CFU-GM, (b) BFU-E and (c) CFU-MIX was calculated by dividing the number of progeni-tors in cultures initiated with panned cells by the numberof progenitors in cultures initiated with plated cells.Results are expressed as Mean±SEM. Significance lev-els: comparison of adhesion of progenitors with andwithout interferon-a treatment; * P < 0.005, ¶P < 0.05.

Statistical analysisResults of experimental points obtained from multiple experiments werereported as mean±SEM. Significance levels were determined by two-sided t test analysis.

Results

Interferon-a treatment of CMLprogenitors restores their adhe-sion to stroma. The effect of interferon-a on the adhesion ofCMLand normal progenitors to normal stroma was assessedby incubating CMLprogenitors for 48 h in the presence orabsence of interferon-a before performing adhesion assays. Aswe have previously demonstrated, adhesion of malignant LTC-IC present in the CMLDR+population to normal bone marrowstroma is significantly decreased (18). Similarly, CFCpresent

Interferon Restores /31 Integrin Function in Chronic Myelogenous Leukemia 387

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in the CMLDR+population adhere significantly less to normalstroma compared with normal CFC. Interferon-a treatment re-sulted in a significant increase in the adhesion of CMLLTC-IC to normal bone marrow stroma (P < 0.05, n = 5) (Fig. 1a). Similarly, significantly more committed CMLprogenitors[BFU-E (P < 0.001, n = 13), CFU-GM(P = 0.001, n = 13)and CFU-MIX (P < 0.001, n = 10)] (Fig. 1 a) adhered tonormal stroma after interferon-a treatment. In contrast, adhesionof normal bone marrow derived LTC-IC, BFU-E, CFU-GMand CFU-MIX was not significantly increased after interferon-a treatment (Fig. 1 b). Although growth of CMLprogenitorscould be suppressed following pretreatment with interferon-a[progenitor growth after interferon-a treatment compared withgrowth of controls not exposed to interferon: BFU-E,77.9+7.3% (n = 13); CFU-GM, 94.8±7.3% (n = 13); CFU-MIX, 92.1±4.3% (n = 10) and LTC-IC, 89.2+8.5% (n = 5)],this did not affect the results of adhesion assays, since thepercent adherent progenitor cells was calculated by dividing thenumber of colonies generated in cultures initiated with pannedcells by the number of colonies in cultures initiated with platedcells under identical conditions.

The increased adhesion of interferon-a treated CMLprogen-itors to bone marrow stroma was dose-dependent (Fig. 2).Adhesion to stroma of both CMLLTC-IC and CFC did notdiffer significantly from that of normal progenitors once CMLprogenitors were pretreated with 1 X 103 to 1 X 104 U/mlinterferon-a. At least 12 h of incubation with interferon-a wasrequired before significantly increased adhesion of CMLpro-genitors to bone marrow stroma was observed (Fig. 3) althougha further increase in adhesion could be seen after 48 h of incuba-tion.

Receptors involved in the adhesion of interferon-a-treatedCMLprogenitors to stroma. The role of a4/31, a5/lI andVCAM, known to be involved in the adhesion of normal com-mitted progenitors to BMstroma, in the adhesion of interferon-a-treated CMLprogenitors was evaluated by studying the abil-ity of antibodies directed against these receptors to block adhe-sion. CMLDR+ cells, incubated with or without interferon-a(10,000 U/ml) for 48 h, were washed and allowed to adhereto normal BMstromal layers for 2 h in the presence of antibod-ies against a4, aS, /B1, CD44, VCAM, and ICAM-1.

The low level adhesion to stroma of CMLCFC, in theabsence of interferon-a, was not significantly inhibited by anti-bodies against a4, aS, /31, and VCAM[BFU-E: control adhe-sion 25.9±4.8%, with anti-a4 25.6+5.6%, anti-aS 25.8±4.8%,anti-,31 20.8±5.0%, anti-VCAM 30.3±5.13%; CFU-GM: con-trols 22.3±+3.3%, anti-a4 20.8±2.5%, anti-aS 21.3±3.2%, anti-/61 26.1±3.0%, anti-VCAM 25.4±2.5%; (n = 4)]. This con-firms previous observations that /-1 integrin receptor-mediatedadhesion mechanisms are deficient in CML. However, as shownin Fig. 4 a, adhesion of CMLBFU-E, treated with interferon-a,to normal BMstroma was significantly inhibited by antibodiesagainst the a-4 (P < 0.01), a-5 (P = 0.005), and 6-1 (P< 0.001) integrin receptors as well as to VCAM(P < 0.05, n= 4). Antibodies against the CD44 receptor (5OB4 and Hermes-3) and ICAM-1 did not have a significant effect on adhesion.Similar results were obtained for CMLCFU-GM (Fig. 4 b).The effect of these antibodies on the adhesion of interferon-a-treated CMLprogenitors was similar to their effect on the adhe-sion of normal progenitors to normal BM stroma. We havepreviously demonstrated that adhesion of normal BFU-E to BMstroma in the presence of anti-a4 antibody is 65.8±5.6% of

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3 < < 4Xw 0 Qx ~~~uFigure 4. Increased adhesion of interferon-a treated CMLprogenitorsto normal stroma involves a4f31 and a5,/1 integrin receptors. CMLDR+ cells (n = 4) were treated with interferon-a (10,000 U/ml) for48 h, washed and subsequently plated and panned on normal stromallayers in the presence of blocking antibodies to the indicated receptorsand control mouse IgG. Cells were replated in methylcellulose progeni-tor culture and the effect of these blocking antibodies on the adhesionof BFU-E (a) and CFU-GM(b) was assessed. Results are shown as

the adhesion of progenitors in the presence of antibody expressed as

the percentage of adhesion in controls without antibody. Significancelevels; *P < 0.01, **P < 0.001, ¶P < 0.05 (n = 4). The number(and percent) of adherent BFU-E and CFU-GMin 5,000 CMLDR+cells was 71.4±15.8 and 28.8±3.3, respectively.

control (P < 0.05); anti-aS, 24.3±12.6% (P < 0.001), andanti-/31, 28.0±10.4% (P < 0.001) (n = 4). Similarly, adhesionof normal CFU-GMin the presence of anti-a4 is 65.8±6.9%of control (P < 0.01); anti-a5, 42.3±12.1% (P < 0.01) andanti-/31, 45.3±13.1% (P < 0.01) (n = 4) (16). Adhesion ofnormal BFU-E and CFU-GMto BMstroma in the presence ofanti-VCAM was 62.9±3.7% (P < 0.001) (n = 7) and58.7+6.3% (P < 0.001) (n = 7) of control, respectively. Theseresults indicate that interferon-a treatment of CMLprogenitorsresults in enhanced adhesion to stroma by restoring /31 integrinreceptor-dependent mechanisms.

Expression of cell surface adhesion receptors on CMLpro-genitors after interferon-a treatment. To determine if the in-creased adhesion through a4,81 and a5,81 adhesion receptorswas the result of increased expression of these receptors, lineagenegative cells from three CMLpatients were incubated withinterferon (10,000 U/ml) for 48 h. Cells were washed and

388 Bhatia, Wayner, McGlave, and Verfaillie

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labeled with antibodies against integrin receptors and CD34andanalyzed using FACS@.Cells with low vertical and horizontallight scatter properties and expressing high levels of CD34 anti-gen were evaluated for expression of integrin receptors by fre-quency histogram. Fig. 5 shows representative histograms ob-tained from one such patient. As we have previously demon-strated, CMLCD34+ cells express similar levels of the a4, a5,and /I integrins as normal CD34+ cells (18). No change inreceptor expression was observed after interferon-a treatment,suggesting that interferon-a may restore /3-1 integrin receptorfunction rather than up-regulating the level of receptor expres-

sion.

Discussion

Interferon-a treatment of CMLcan result in the selective sup-

pression of malignant hematopoietic progenitors and resump-

i Ja~SUIT Figure 5. Interferon-a treatment does not changethe level of expression of a4, a5 and ,l1 integrinreceptors on CMLprogenitors. Lineage negativeCMLcells incubated with or without interferon-a (10,000 U/ml) for 48 h, were labeled withantibodies to the a4, a5, and ,/1 integrin recep-

tors in conjunction with antibodies to CD34. Theexpression of adhesion-receptors on cells coex-

pressing CD34 was measured using FACSO.Representative results from one patient are

shown. Three patients were studied in this man-

ner. No change in a4, a5, and ,/1 receptor ex-

pression was observed after interferon-a treat-ment.

tion of normal hematopoiesis (19). The mechanism(s) wherebyinterferon achieves this effect are not clear. It has been demon-strated that CMLprogenitors, which fail to adhere to normalbone marrow stroma, can adhere to normal stromal layers aftertreatment of stroma with interferon-a (20). Wedemonstratehere that interferon-a can increase adhesion of CMLprogenitorsto stroma through a direct effect on the progenitors involvingupregulation of a4/31 and a5l I integrin receptor function. Res-toration of normal adhesive interactions between CMLprogeni-tors and stroma may then result in restoration of normal growthregulatory mechanisms, which in normal individuals are thoughtto be dependent on close progenitor-stroma interactions (5, 6,10, 11).

Integrins are heterodimeric receptors consisting of a andchains which possess a large extracellular domain, a transmem-brane domain and a short intracellular domain (26). The role ofintegrin receptors in hematopoiesis has recently gained interest.

Interferon Restores 61 Integrin Function in Chronic Myelogenous Leukemia 389

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a4/31 and a5p1 integrin receptors are present on normal progen-itors and are involved in the adhesion of these progenitors tostroma (12-16). Wehave previously demonstrated that CMLprogenitors express similar levels of cell surface a4fi1 anda5lI1 integrin receptors as normal progenitors (18). However,CMLprogenitors are deficient in their ability to adhere to stromaor to purified fibronectin and fibronectin proteolytic fragments(18, 19) suggesting that these receptors are present in a func-tionally inactive state. A variety of studies have demonstratedthat the mere presence of integrin receptors does not necessarilyindicate that the receptor has functional significance (26). Inte-grins can be present in a nonfunctional low affinity state thatcan be switched to a high affinity state after stimulation of thecell with cytokines (9), through other cell surface receptors(27) or through its own ligands (28). The mechanisms underly-ing the decreased affinity of a4fi1 and aS/il integrins on CMLprogenitors are not clear.

One possibility is that the presence of the p210""abI tyrosinekinase in CMLprogenitors results in phosphorylation and deac-tivation of /I1 integrins. Similar mechanisms have been sug-gested for other malignancies. Transformation of fibroblasts bythe Rous sarcoma virus results in their attachment independentgrowth (29). It has been demonstrated that colocalization ofpp6Wm with/31 integrins at the cell membrane is associated withphosphorylation of the /31 chain, which may result in decreasedaffinity of the /31 integrin receptor (30). Similarly the abnormalp210lcr1abl phosphotyrosine kinase is also located in the cyto-plasm (31, 32). Colocalization of p21Ojroabl and /1 integrinsmay then lead to deactivation of the /31 integrins through abnor-mal phosphorylation. In addition, impaired /31 integrin functionmay result from abnormal phosphorylation of cytoskeletal orother proteins closely associated with the integrin receptor ashas recently been demonstrated for bcr/abl transfected hemato-poietic cell lines (33). Recent studies have demonstrated thatinterferon-a may prevent transcription of the bcr/abl mRNA,and hence expression of the p21Olcr/abI tyrosine kinase (34).Removal of the p210bCr'abl neoprotein and its possible effecton integrin affinity may then explain the restored adhesion ofprogenitors following interferon-a treatment. The time requiredfor interferon-a to cause enhanced adhesion of CMLprogeni-tors to stroma is consistent with an effect mediated by alterationin mRNAand/or protein synthesis. It is also possible that inter-feron-a may act on CMLprogenitors to upregulate certain otheradhesion receptors, which we have not evaluated, such as mem-bers of the selectin family (35). Stimulation of CMLprogeni-tors through these receptors could subsequently result in en-hanced affinity of /31 integrin receptors (27).

Adhesion through integrins serves not only to localize cellswithin specific microenvironments, but may also have profoundeffects on cell growth and differentiation. Engagement of inte-grins results in cytoskeletal rearrangement (36), tyrosine phos-phorylation of certain cytoplasmic proteins (37), and, de-pending on the cell type, in either increased proliferation (38),with associated increased expression of AP-1 (fos/jun) (39),or reduced proliferation and induction of differentiation (40).The importance of signal transduction through integrin receptorson hematopoietic progenitors has been less well studied. Normalprimitive progenitors adherent to bone marrow stroma are usu-ally quiescent (5). Recent studies from our laboratory suggestthat inhibition of proliferation of normal progenitors in contactwith stroma may result from the adhesive interaction itself withstroma and extracellular matrix, specifically fibronectin (10,

11). It is therefore possible that impaired integrin receptor func-tion on CMLprogenitors (18) may lead to reduced growthinhibition, thereby providing an explanation for the observationthat CMLprogenitors present within the adherent layer of stro-mal cultures are continuously proliferating (1). Modulation ofa4/31 and aS/p3 function on CMLprogenitors by interferon-amay then restore normal growth inhibitory signal transductionpathways, leading to normal regulation of proliferation. In addi-tion, inhibition of proliferation of normal progenitors as a resultof adhesion to stroma may result from colocalization of progeni-tors with negative growth regulatory factors, such as trans-forming growth factor-/ or macrophage inflammatory protein-la, which are bound and concentrated by the extracellular ma-trix (6-9). Such interactions would be expected to be impairedin CMLbecause of impaired adhesion to stroma. Restored adhe-sion induced by interferon-a may then allow normal regulationof CMLprogenitors by these negative growth regulatory factors.

In conclusion, the present study demonstrates that interfer-on-a restores normal adhesive interactions between CMLpro-genitors and the bone marrow microenvironment through upreg-ulation of /31 integrin function. Further studies will be requiredto characterize the functional defect of abnormal /1 integrinreceptors in CMLand the mechanisms by which interferon-amodulates receptor function. This knowledge may help in thedevelopment of improved treatment strategies for this disease.

Acknowledgments

The authors acknowledge the excellent technical assistance of B. Ander-son, P. Catanzaro, B. Hoffstrom, and Wen-na Li.

Supported in part by National Institutes of Health grants RO1-CA-4581401, POI-CA-21737 and American Cancer Society grant IM69879. Wealso acknowledge the support of the Gamble-Skagmo Foun-dation, the Paul Christiansen Foundation, the University of MinnesotaBone Marrow Transplantation Research Fund, the Minnesota MedicalFoundation, the Leukemia Task Force, the Children's Cancer ResearchFund, the Graduate School of the University of Minnesota, the AmericanCancer Society and the University of Minnesota Hospitals and Clinic.C. Verfaillie is a Special Fellow of the Leukemia Society of Americaand a Special Fellow of the "Fundacion Internacional Jose CarrerasPara La Lucha Contra La Leficemia."

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