Experimental Hematology 28 (2000) 916–923

0301-472X/00 $–see front matter. Copyright © 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.PII S0301-472X(00)00493-8

Lisofylline suppresses ex vivo release by murine spleen cells ofhematopoietic inhibitors induced by cancer chemotherapeutic agents

Peter de Vries and Jack W. Singer

Cell Therapeutics, Inc. Seattle, Wash., USA

(Received 31 January 2000; revised 30 March 2000; accepted 18 April 2000)

Objective.

Many cytotoxic cancer therapeutic drugs activate stress response signaling path-ways that transcriptionally activate a variety of genes. We decided to determine if cytotoxictherapies induce inflammatory cytokines with inhibitory effects on hematopoiesis and iflisofylline (LSF), a novel antiinflammatory compound, suppresses this induction.

Materials and Methods.

Mice were treated with cytosine

b

-d-arabinofuranoside (AraC), cis-platinum(II)diammine-dichloride (CisP), etoposide (VP-16), or melphalan at clinically rele-vant doses, with or without LSF.

Results.

Spleen cell conditioned media (CM) derived from mice treated with cytotoxic agents,but not from control or LSF treated mice, reduced colony formation by murine bone marrowprogenitors belonging to the myeloid, erythroid, megakaryocytic, and B-lymphoid lineages.LSF (100 mg/kg), administered either simultaneously with or up to 48 hours before the cyto-toxic agents, suppressed the release of this inhibitory activity. Treatment of inhibitory CMwith neutralizing antibodies against known growth inhibitory cytokines, including tumor ne-crosis factor

a

, transforming growth factor

b

, and macrophage inflammatory protein-1

a

, re-sulted in enhanced colony growth.

Conclusion.

We conclude that treatment of mice with chemotherapeutic drugs induces the exvivo production of multilineage hematopoietic inhibitors and that induction of these inhibitorscould be abrogated by treatment with LSF. These findings suggest a mechanism whereby LSFcan accelerate recovery of hematopoiesis following cytotoxic therapies. © 2000 InternationalSociety for Experimental Hematology. Published by Elsevier Science.

Keywords:

Hematopoietic inhibitors—Lisofylline—Cancer chemotherapeutic agents—Spleencell conditioned media

Introduction

Hematopoiesis is a dynamic, complex process by whichvery large numbers of mature blood cells with specific func-tions and limited lifespans are generated on a daily basis [1].Hematopoietic cell proliferation, differentiation, and celldeath are tightly regulated by the balance between growthstimulatory and growth inhibitory cytokines. Growth stimu-latory molecules include the colony-stimulating factors(CSFs) such as granulocyte/macrophage (GM)-, granulo-cyte (G)-, and macrophage (M)-CSF, erythropoietin (Epo),thrombopoietin, interleukins (IL) such as Il-3, and the co-stimulatory cytokines such as stem cell factor (SCF) andFlt3 ligand. Growth inhibitory molecules include transform-ing growth factor

b

(TGF-

b

) [2–7], tumor necrosis factor

a

(TNF-

a

) [8–10], interferon

g

(IFN-

g

) [11,12], and chemo-kines such as macrophage inflammatory protein-1

a

(MIP-1

a

) [13–16] and platelet factor 4 [17–20].Under the stress of cytotoxic therapy for cancer, the nor-

mal homeostatic balance between positive and negative reg-ulatory molecules is temporarily disrupted to allow a physi-ologic response to cell depletion [18,21]. Under normalcircumstances, growth stimulatory molecules cannot be de-tected in the serum of mice [22]. Increased levels of(co)stimulatory cytokines such as G-CSF, IL-6, Epo, andSCF are induced following cancer treatment [23–30]. How-ever, the status of negative regulators of hematopoiesis fol-lowing cancer therapy has been less well studied.

Common treatments for cancer, such as ionizing irradia-tion, cis-platinum(II)diamminedichloride (CisP), and cy-tosine

b

-d-arabinofuranoside (AraC), activate stress re-sponse signaling pathways, such as the stress-activatedprotein kinase (SAPK or JNK-1) and mitogen-activated

Offprint requests to: Peter de Vries, Ph.D., Cell Therapeutics, Inc., 201Elliott Avenue West, Seattle, WA 98119; E-mail: pdevries@ctiseattle.com

P. de Vries and J.W. Singer/Experimental Hematology 28 (2000) 916–923

917

protein kinase (MAPK) pathways [31–35]. Signal propaga-tion in these pathways leads to activation of transcriptionfactors that regulate the expression of a wide variety ofgenes, including those encoding proinflammatory andgrowth inhibitory cytokines such as TGF-

b

, TNF-

a

, and IL-1[36–39]. Such cytokines also stimulate secondary mediatorsof inflammation including MIP-1

a

and IFN-

g

[40], whichmay inhibit and delay the recovery of rapidly dividing tis-sues such as the hematopoietic system and the mucosal stemcells in the gastrointestinal tract. In addition to causingDNA damage, radiation and radiomimetic drugs induce freeoxygen radicals that oxidize cellular constituents, which areimportant in propagation of oxidative damage and associ-ated signaling [41–45].

Lisofylline (LSF), 1-[5-R-hydroxyhexyl]-3-7-dimethyl-xanthine], is a novel antiinflammatory agent that protectsagainst a broad variety of insults [40,46–48]. Clarke et al.[49] showed that LSF enhanced trilineage hematopoietic re-covery in mice after 5-fluorouracil or tiothepa treatment,possibly by inhibiting the release of TGF-

b

. In murine au-tograft models, LSF decreased the duration of cytosuppres-sion following high-dose 5-fluorouracil, thiotepa, BCNU(1,3-bis(2-chloroethyl)-1-nitrosourea), cyclophosphamide, or

g

-irradiation treatment [50,51]. In addition, LSF was shownto be highly protective against lung damage associated withbacterial sepsis, direct oxidative injury, cytokine- and neu-trophil-induced injury, and reperfusion injury in rats andmice [40,46,48,52]. LSF also has been reported to suppressTh1-mediated diseases such as experimental allergic en-cephalomyelitis by inhibiting IL-12 signaling and thus IL-12-mediated Th1 cell differentiation and secretion of the in-flammatory cytokine IFN-

g

[53,54].Based on these prior studies, we determined whether

treatment of mice with mechanistically and structurally dif-ferent cancer chemotherapeutic drugs would result in the exvivo release of inflammatory and growth inhibitory mole-cules from murine spleen cells and whether or not treatmentwith LSF could abrogate release of such cytokines.

Materials and methods

Mice

In this study, 6- to 8-week old female BALB/c mice (Charles RiverRaleigh Laboratories, Raleigh, NC) were used as spleen cell do-nors. Female, 8- to 12-week old BALB/c mice (B&K Universal,Kent, WA) were used as bone marrow cell donors. The mice werehoused in conventional animal quarters.

Treatment of mice

Groups of 10 mice were injected intraperitoneally with LSF 100mg/kg (Cell Therapeutics, Inc., Seattle, WA) or control vehicle(0.2% HC1/10% ethanol) on the left side of the peritoneal cavity,followed by a second injection of control vehicle, or an injection ofAraC 800 mg/kg, CisP 8 mg/kg, melphalan 40 mg/kg (Melph), oretoposide 75 mg/kg (VP-16) (Sigma, St. Louis, MO) on the rightside of the peritoneal cavity. Each injection volume was approxi-

mately 0.2 mL. LSF or control vehicle was injected either simulta-neously with the cancer chemotherapeutic agents, or 1, 3, 5, 8, 24,or 48 hours before injection of the chemotherapeutic agents. In allexperiments, the mice were sacrificed by cervical dislocation andtheir spleens removed 24 hours after the last injection.

Preparation of spleen cell conditioned medium

Spleen cell suspensions were obtained by grinding spleens be-tween the frosted ends of two glass slides, wetted with RPMI-1640medium, supplemented with 10% fetal bovine serum (FBS) and1% penicillin/streptomycin (RPMI) (Gibco Laboratories, GrandIsland, NY). Individual spleen cell suspensions (5 mL of cell sus-pension in RPMI/spleen) were transferred to wells of a six-welltissue culture plate (Nunc, Napervill, IL) and incubated for 24hours at 37

8

C in a fully humidified atmosphere of 5% CO

2

in air.After 24 hours, the contents of the wells were collected, trans-ferred to a 15-mL centrifuge tube, and centrifuged for 15 minutesat 1,200

g

to remove particulate matter. The conditioned media(CM) were collected and dialyzed (Spectra/Por6 dialysis mem-brane, molecular weight cutoff 3,500; Spectrum, Houston, TX)against phosphate-buffered saline (PBS) for 24 hours with threePBS changes. The dialyzed CM were subsequently filtered, ali-quoted, and frozen (

2

20

8

C) until further use.

Preparation of bone marrow cell suspensions

Bone marrow cell suspensions were obtained by flushing the fem-oral shafts with 1 mL of ice-cold RMPI. The cells were washedonce with RPMI, counted using a hemocytometer, and maintainedat 4

8

C until used in the colony forming assays.

Colony forming cell assays

Spleen cell CM were added to achieve a 10% v/v final concentra-tion in methyl cellulose (MC) supplemented with previously deter-mined (not shown) optimal combinations and concentrations ofcytokines for assessment of multiple normal murine bone marrowprogenitor cells within a single dish. CFU-GM, CFU-MEG, BFU-E, CFU-MIX, and HPP-CFC were cultured in a mixture (1:1) ofMethocult M3230 and growth factors containing MethocultM3434 (Stem Cell Technologies, Vancouver, British Columbia,Canada). This mixture was supplemented with Epo (Amgen,Thousand Oaks, CA) and hemin (Sigma), resulting in a final con-centration of 25 ng/mL SCF, 5 ng/mL IL-3 and Il-6, 2 U/mL Epo,and 0.2 mM hemin. CFC-preB were cultured in Methocult M3220(Stem Cell Technologies), supplemented with murine SCF (80 ng/mL) and murine IL-7 (60 ng/mL) (R&D Systems, Minneapolis,MN). To evaluate the effect of each CM, three 1-mL aliquots ofMC with the appropriate growth promoting cytokines, 10% (v/v)CM, and 2

3

10

4

bone marrow cells were dispensed in 35-mm tis-sue culture dishes (Nunc). The cultures were incubated at 37

8

C in afully humidified atmosphere of 5% CO

2

in air. Colonies werescored using an inverted microscope. CFU-GM, CFU-MEG, andBFU-E were scored after 7 days. The dishes were placed back inthe incubator and CFU-MIX and HPP-CFC in the same disheswere scored after an additional 7-day culture period. CFC-preBcolonies were scored after 7 to 9 days.

Treatment of CM with neutralizing antibodies

Pools of CM (equal volumes of each individual CM from a spe-cific treatment group) were treated with neutralizing polyclonalantisera to MIP-1

a

, TNF-

a

, IL-1

a

, and TGF-

b

(R&D Systems), ora neutralizing monoclonal antibody to mouse IFN-

g

(PharMingen,

918

P. de Vries and J.W. Singer/Experimental Hematology 28 (2000) 916–923

San Diego, CA), all at 10

m

g/mL final concentration, either indi-vidually or in combination. As a control, CM were treated withRPMI. In some experiments, nonspecific species and/or isotypematched antisera (10

m

g/mL) were added: normal goat serum(R&D Systems) as a control for anti–MIP-1

a

, anti-TNF-

a

, andanti-IL-1

a

, normal rabbit serum (R&D Systems) as a control foranti–TGF-

b

, and rat IgG

1

(PharMingen) as a control for anti–IFN-

g

. To these mixtures, GammaBind Plus Sepharose (Pharma-cia Biotech, Alameda, CA) was added. The mixtures were incu-bated for 4 to 14 hours at 4

8

C on a rocking platform to remove im-mune complexes. After the incubation, the suspensions werecentrifuged for 30 minutes at 12,000

g

at 4

8

C. The neutralized CMwere collected and subsequently tested in colony forming assays.

Statistical analysis

The mean colony counts (results of triplicate dishes) obtained foreach of the 10 spleen CM per treatment group and the standard de-viation (SD) were calculated. An unpaired Student’s

t

-test was per-formed to determine statistical differences. Statistical significanceis indicated at

p

,

0.05.

Results

Treatment of mice with cancer chemotherapeuticagents results in ex vivo release of hematopoietic inhibitors

Results of an experiment in which AraC, CisP, and Melphwere injected simultaneously with LSF or control vehicleare depicted in Table 1. CM from spleens of mice treatedwith AraC, CisP, or Melph

1

control vehicle inhibited col-ony formation by all the progenitor types studied except

CFU-GM, when compared to CM from mice treated withcontrol vehicle. For CFU-MEG, BFU-E, and CFC-preB, theinhibition of colony formation after treatment with cyto-toxic agent CM was statistically significant (except for theAraC CM on BFU-E, Table 1). Growth inhibition rangedfrom 30% to 37% for CFU-MEG, 8% to 39% for BFU-E,and 14% to 28% for CFC-preB. These CM also numericallyreduced growth of CFU-MIX and HPP-CFC by 17% to35%, and by 12% to 28%, respectively, but the effects didnot reach statistical significance (Table 1).

Lisofylline suppresses ex vivo release of hematopoietic inhibitors induced by cancer chemotherapeutic agents

Table 1 shows that CM from mice treated simultaneouslywith LSF and AraC, CisP, or Melph had less inhibitory ac-tivity than CM from mice treated simultaneously with con-trol vehicle and AraC, CisP, or Melph. In some instances,CM from mice treated with LSF

1

a chemotherapeuticagent significantly enhanced the number of colonies, notonly as compared to CM from chemotherapeutic agenttreated mice, but also as compared to Cm from control mice(Table 1). CM from mice treated with LSF by itself had nei-ther a stimulatory nor inhibitory effect.

The duration of the suppressive effect for a single dose ofLSF on chemotherapy induction of hematopoietic inhibitorswas tested. To this end, groups of 10 mice each were firstinjected with vehicle control or LSF followed after 1, 3, 5,8, 24, or 48 hours by an injection of VP-16, CisP, or Melph.Twenty-four hours after the last injection, the mice were

Table 1.

Effect of spleen cell CM on progenitor cell growth after simultaneous treatment with LSF or control vehicle and cancer chemotherapeutic agents

Colonies per 10

5

cells

CFU-GM CFU-MEG BFU-E CFU-MIX HPP-CFC CFC-preB

Experiment AControl 103

6

24 54

6

25 24

6

10 12

6

7 57

6

19 219

6

12LSF 105

6

20 57

6

30 30

6

15 13

6

9 61

6

23 224

6

13Percent of control 102 106 125 108 107 102AraC 103

6

23 35

6

15* 22

6

8 10

6

5 50

6

14 184

6

38*Percent of control 100 65 92 83 88 84LSF

1

AraC 120

6

26 57

6

34

†

35

6

13*

,†

12

6

7 68

6

22* 220

6

35

†

Percent of control 117 106 146 100 119 100Experiment B

Control 124

6

28 57

6

17 31

6

13 17

6

9 65

6

25 219

6

12LSF 133

6

32 60

6

15 36

6

19 19

6

10 72

6

33 224

6

13Percent of control 107 105 116 112 111 102CisP 118

6

20 40

6

6* 19

6

7* 12

6

6 47

6

9* 188

6

16*Percent of control 95 70 61 71 72 86LSF

1

CisP 143

6

34

†

79

6

16*

,†

35

6

8

†

14

6

5 62

6

19

†

226

6

13

†

Percent of control 115 139 113 82 95 103Melph 115

6

11 36

6

13* 19

6

5* 11

6

6* 53

6

17 158

6

27*Percent of control 93 63 61 65 82 72LSF 1 melph 138 6 23† 74 6 20*,† 39 6 6*,† 14 6 7 72 6 2† 184 6 28*,†

Percent of control 111 130 126 82 111 84

*p , 0.05 vs control vehicle; † p , 0.05 vs cancer chemotherapeutic agent only group.

P. de Vries and J.W. Singer/Experimental Hematology 28 (2000) 916–923 919

sacrificed, their spleens removed, and spleen cell CM pre-pared and tested. Figure 1 shows that CM from mice treatedwith VP-16, CisP, or Melph after pretreatment with controlvehicle in most instances significantly inhibited BFU-E col-ony formation and that CM from mice treated with VP-16,CisP, or Melph after pretreatment with LSF did not demon-strate this inhibition. The LSF effect was observed when itwas given up to 48 hours before the chemotherapeuticagents. Suppression by LSF of the inhibitory activity in-duced by chemotherapeutic agents was likewise observedfor CFU-GM, CFU-MEG, CFU-MIX, and HPP-CFC (datanot shown).

Effect of treatment of CM with neutralizingantibodies against growth inhibitory cytokinesTo study the effect of cytokine depletion on colony forma-tion, pooled CM were treated with neutralizing antibodiesdirected against known growth inhibitory cytokines. Treat-ment of the control vehicle CM with neutralizing antibod-ies, either individually or as a mixture, did not enhance col-ony growth. In contrast, all antibody treatments of the AraCCM resulted in enhanced growth of HPP-CFC colonies

compared to the no-antibody control, which inhibited HPP-CFC growth by 20% compared to control vehicle CM (Fig.2, top left). Removal of MIP-1a from AraC CM was mosteffective at enhancing growth. This concurs with data re-ported by others that MIP-1a predominantly inhibits moreprimitive stem and progenitor cells such as HPP-CFC, butnot the more mature progenitors [13–16,55].

Figure 3 shows a similar experiment in which the effectof treatment of AraC and Melph CM with neutralizing anti-bodies on CFU-MIX colony formation was studied. Treat-ment of control vehicle CM with neutralizing antibodies, ei-ther alone or as a mixture, did not enhance colony growth.Control CM from AraC- and Melph-treated mice inhibitedCFU-MIX colony formation by approximately 50% and30%, respectively (top left). Treatment of AraC and Mel-phalan CM with some of the neutralizing antibodies en-hanced CFU-MIX colony growth, whereas treatment withothers did not. However, the patterns were different for CMfrom AraC- and Melph-treated mice. Thus, treatment ofAraC CM with anti–TNF-a and anti–TGF-b enhanced col-ony growth, whereas this had no effect on Melph CM.Treatment of AraC CM with anti–TNF-a had the greatest

Figure 1. Pretreatment with LSF abrogates BFU-E growth inhibitory activity induced by cancer chemotherapeutic agents. Effect of spleen cell CM frommice treated with VP-16 (75 mg/kg), CisP (8 mg/kg), or Melph (40 mg/kg), either with control vehicle or in combination with LSF (100 mg/kg) on BFU-Egrowth, is shown. Control vehicle and LSF were administered at the noted time intervals before administration of the chemotherapeutic agents. Each bar rep-resents the mean colony counts obtained from 10 individually tested spleen cell CM (each tested in triplicate) 6 1 SD. p , 0.05 vs *control CM or OChemo-therapeutic agent alone CM.

920 P. de Vries and J.W. Singer/Experimental Hematology 28 (2000) 916–923

effect, whereas anti–IL-1a and anti–INF-g had the greatesteffect on Melph CM.

Comparison of the effects of treatment with neutralizingantibodies against hematopoietic inhibitors of AraC CMshown in Figures 2 and 3 also indicates differential effectson progenitors. For example, anti–MIP-1a treatment ofAraC CM enhanced HPP-CFC growth, but had no effect onCFU-MIX colonies.

Figure 4 shows the effect on BFU-E growth by treatingMelph CM or Melph 1 LSF CM with neutralizing antibod-ies. Melph CM inhibited BFU-E colony formation by ap-proximately 30%, whereas Melph 1 LSF CM was not in-hibitory (top left). Treatment of control vehicle CM withneutralizing antibodies did not result in higher colony num-bers. In contrast, treatment of Melph CM with neutralizingantibodies to cytokines other than MIP-1a increased colonynumbers. Treatment of Melph 1 LSF CM with neutralizingantibodies showed a similar pattern to control CM.

DiscussionTreatment of mice with four commonly used, structurallyand mechanistically unrelated, cancer chemotherapeuticagents at clinically relevant doses evokes the ex vivo release

of hematopoietic progenitor growth inhibitory activity byspleen cells. Such inhibitory activity may delay hematopoi-etic recovery after cytotoxic therapy. Following cytotoxictherapy, elevated levels of endogenous growth stimulatorycytokines, such as G-CSF, Flt3 ligand, and Epo, can be de-tected in serum [23–30,56]. Nevertheless, hematopoietic re-covery in animals and humans can be accelerated by the ad-dition of pharmacologic doses of cytokines [57–61]. It ispossible that a high percentage of receptor occupancy bygrowth stimulatory cytokines may be needed to override theeffects of the chemotherapy-induced, lineage nonspecificnegative regulators.

In this study, we found that the novel antiinflammatoryagent LSF abrogated progenitor growth inhibitory activityinduced by treatment with chemotherapeutic agents. This

Figure 2. Treatment with AraC leads to release of cytokines at concentra-tions and combinations that inhibit HPP-CFC colony formation. PooledCM from five mice treated with control vehicle (top right) or AraC (bottomright) were treated with RPMI-1640 supplemented medium (control, noantibodies) (top left) or neutralizing antibodies (Abs) against knowngrowth inhibitors (panels on the right). Each bar represents the mean HPP-CFC number 6 1 SD. The horizontal bar represents the mean number ofcolonies in the control without Abs 6 1 SD.

Figure 3. Treatment with AraC or Melph leads to release of cytokines atconcentrations and combinations that inhibit CFU-MIX colony formation.Pooled CM from five mice treated with control vehicle (top right), AraC(middle right), or Melph (bottom right) were treated with RPMI-1640 sup-plemented medium (control no antibodies) (top left) or neutralizing anti-bodies (Abs) against known inhibitory molecules (panels on the right).Each bar represents the mean CFU-MIX number 6 1 SD. The horizontalbar represents the mean number of colonies in the control without Abs 6 1 SD.

P. de Vries and J.W. Singer/Experimental Hematology 28 (2000) 916–923 921

effect of LSF was not only observed when LSF was givensimultaneously with the cytotoxic agents (Table 1), but evenwhen LSF was given up to 48 hours earlier (Figs. 1 and 4).The latter finding was surprising, due to the rapid and com-plete clearance and excretion of LSF. The plasma half-lifeof LSF in mice after an intraperitoneal injection at a concen-tration of 100 mg/kg has been reported to be approximately7 minutes [49]. The data presented here suggest that LSFhas a biologic effect that persists long after drug levels areno longer detectable. Inhibitory molecules such as MIP-1a,TGF-b, and platelet factor 4 can be induced by other in-flammatory proteins activated by cytotoxic drugs [62–64].Thus, LSF may act proximally to prevent production or acti-vation of a cascade of cytokines, chemokines, and interfer-ons that suppress hematopoiesis. Although the biomoleculartarget for LSF is unknown, it has been reported to inhibitsignaling through the IL-12 receptor on murine and human

Th1 cells by suppressing STAT4 phosphorylation and thussuppressing IFN-g production [53,54].

LSF abrogated the growth inhibitory activities inducedby cancer chemotherapeutic agents for myeloid, erythroid,megakaryocytic, B-lymphoid lineage, and multipotentialprogenitor cells. In contrast to growth stimulatory cyto-kines, which are relatively lineage specific, negative regula-tors of hematopoiesis lack lineage specificity [15,20]. Thissuggests that the observed multilineage effect of LSF is theresult of suppression of the release of one or more nonlin-eage restricted negative regulator(s). In contrast, LSF doesnot induce or inhibit the release of G-CSF by LPS (li-popolysaccharide) stimulated human mononuclear cells invitro, or by murine or human bone marrow cells treated invitro with various cancer therapeutic agents [49,65,66]. Thesame principle may apply to and explain the effects of LSFon other cells with similar rates of turnover, such as gas-trointestinal mucosal crypt cells or the basal layer cells ofthe oral mucosa, and thereby may have an impact on mu-cositis [40,46,48,52].

Neutralizing antibodies against selected known negativeregulators removed inhibitory activity from CM from micetreated with cancer chemotherapeutic agents alone (Figs. 2–4). This is especially well demonstrated for HPP-CFC aftertreatment with AraC CM (Fig. 2) where removal of MIP-1aresulted in a large increase in HPP-CFC colony formation.In vivo, MIP-1a suppressed cycling rates of stem and pro-genitor cells and was myeloprotective for AraC and hydroxy-urea [21]. Our data are in agreement with these observa-tions. ELISAs for MIP-1-a using CM before and aftertreatment with neutralizing antibodies showed that, aftertreatment, MIP-1a could no longer be detected (data notshown). These findings suggest that the observed effects inthe colony assays were due to the removal of MIP-1a to alevel below the detection limits of the ELISA (2-5 pg/mL).Also, treatment of LSF 1 Melph CM with neutralizing anti-bodies did not increase colony growth, indicating that LSFsuppressed elaboration of the hematopoietic inhibitors (Fig.4). However, ELISAs performed on the CM did not alwaysshow the highest levels of inhibitory cytokines in CM frommice treated with cytotoxic agents alone and/or reduced lev-els of inhibitory regulators in CM from LSF 1 cytotoxicdrug treated mice and thus were in contrast with the colonyforming assay results. Nonetheless, because most of theknown inhibitors of hematopoiesis are pleiotropic and act inthe presence of other cytokines (positive and negative), it isnot unexpected that their activities might be nonlinear withrespect to concentration, or under certain circumstances,even biphasic. For instance, it has been demonstrated thatMIP-1a, TGF-b, and TNF-a can each inhibit or stimulatethe growth of more mature progenitors such as CFU-GM,depending on their concentrations and the cytokines used tostimulate growth [6,13,14,67,68]. In addition, inhibitory cy-tokines and chemokines can synergize with each other orother molecules, such as vascular endothelial growth factor,

Figure 4. Pretreatment with LSF abrogates the induction of negative regu-lators for BFU-E colony formation by Melph. Pooled CM from five micetreated with control vehicle (top right), Melph (middle right), or LSF 1Melph (bottom right) were treated with RPMI-1640 supplemented medium(control, no antibodies) (top left) or neutralizing antibodies (Abs) againstknown growth inhibitors (panels on the right). Each bar represents themean BFU-E number 6 1 SD. The horizontal bar represents the meannumber of colonies in the control without Abs 6 1 SD.

922 P. de Vries and J.W. Singer/Experimental Hematology 28 (2000) 916–923

in a manner analogous to stimulating cytokines [15]. For in-stance, TGF-b can interact with TNF-a and IFN-g to inhibitprogenitor growth, whereas each of the individual mole-cules did not [19].

CM obtained from mice treated with AraC and Melphcaused different patterns of CFU-MIX growth inhibition(Fig. 3). This may occur because these drugs induce differ-ent patterns or concentrations of hematopoietic inhibitorymolecules, as implied by the biologic effect of pretreatmentwith neutralizing antibodies. In addition, studies with neu-tralizing antibodies showed that a certain induced negativeregulator, for instance MIP-1a, has different effects onHPP-CFC and CFU-MIX colony formation (Figs. 2 and 3).

Thus, the findings of this study are that cytotoxic drugsin various classes induced multiple hematopoietic inhibitorymolecules that differentially affect growth of various he-matopoietic progenitors. Furthermore, treatment of the micewith LSF preceding or concurrent with the cytotoxic agentssuppresses the release of hematopoietic inhibitors. The lat-ter finding suggests a basis for the enhancement by LSF ofhematopoietic recovery in mice treated with cancer chemo-therapeutic agents and in patients with acute myelogenicleukemia undergoing standard induction chemotherapy [69].

AcknowledgmentsThe authors thank Dr. Robert A. Lewis for critical reviewing of themanuscript and helpful comments and Ms. Zhenna Xu for excel-lent technical assistance.

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