Transforming growth factor-3 is the major mediator of natural suppressor cells derived from normal bone marrow Steven

C. Moore,

Department

Michael

of Microbiology

and

Abstract: We previously reported marrow cells activated by interleukin-3 locyte-macrophage colony-stimulating

A. Shaw, Immunology,

and

Lee

University

that

murine bone (IL-3) or granufactor (GM-CSF)

Biol. 52: 596-601;

Key Words: stimulating factor

interleukin-3 . regulation

tent

colon

have reported that detectable suppressor

NS

cells factors

function [11, 13].

Journal

of Leukocyte

Biology

Arkansas

to nylon wool or plastic, radiation to lysis with leucine methyl ester we report that cytokine-activated

elaborated soluble

a soluble factor had

of transforming

AND

Rock,

growth

factor properties

with

factor-f3

supconsis-

(TGF-/3).

METHODS

of NS cells

cells/ml with 40 U/mi IL-3 (Genzyme Corp., Boston, MA) or 50% concanavalin A (Con A) spleen cell supernates (CAS). The CAS were prepared [3] as 24 h supernates of 5 x 106 C57BL/6 spleen cells/mi with 2.5 tg/ml Con A (Sigma Chemical Co., St. Louis, MO). Before use, 10 mg/mi a-methyl-D-mannoside (Sigma Chemical Co., St. Louis) was added and the CAS was filter sterilized and stored at - 70#{176}C.Following the 72 h incubation with cytokines, the cells were washed three times to remove exogenous cytokines

-

and assayed for NS activity or cultured for an additional 24 h in serum-free medium at 10 cells/mi to prepare NS cell supernates. The NS cell-conditioned supernates were filter sterilized and stored at - 70#{176}C. Cell viability was determined

by

trypan

blue

exclusion.

Natural Suppressor Normal washed,

assays

C57BL/6 and placed

spleen cell in microculture

suspensions were prepared, at 2 x 10 cells/well with

2.5 ig/ml Con A. Dilutions of cells/well) or NS supernates (5-75%) spleen cell cultures. Cultures were

NS cells (0.7-20 x 10 were added to triplicate labeled with 1 sCi/ml

reported to be other investigain

the

absence

of Abbreviations:

We have found that nylon wool nonadherent murine bone marrow cells activated with interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GMCSF), but not IL-2 or y-interferon, acquired potent NS activity [14]. The activated NS cells nonspecifically suppressed T and B cell proliferative responses to mitogens and mixed lymphocyte reactivity. These bone marrow-derived NS cells

596

those

cells The

Little

nonadherent cells were suspended in RPMI 1640 containing 10% fetal calf serum (HyClone Laboratories, Logan, Utah), 50 tM 2-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 0.25 sg/ml fungizone. Cell suspensions were incubated for 72 h at 4 x 106

defined. The production of prostaglandins has been implicated in NS cell activity in some studies [5, 9], but other studies have found that NS cells do not elaborate prostaglandins [3, 10, 11]. Unidentified soluble suppressor factors dis-

tors any

Sciences,

Untreated, 6-wk to 6-months-old, female C57BL/6 mice (Charles River Breeding Laboratory, North Wilmington, MA) were used as a source of normal bone marrow cells for all studies. Washed bone marrow cells were separated on nylon wool columns as described by Morecki et al. [16]. The

Natural suppressor (NS) cells nonspecifically suppress lymphocyte proliferative responses [1] and antibody induction [ 2]. Natural suppressor cells are prevalent in the spleens of neonatal mice [3] and of adult mice exposed to a variety of conditions, including total lymphoid irradiation [2, 3], chronic graft vs. host disease [4], and certain tumors [5]. In addition, NS cells have been found in normal adult bone marrow [1, 6-8], suggesting that NS cells might migrate from the bone marrow to peripheral lymphoid organs in response to these insults [2]. The mechanism of NS suppression has not been clearly

have also been 10, 12]. However,

with

Preparation

y

[6,

Medical

MATERIALS

INTRODUCTION

tinct from prostaglandin produced by NS cells

for

bone marrow NS pressive activities.

1992. granulocyte-macrophage

Soderberg

F.

were Thy-1, nonadherent resistant, and susceptible [15]. In the present study,

had potent nonspecific natural suppressor (NS) cell activity. In the present study, we demonstrated that these activated NS cells released a soluble factor (or factors) capable of nonspecifically inhibiting T cell mitogenic responses. Consistent with the properties of transforming growth factor-13 (TGF-/3), treatment of the NS supernates with heat failed to denature the factor, and in fact significantly increased its suppressive activity. The NS suppressor factor strongly inhibited proliferation of the TGFf3-sensitive tumor cell line, A549. Cytokine activation of suppressive activity correlated with the production of a 10- to 13-kDa protein, consistent with the size of TGF-f3 and rIL-3 induced a sevenfold increase in TGF-f3 transcription. Finally, neutralizing anti-TGF-/3 antibody inhibited the suppressive activity of the supernates, mdicating that TGF-f3 was responsible for most, if not all, of the suppression expressed by these bone marrow NS cells.

J. Leukoc.

S.

of Arkansas

Volume

52,

December

NS,

natural

granubocyte-macrophage growth

factor-fl;

nates;

SDS-PAGE,

suppressor,

IL-3,

colony-stimulating Con

A,

concanavalin

sodium

A;

dodecyl

interleukin-3;

TGF-3

factor; CAS,

Con

A

sulfate-polyacrylamide

GM-CSF,

,

transforming

spleen gel

cell

super-

electropho-

resis. Reprint Immunology,

W.

1992

Markham Received

requests: Slot

St., August

Lee 511,

Little 13,

S.F.

Soderberg,

University

Rock, 1991;

of

Department Arkansas

Arkansas accepted

of for

72205. July

1, 1992.

Medical

Microbiology Sciences,

and 4301

( Hoefer [3H] thymidine (ICN Radiochemicals, Irvine, CA) over the final 4 h of 72 h incubation. Cells were then harvested and radioactivity was measured in a liquid scintillation spectrometer (Packard, Downers Grove, IL). Significance was determined by Student’s t test. Results are representative of at least two separate experiments and consistent with many similar The tumor

experiments. A549 human cell line, kindly

Medical essential subcultured medium measured

lung carcinoma, provided by Dr.

a TGF-3-sensitive Y.C. Yeh (Univ

Systems, 1 iCi/ml

Minneapolis, [3H] thymidine

MN). over

Cultures the final

TGF-f3

RNA

slot

by

Instruments,

San

Francisco,

CA)

and

ana-

autoradiography.

Transforming

growth

factor-fl

neutralization

assay

The TGF-13 neutralizing antibody (R & D Systems, Minneapolis, MN) was a purified IgG preparation with neutraiizing activity against TGF-/31, TGF-f32, and TGF-f31.2, but not TGF-f33. Anti-TGF-/3 antibody (75 g/ml) was incubated with 2 ng/ml purified TGF-f31 or 50% NS supernates for 3 h at 37#{176}C.The mixtures were then assayed for suppression of spleen cell mitogenic responses to Con A or of A549 cell proliferation, with final concentrations of 1 ng/ml TGF-fll and 25% NS supernate.

AR

A549 cells were After 24 h, the minimal essenwith or without

RESULTS Activated

(R & D

Nylon

were labeled with 4 h of 48 h incuba-

tion. Cells were trypsinized for 5 mm prior to harvesting optimize the recovery of adherent cells. Radioactivity measured as described previously.

TGF-f3

lyzed

Sciences), was maintained in Dulbecco’s minimal medium with 10% fetal calf serum. The cells were by trypsinization every 4 days with a change in after 48 h. The presence of TGF-f3-like activity was by suppression of A549 cell proliferation, as

described by Zarling et al. [17]. Briefly, placed in microculture at 1 x 10 cells/well. culture medium was replaced by Dulbecco’s tial medium containing 0.1% fetal calfserum dilutions ofthe NS cell supernates or purified

Scientific

NS cells wool

suppress

nonadherent

T cell

bone

proliferation

marrow

cells

were

incubated

for 3 days in media containing 50% murine CAS. The cells were then collected and assayed for suppression of spleen cell responsiveness to Con A. As we have reported before [14], these cells acquired potent, dose-dependent suppressive activity (Fig. 1). T cell responses to Con A were suppressed by nearly 90% when 2 x 10 CAS-treated bone marrow cells were added to 2 x 10 spleen cells. As few as 6 x 10 NS cells significantly suppressed the T cell proliferative response by 35%. Over 9 experiments, an average ratio of 1 NS cell per 7 spleen cells reduced responsiveness to Con A by 50%. Bone marrow cells incubated in the absence of CAS did not suppress proliferation, except at higher NS:spleen cell ratios

to was

blot

The RNA slot blots were performed as described [14]. Briefly, fresh nylon-wool nonadherent bone marrow cells at 4 x 106 cells/ml were incubated for 10 h with medium, 50 U/ml rIL-3, or 1000 U/ml Wy-interferon. Total cellular RNA was then extracted and quantified by absorbance at

and not

260 nm. The RNA (3.5 pg/sample) was denatured at 60#{176}C for 1 h in 1 M glyoxal, 10 mM sodium phosphate buffer, and transferred onto a nylon membrane using a Bio-Dot SF (BioRad, Richmond, CA) blotting apparatus. The membrane was then baked for 1 h at 80#{176}Cin vacuo. The human TGF-

then by less substantially

than 25%, contribute

suggesting that cell to the suppression.

periments, irradiated syngeneic % Suppression cell numbers were used in place

f31 oligomer probe (R & D Systems, Minneapolis, MN), which is homologous with 27 of 28 murine bases, was 5-end labeled with ‘y[32P]ATP (ICN, Irvine, CA) and T4 polynucleotide kinase. Unincorporated label was removed by ethanol precipitation. After overnight hybridization at 37#{176}C, the membrane was washed twice in 1 x prehybridization buffer containing 0.1% SDS for 30 mm at room temperature. The membrane was exposed for 20-48 h at - 80#{176}Cto Kodak X-AR5 film using intensifying screens. Densitometric tracings of the developed film were performed using a Fast Scan 300A Computing Densitometer (Molecular Dynamics, Sunnyvale, CA). Total RNA levels per slot were assessed by the expression of f3-actin mRNA.

crowding In other

spleen cells at of NS cells, they,

comparable too, did

did exnot

100

CAS

80

Me

ium

-

60-

40

SOS-PAGE

analysis

of NS supernates

The NS cells were cultured for 72 h with medium alone or medium containing 50% CAS or 40 U/ml IL-3. The cells were extensively washed and placed in serum-free methioninedeficient medium (Gibco Laboratories, Grand Island, NY) containing 100 tCi/ml [355] methionine cals, Irvine, CA). After incubation at 15 hr, cell-free supernates were collected. 35S-labeled NS supernates and loading

20

(ICN Radiochemi1 x 10 cells/mi for Equal volumes of buffer containing

2-mercaptoethanol were boiled for 5 mm and 30 l assayed with 17% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Low-molecular-weight standards (Bio-Rad Laboratories, Richmond, CA) were added to one lane. The samples were separated at 135 v for 5 h and stained with Coomassie blue. The gel was dried using DryGel Jr.

4

.....+ -

I

0 6300

1 2500

25000

1 00000

50000

200000

NS cells/culture

-

Fig. 1. CAS-activated marrow cells were washed and added

bone marrow NS cells. Nylon wool nonadherent incubated for 72 h in 50% CAS. The cells were at various concentrations to 2 x l0 normal spleen

plus

2.5

sg/ml

A.

over

the

final

pression

Moore

±

et al.

Con

Proliferation

was

measured

4 h of 72 h cultures. The data represent standard error of 9 separate experiments.

TGF-/3-mediates

bone

marrow

by

3Hthymidine

the

NS

mean

activity

bone then cells uptake

percent

sup-

597

affect the proliferation assay except at very high cell numbers [14]. To determine if the CAS-activated bone marrow NS cells produced a soluble suppressor factor, nylon wool nonadher-

100

80

ent bone marrow cells were incubated for 72 hr in 50% CAS, washed 3 times to remove exogenous cytokines, and suspended in serum-free medium at 10 cells/mi. After 24 h incubation, the NS-conditioned supernates were collected and dilutions were assayed for suppression of responses to Con A. As shown in Fig. 2, the NS supernates markedly reduced cell proliferation in a dose dependent manner. As little as 10% supernate suppressed T cell responses by 40%, more supernate totally blocked proliferation. was not affected by the supernates.

while Cell

/!

0 Cl) (9

0)

50% or viability

0.

40 C,) .‘.-o O

Characterization Because

of the suppressor

production

factor

of prostaglandins

mediate nonspecific the effects of blocking

immunosuppression prostaglandin

has

been

reported

20

to

[5, 9], we examined production on the sup-

0

pressive activity of these cells. The CAS-activated NS cells were assayed for suppression ofCon A responsiveness at a 1:1 NS:spleen cell ratio in the presence of 0.01-100 g/ml indomethacin. Indomethacin at 10 g/ml has been previously shown to effectively reduce nonspecific suppression in other systems (Soderberg, L.S.F., unpublished data). As shown in Fig. 3, suppression was not reduced at any ofthe indomethacm concentrations tested, suggesting that prostaglandins did

0

0.01

0.1

1.0

10

Indomethacin Fig.

3. Prostaglandins

immune Fig.

1, were

cells/well

not mediate suppression. In fact, indomethacin at 10 and 100 g/ml slightly, but significantly, increased suppression, suggesting that prostaglandins present in the cultures marginally reduced NS activity. To characterize the heat-stability of the suppressor factor, NS supernates were heated to 100#{176}Cfor 3 mm prior to assay. This treatment not only failed to negate the suppressive ac-

0.01

were

suppression.

to

Control of36,426

washed, and

100 sg/ml.

Con

not

Natural and A. Data

cultures incubated ± 1401.

cultured Indomethacin are

(ug/mi)

responsible

for

suppressor

reported

in the

at

cells 2

x l0

was as the

absence

100

marrow

activated at

with final

percent

of NS cells

NS-mediated with

cells/well

added mean

bone

2

CAS, x

l0

concentrations suppression

had

as

an average

of

SD. CPM

±

tivity of the supernate but consistently enhanced the suppressive activity in the Con A assay (data not shown). Because suppression following such heat treatment is a property characteristic of TGF-f3 [18], NS supernates were tested for suppressive activity using the TGF-/3-sensitive tumor cell line, A549. As shown in Fig. 4B, proliferation of A549 cells was inhibited by the NS cell supernate in a dose-

100

dependent manner, suggesting that the supernates had 1XW/3-like activity. Purified TGF-f3 likewise suppressed A549 cell growth (Fig. 4A) and spleen cell blastogenesis to Con A (1 ng/ml F-fl suppressed Con A responses by about 50%). The suppressive activities of purified ‘1DF-j3 and the NS supernates were both substantially reduced if added 24 h after initiation of the assay cultures. Based on the activity of purified TGF-f3 as shown in Fig. 4A, this suppression, if totally due to 1DF-f3, would be consistent with the presence of approximately 25 ng/ml TGF-13 in the undiluted NS

80

6O

supernates. As with the spleen cell Con A assays cited before, heat treatment known to convert the latent form ofTGF-/3 to the active form [18] roughly doubled the suppressive activity of the supernates in the A549 cell proliferation assay (Fig. 4B). This would suggest that the latent and active forms of

4o 20

TGF-/3

were both present in the supernates. To determine if ].13F-fl synthesis was induced by IL-3, nylon wool nonadherent bone marrow cells were incubated for 10 h in the presence of medium or 50 U/ml rIL-3. Cellular RNA was then extracted and probed for TGF-f3 mRNA by slot blot. As shown in Fig. 5, 1.DF-f3 transcription in cells incubated in medium was minimal. Incubation of the cells with rIL-3 increased specific ‘TtW-f3 transcription by sevenfold. Incubation of the cells with ‘y-interferon did not affect TGF-fl expression.

0 0

25

50

75

% NS Supernate Fig. 2. Bone marrow NS cells released a soluble suppressor wool nonadherent bone marrow cells were cultured in 50% The cells were then washed and incubated at 1 x l0 cells/ml medium

pression percent absence

598

for

in

spleen

24

h.

Dilutions

of the

culture

supernates

were

factor. Nylon CAS for 72 h. in serum-free assayed

for

sup-

To characterize the proteins released by NS cells into the supernates, nylon wool nonadherent bone marrow cells were labeled with [35S]methionine during NS cell activation with

of spleen cell responses to Con A. Data are reported as the mean suppression ± SD. Control cultures stimulated with Con A in the of NS supernate had a mean CPM of 11,621 ± 679.

Journal

of Leukocyte

Biology

Volume

52,

December

1992

anti-TGF-/3 antibody. As shown in Fig. 8, neutralizing antibody to TGF-13 significantly reduced the suppressive capability of the NS cell supernates. The anti-TGF-f3 completely abolished the suppressive activity of 1 ng/ml purified TGF-f3 and reduced the suppressive control cultures. These data

60

C 0 (I)

40

TGF-f3

suggest that ble suppressive bone marrow

U)

a) a a.

?/

C/)

20

is responsible, activity elaborated NS cells.

in large by the

part, for the cytokine-activated

solu-

DISCUSSION

A These

0

10

1.0

studies

show

that

NS

cells

present

in the

bone

marrow

could be activated by hemopoietic cytokines and that these activated cells mediated their suppressive activity, at least in part, through the release of a soluble factor. The suppressor factor was characterized as a highly stable, low-molecularweight protein believed to be the active form of 1tF-3, a cytokine known to inhibit T cell responses [19]. The suppressive activity in the NS supernates was increased by heat treatment, closely reflecting the properties of purified TGF-

.

(ng/mI)

/3 Activation

80

0- -O

. of the bone marrow NS cells with CAS or IL-3 generated increased levels of a 10- to 13-kDa protein consistent with the reported size of active TGF-j3 [20]. Interleukin-3 activation of these cells increased TGF-f3 transcription by sevenfold. In addition, anti-TGF-f3 antibody

0

#{149}-#{149} Untreated 100#{176}C,3min

-

60

.-

40

I

-

0

2,000

‘1

;>#{149}

I I

20 0 0

B

.,#vTv

I

C/)

c

*

0

10

20

30

40

1,500

50

% NS Supernate Fig.

4. The

trations cell

cultures

further line).

±

medium, nates

suppressor

TGF-j3

(1 x l0

factor

(solid

Proliferation

was

were

CAS, analyzed

or

determined

or

TGF-/3-like cell

The

line)

48 h cultures. Data SD. Control cultures

50%

had

(A) or NS

cells/well).

treatment

4 h of sion

NS

of purified

are had

NS

after

(B)

supernates

heating by

activity.

supernates

to

as

were

assayed 3

±

447

40 U/ml IL-3. by SDS-PAGE

uptake

the

mean

(A)

and

concen-

added

100#{176}C for

[3H]thymidine

reported 20,810

Various were

to A549 without

mm over

percent 25,100

the

final

suppres±

1157

(B).

The cell-free superin the presence of

2-mercaptoethanol. As shown in Fig. 6, untreated cell supernates (designated U) contained a variety teins over a broad range of molecular weights. Few

1,000

-

(dashed

z E

LL 0 I-

500

15 h NS of proapparent

differences were observed whether cells were incubated in medium, CAS, or IL-3. To eliminate most proteins, the [355] methionine-labeled supernates were treated with agarosecoupled proteinase K before SDS-PAGE analysis (designated K). As shown in the figure, the majority of proteins present in all the supernates were degraded. A 10- to 13-kDa protein, consistent with the molecular weight of the TGF-fl monomer (12.5 kDa), survived proteinase K treatment. This band was present in the CAS and IL-3-activated NS supernates, but greatly reduced in supernates of cells incubated in medium alone. Quantitation of the bands by densitometer scanning showed that incubating cells with IL-3 or CAS increased the amount of the 10- to 13-kDa protein by more than fivefold (Fig. 7). To verify the presence of TGF-/3 in the NS supernates, supernates were treated with neutralizing

Medium

lL-3

Medium

y-IFN

IL-3

i-IFN

TGF-

fl-actin

.-

Fig. 5. Interleukin-3 bone marrow cells 1000

1tF-

U/ml

RNA

computing

Moore

induces 113F-fl transcription. Nylon wool nonadherent were incubated for 10 h in medium, 50 U/mI rIL-3, or ry-interferon. Cellular RNA was extracted and probed for by slot blot. The blots were scanned on a Molecular Dynamics

laser

et al.

densitometer.

TGF-f3-mediates

bone

marrow

NS

activity

599

IL-3

CAS

MEDIA

UKUKU

600

K

974) 66.2)

400 0)

45.0)

E 0

31.0

0) >

21.5 0 14.4,

IL.-3 Fig.

7. Interleukin-3

protein.

The

Molecular of

the

6. Analysis marrow

medium free,

alone

for

72

h.

The

methionine-deficient

methionine

were

by SDS-PAGE of “S-labeled NS supernates. cells were cultured with IL-3 (40 U/mI),

for

separated

cells RPMI

15 h. Untreated

under

were 1640

(U)

reducing

and

then

washed

containing

100

proteinase

conditions

and

Nonadherent 50% CAS, placed

in

zCi/ml

K-treated

medium

or

serum-

supernates

by SDS-PAGE.

neutralized almost all of the suppressive activity of the NS supernates, suggesting that TGF-f3 was the principal active factor. Clark et al. [21] reported that nonspecific suppressor cells present in the uterine decidua of allogeneically pregnant mice released a soluble suppressor factor that was neutralized by anti-’ItF-f3. It is possible that similar types of nonspecific suppressor cells are present in the bone marrow and in the allopregnant uterine decidua. The physical properties of TGF-/3 are also consistent with those of a heatstable cells

suppressor [6]. Because

factor produced by rabbit anti-TGF-/3 in the present

bone study

marrow did not

not

affect

the

synthesis

of

0

Journal

of Leukocyte

suppressive

a relative

activity

Volume

52,

of

Fig.

6 were

densitometer.

The value

of

following

10-

to

13-kDa

scanned

volume

on

a

intensity

100%.

activation

with

moiety is synthesized in a latent form due to precursor peptides [26]. In addition, complex with 1DF-/3-binding proteins of latent TGF-f3 to the active form is not but is believed to occur by acidic or enof the precursor peptides and TGF-j3in microenvironments within the cells or

30

0) 0. 0.

20

C/)

10

0

TGF-f3

TGF-B

Biology

in

Cl)

NS en-

Most cells can be induced to produce TGF-/3, generally in a biologically inactive form [23]. Appropriate activation of T [19] and B lymphocytes [24] and macrophages [25] has been shown to greatly increase TGF-f3 synthesis from low to undetectable background levels. This is consistent with the observed increases in NS cell TGF-f3 mRNA, 10- to 13-kDa

600

assigned

expression

as

Cl)

Prostaglandins, at least, were not involved in the suppression by these cells, as indomethacin over a broad range of concentrations did not reduce their suppressive activity. Interferon.y, which has been reported to be involved in NS cell activity [22], is not believed to play a facilitating role in the present system because excess anti-interferony present during the induction and/or assay of NS cells did not diminish their suppressive activity [14]. In addition, the present study showed did cells.

laser

was

the

obtained

medium

40

tirely eliminate suppressive activity in the NS supernates, the presence of other suppressor factors cannot be ruled out.

that interferon-y mRNA in these

increased

bands

computing

control

and

CAS

l3-kDa

IL-3. The 1DF-/3 to covalent binding latent TGF-f3 may [26]. Conversion well understood, zymatic removal binding proteins

35S-labeled (K)

to

Dynamics

protein,

Fig. bone

and

10-

cAS

December

Fig.

Natural

were

treated

assayed

Data had

1992

8. Neutralizing

sion.

for

anti-’mF-3

suppressor

with

cell

anti-TGF-/

suppression

are reported as the 16,903 ± 1617 CPM.

NS Supernate antibody

supernate

(75

zg/ml)

of spleen

cell

mean

percent

eliminates

Con

NS

mediated

1.DF-3 for 3 h at 37#{176}C.Samples

(25%)

and

purified

A responses

suppression

as described ±

SD.

Control

suppres(1 ng/ml)

were

then

in Fig. cultures

1.

through interactions with cell surface molecules [27, 28]. Enzymes proposed to mediate 1DF-13 activation by proteolytic cleavage include cathepsin D, plasmin [29], and sialydase [28]. Activation could modulate the enzymes that convert TGF-fl, which might be produced by the lymphocytes in culture, from the latent to the active form. The model is consistent with the observation by Oreffo et al. [30] that untreated, nonadherent avian bone marrow cells produced latent TGF-

9.

Choi,

Maier,

T,

T-cell

generation

is partially

reversed

K.L.,

ofcytotoxic

GVHD 10.

Mortari,

F.,

and

suppressor proliferation.

cell

Weigensberg, Suppression radiation

12.

phocyte reaction. Hertel-Wulff, B.,

13.

derived from Hertel-WuIff,

nor active TGF-f3 was produced by these cells. It is, thus, likely that cytokine activation induced 11F-j3 synthesis/secretion rather than regulating enzymes involved in its conversion from latency. Finally, the bone marrow NS cells were shown to inhibit the growth of A549 human lung carcinoma cells, probably through the known effects of TGF-f3 [31]. Although the immunosuppressive activity of these NS cells may indirectly promote the growth of some types of tumor cells, these cells

14.

may directly inhibit the growth ofother tumor cells, and thus participate in immune surveillance. Recently, Sugiura et al. [32] likewise found that bone marrow NS cells, through the production of an uncharacterized soluble factor, inhibited the proliferation of the tumor cell lines WEHI-164, EL-4, P815, and X5563. As IL-3 and GM-CSF activate NS cells (14), the induction of these cytokines-whether to meet increased hemopoietic demands or as a result of immune stimulation apparently plays a regulatory role in TGF-j3 production, and consequently could modulate the growth of at lease some types of tumor cells.

Moore,

Theus,

suppression

Bone

marrow

genitor

cells

j

S.A.,

suppressor

the

mmmunol.

15,

and

J.B.,

cells

M.,

18.

and

and

Lawrence,

Shoyab,

G.J.

5.

6.

7.

8.

Maier,

T,

M.,

Marquardt,

(1986)

Oncostatin lymphoma

Pircher,

19.

Kehrl,

J.H.,

Mon,

R.,

Wakefield,

M.,

transforming

role 20. 21.

R.,

growth

in the

Roberts,

Adv.

22.

and

Claman,

H.N.

(1985)

Graft-vs-host

23.

Cancer

(1992)

activity in 398408.

L.S.F.

(1992)

Lectin

lymphoid

H.,

Hanson,

separation

of

Eur.

irradiation.

M: a growth cells. Proc.

MB.,

Lioubin,

regulator

Nail.

produced

Acad.

Sci.

USA

P. (1985)

fi

factor

by

Sporn,

51,

A.B.,

and

ofa

high

its

163,

Transforming

Alvarez-

Production

and

Exp. Med.

j

(1988)

1037-1050. factor-fl.

107-145. Stedronska-Clark, in pregnant

Ix.

Soluble

allopregnant

mouse

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