Release of interleukin-1β by dermatan sulfate suppresses hepatocyte growth

Comp. Biochem. Physiol. Vol. 113C, No. 1, pp. 31-36, Copyright 0 1996 Elsevier Science Inc.

1996

ISSN 0742-8413/96/$15.00 SSDI 0742-8413(95)02047-O

ELSEVlER

Release of Interleukin-lp by Dermatan Suppresses Hepatocyte Growth

Sulfate

lsami Tsuboi, ’ Masashi Suzuki, 2 and Youji Mitsui2 DIVISION, BML GENERAL LABORATORY,MATOBA 1361-1, KAWAGOESHI,SAITAMA 350-l 1, JAPAN; AND ‘NATIONAL INSTITUTEOF BIOSCIENCEAND HUMAN TECHNOLOGY, AGENCY OF INDUSTRIALSCIENCESAND TECHNOLOGY, HIGASHI 1-1, TSUKUBA SCIENCECITY, IBARAKI 305, JAPAN

~REAGENT

Among

glycosaminoglycans,

in adult rat hepatocytes

in primary culture

ABSTRACT: also inhibited

DNA

synthesis,

synthesis

in hepatocytes.

revealed

that interleukin-1

hepatocytes

of DNA

(IL-l)

synthesis,

was required.

was detected.

These

WORDS.

sulfate,

and epidermal

4-sulfate

was released

into the medium.

more than

Similarly,

chain reaction,

10 hr of exposure

IL-lp

or heparin culture

mRNA

Interleukin

COMP

had no effect

on DNA

medium treated with DS

was detected

in DS-treated

hepatocytes.

For a marked

to DS before cultured

hepatocytes

started

BIOCHEMPHYSIOL113C, 31-36,

lp, glycosaminoglycan,

synthesis

Hyaluronate

but not in untreated

more than 10 hr was required after the addition

in hepatocytes.

of DNA

growth factor.

dermatan sulfate,

DNA

of DS before IL-1 /3 mRNA

findings suggest that DS in the medium induced the production

reduced DNA synthesis KEY

chondroitin-6

with insulin

Analysis of growth regulatory factors in hepatocyte

by reverse transcriptase-polymerase

inhibition synthesis,

whereas

dermatan sulfate (DS) is the strongest inhibitor stimulated

of !L-l/? which,

in turn,

1996

RT-PCR,

hepatocytes,

primary culture,

time course

INTRODUCTION Glycosaminoglycan (GAG) and proteoglycan play important (6) and differentiroles in cell proliferation (5)) inflammation ation (4). It was reported that GAGS induced interleukin-1 which is one of the mediators of inflammation (IL-l), (20,3)and

heparin

reportedly

stimulated

IL-1 production

from

human monocytes (20). IL-1 is produced by various types of cells, such as lymphocytes (9), astrocytes (2), endothelial cells (10) and hepatocytes (19) although the biological roles that lymphocytes play in each of these cell types are not yet known. Two forms of IL- 1, IL- 1 (Y and IL- l/3, have been identified by cDNA cloning (8). IL-1 (Y and IL- l/3 have various biological activities including stimulatory and inhibitor effects

DNA synthesis in adult rat hepatocytes and its relation release of IL-l@ into the culture medium.

MATERIALS

AND

to the

METHODS

Materials Hyaluronate (umbilical cord grade I), heparin, RNAsin, dermal growth factor (EGF) and insulin were obtained

epifrom

Sigma Chemical Co. (St Louis, MO). Chondroitin-6 sulfate, Chondrotin-4 sulfate, Dermatan Sulfate (DS) and phytohemagglutinin-p (PHA-P) were purchased from Seikagaku Kogyo Inc. (Tokyo, Japan). ES-Limulus obtained from Wako Pure Chemical

test and endotoxin were Industries, Ltd. (Osaka,

on cell growth. Although the addition of IL-l@ into culture medium has been reported to inhibit DNA synthesis in hepatocytes ( 1 l), IL- 1 cx has no effect on DNA synthesis in hepato-

Japan). Human recombinant interleukin1 p was purchased from Cosmo Bio (Tokyo, Japan). Female wistar adult rats and

cytes (7). Therefore,

C3H/HeJ mice were obtained from Clea Japan Japan). [3H]Thymidine and a cell proliferation

we investigated

the effects

of various

GAGS

on

chased Address reprint requests to: Y. Mitsui, Nat’1 Institute Bioscience & Human Technology, Agency of Industrial Sciences &aTechnology, Higashi 1-1, Tsukuba Science City, lbaraki 305, Japan. Abbreviations -IL, interleukin; GADPH, glyceraldehyde-3 phosphate dehydrogenase; GAG, glycosaminoglycan; DS, dermatan sulfate; rIL, recombinant interleukin; RT-PCR, reverse transcriptase-polymerase chain reaction; M-MLV, Maloney murine leukemia virus. Received 15 June 1995; revised 17 October 1995; accepted 20 October 1995.

from

Amersham

International,

Inc. (Tokyo, kit was pur-

(Buckinghamshire,

U. K.). Vent polymerase and Moloney murine leukemia virus (M-MLV) reverse transcriptase were obtained from New England Biolabs (Beverly, MA). William’s medium E and RPMI1640 medium were obtained from Dainippon Pharmaceutical (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Kantokagaku (Tokyo, Japan). Ninety-six-well microplates and 24-well plates coated with type I collagen were obtained from Sumitomo Bakelite (Tokyo, Japan).

32

Tsuboi et al.

Assay of DNA Synthesis and the LubeZing Index of Hepatocytes

of RNAsin

in a tube. For PCR,

the sample was incubated at

Hepatocytes were isolated from the liver of adult rats (7-9 week old) by in situ perfusion with collagenase, according to the method of Seglen (17). Next, 8 x 106 cells were plated

68°C for 5 min (denaturation), then cooled at 37°C for 60 min (annealing), and then heated at 95°C for 3 min (extension). The cDNA products were stored at - 80°C until assay by PCR.

into each well of a 24-well plate coated with type I collagen. The cells were cultured in 0.5 ml of William’s medium E

Oligonucleotide Primer

(WE) supplemented with 10e7 M insulin and 10 ng/ml EGF under 5% CO, in air at 37°C for 5 hr. The culture medium was then replaced with WE supplemented with 10e7M insulin and 10 ng/ml EGF containing each kind of GAG. [3H]Thymidine (9.25 kBq/ml, 6.18 GBq/mmol) was added for 30 hr after plating. After incubation for another 24-hr, the cells were washed once with cold phosphate-buffered saline [PBS( --)I and lysed with 1 N NaOH at 37°C for 30 min. The cell lysate was trapped on filter paper and washed with distilled water using a cell harvester (LABO-MASH Labo Science, Tokyo). The levels of incorporated radioactivity were measured with a liquid scintillation counter (Beckman Model LS1801). For determination of the labeling index, the hepatocytes were incubated with bromodeoxy uridine (BrdU) for 24 hr and later growth factors and GAGS were added. The cells were washed twice with cold PBS( - ), fixed in Camoy’s solution for 20 min and stained using a cell proliferation kit. The labeling index is expressed as the percentage of labeled nuclei. Assay of IL-1 Activity in Hepatocyte

plating of hepatocytes, the culture medium was replaced with fresh WE supplemented with GAGS. After 48 hr of incubation, the culture medium was collected. Then, 1 x lo6 cells of C3IUHeJ mouse thymocytes in 0.2 ml of RPMI-1640 medium and 5% FBS containing 2 pg/ml PHA, pyruvate, 4 mM glutamine, and 5 x lo-’

RNA Isolation

1 mM sodium

M 2-mercap-

distributed into each well of a 96-well mi20 ~1 of the hepatocyte culture medium was well. After 65 hr incubation, the cells were &i of [3H]Thymidine for 7 hr. Total radioac-

tive thymidine incorporation scintillation counter.

and cDNA

to match

conserved

regions of the

GG-3’

and 5’-GATGTGAAGTAGTTCTT

AG-3’

IL-l@: 5’

-GACCTGTTCTTTGAGGCTGA-3’ and 5’-GATGTGAA GTAGTTCTT AG-3’ GADPH: 5’-CTCAACTACATGGT CTACATG-3’ and 5’-GCTGTAGCCATATTCAT TG TC-3 ’

Enzyme Amplification Total RNA (lpg)

Methods

(10 ~1 of RT reaction)

was dissolved in 10

~1 Vent buffer containing, 1 pg of each sense and reverse primers, 0.2 ~1 Vent polymerase, and 1.25 mM dNTP. The reaction cycles were as follows: 94°C for 1 min, 55°C for 2 min, 72°C for 3 min for 50 cycles. The products of the amplification were analyzed on a 1.5% agarose gel and stained with ethidium bromide.

Culture Medium

IL-1 was measured by the standard thymocyte costimulator assay, as described by Hiro et al. (3). Briefly, at 5 hr after

toethanol were croplate. Next, added to each pulsed with 0.5

Primers were designed

known rat interleukin 1 a (IL-la) (14), rat interleukin-l@) (IL- 1p) ( 13)) and glyceraldehyde-3-phosphate dehydrogenase (GADPH) ( 18). IL- 1 a: 5’AAGATTCTGAAGAAGAGAC

was determined

with a liquid

Synthesis

Total RNA was isolated from 0.8 x lo5 cells by the guanidinium-thiocyanate-chloroform extraction method as described by Schmidt et al. (16). First single-strand cDNA was synthesized using M-MLV reverse transcriptase according to the method described by Schmidt et al. (16). One microgram of total RNA was reverse transcribed with 1000 U of cloned M-MLV reverse transcriptase in a volume of 20 ~1 containing 1 x polymerase chain reaction (PCR) buffer [50 mM TrisHCl (pH 8.3) containing 74 mM KC1 and 3 mM MgCl,], 1.25 mM of each dNTP, 50 pmol of random primers, 1 unit

Delayed Addition of DS

on DNA Synthesis in Hepatocytes WE medium containing

growth factors alone or with 100 pg/

ml of DS was replaced to hepatocyte cultures at 5, 20, 25 or 30 hr after plating. [3H]Thymidine was added for 30 hr after plating. After incubation for another 24 hr, total thymidine incorporation was determined using a liquid scintillation counter. Delayed Addition of DS

on RT-PCR Amplification of IL-I/? mRNA WE medium containing growth factors alone or with 100 pg/ ml of DS was replaced at 5, 15, 20 or 25 hr after plating. Total RNA was isolated at the end of the 30-hr incubation period and the levels of IL-lp mRNA and GADPH mRNA were determined by RT-PCR amplification.

RESULTS Effects of (;A@

on DNA Synthesis in Hepatocytes

The effects of GAGS on DNA synthesis in hepatocytes induced by insulin and EGF were investigated (Fig. 1). We found that DS strongly inhibited DNA synthesis in hepatocytes. Hyaluronate also inhibited DNA synthesis, whereas no effects on DNA synthesis hepatocytes were observed following treatment with chondroitin-6 sulfate or -4 sulfate or with

Effect of Dermatan Sulfate on Hepatocyte

33

Growth

1

TABLE 1.Labelingindex of hepatocytes treated with dermatan-sulfate Labeling Index (%) Control Dertnatan-sulfate

20 + 2 (n = 3)

14 + 2 (n = 3)’

(100 /q/ml)

Values are expressed as the mean of 3 determinations 2 SE. ‘Significantly different from control (p < 0.05).

hepatocytes was significantly decreased as compared with untreated hepatocytes (Table 1).

Effects of CjACjs on Hepatoqte IL-1 Production Since human recombinant

IL-lp

is known to strongly inhibit

DNA synthesis in hepatocytes, we next examined the effects of GAGS on IL-1 production in the cultured medium by the CONT

HA

HP

A

DS

C

FIG. 1. Effect of GAGS on DNA synthesisin hepatocytes. Hepatocytes were treatedwith each kind of GAG ( 100 /.&nl). DNA synthesiswas measured as describedin Materialsand Methods. CONT, control; HA, hyahuonate; A, chondroitin-6 sulfate; DS, dermatan sulfate; C, chondoroitin-4 sulfate; HP, heparin. Values are expressed as the mean of 6 determinations + SE; l, l*; significantlydifferent from untreated control ( p < 0.01 and 0.001, respectively). heparin.

Heparan

sulfate had no effect on DNA

synthesis

(data not shown). As shown in Fig. 2, the inhibitory effect of DS on hepatocytes was detectable at 5 pg/ml and was significant at more than 10 /q/ml. The percentage of labeled nuclei of DS-treated

I

thymocyte costimulator assay. As shown in Table 2, DS stimulated hepatocytes significantly ml of IL-l@

to produce

high levels of IL-I, corresponding to about 7 ng/ At 100 pg/ml of DS, IL-1 activity was approxi-

mately 3.5 times the control level (Fig. 3). It is well recognized that endotoxin can stimulate macrophages to produce IL-1 although such an action of endotoxin on hepatocytes has not been reported. It is crucial to know whether the DS-induced increase in IL-1 production in hepatocytes was due to increased levels of endotoxin in hepatocyte culture medium which may be a contaminant in DS preparation. We measured the endotoxin found that the medium containing

level by a limulus test and 100 pg/ml of DS was con-

taminated by 10 ng/ml of endotoxin. However, medium containing 20 ng/ml of endotoxin did not inhibit DNA synthesis or induce IL-1 synthesis at all (data not shown).

RT-PCR Amplification Because IL-la and 11-lfl are indistinguishable by the thymocyte co-stimulator assay, we measured their respective mRNA levels by RT-PCR amplification. RT-PCR amplification from total RNA from the hepatocytes treated with DS using specific IL-l@ primers yielded one major product of the expected size of 591 bp (Fig. 4). TABLE2. Effect of GAGS on IC 1 activity in hepatocyte culture medium Material Added

1

s

10

50

loo

Dermatan-sulfate @g/ml) FIG. 2. Dose-dependent effect of dermatan sulfate on DNA synthesis in hepatocytes. Values are expressed as the mean of 6 determinations 2 SE.

Control HA A DS C HP IL-lj?

(30 &ml)

ICl Activity ( x l@ cpm) 1.38 3.07 1.50 6.84

+ ? 2 +

0.43 0.69’ 0.84 1.63’

1.51

f

1.36

2.21 2 1.12 25.87

2

0.46

Hepatocytes were treated with each kind of GAG (100 p,g/ml). Values are expressed as mean of 5 determinations + SE. * Signi&antIy different from untreated control (p < 0.01)

34

Tsuboi et al.

6 -

0

l-

1

10

.

loo

Dermatan-sulfate( &ml ) FIG. 3. Dose-dependent effect of dermatan sulfate on IL1 activity in the hepatocyte culture medium. Values are expressed as the mean of 6 determinations f SE. We reamplified the eluted PCR product using an internal primer (IL-l/?: 5’-GAGAGCATCCAGCTTCAA-3’ and 5’CTGTTTTAGGGACACCGGAA-3’). The internal reamplification generated a smaller product corresponding to the 396 bp fragment spanned by two primers on the target IL-ID sequence (data not shown). However, RT-PCR amplification from the RNA of untreated hepatocytes using the specific IL-l/3 primers gave no band. On the other hand, using the specific IL-la primers, equal amounts of PCR products were observed from DS-treated and untreated hepatocytes (Fig. 4).

Effect of Delayed Addition of DS to Hepatocyte Culture on DNA Synthesis and Expression of ILIP mRNA DS inhibited DNA synthesis in hepatocytes only when it was added in the Gl phase of the cell cycle. So, we examined the

Time (h) FIG. 5. Effect of delayed addition of dermatan sulfate on DNA synthesis in hepatocytes. Values are expressed as mean of 5 determinations f SE. 0; control, n ; demnatan-sulfate(100 pg/ ml), q: IL-1 p ( 15 ng/ml) Values are expressed as the mean of 6 determinations + SE; *, l*; significantly different from untreated control ( p < 0.05 and 0.01, respectively). effect of the delayed addition of DS to hepatocyte

culture on

DNA synthesis and on the expression of IL-ID mRNA. The maximum rate of DNA synthesis stimulated by insulin and EGF was found to be between 30 to 54 hr after plating in this assay system (data not shown). IL-l/3 added just before DNA synthesis started at 30 hr after plating strongly inhibited DNA synthesis (Fig. 5). Although the addition of DS to hepatocyte culture at 25 hr after plating slightly inhibited DNA synthesis, the addition of DS to hepatocyte culture at 20 hr produced significant inhibition (Fig. 5). However, the inhibition of DNA synthesis by DS treatment at 20 hr after plating was not as pronounced as when DS was added at 5 hr (Fig. 5). Although no IL-lp mRNA was detected by RT-PCR when DS was added to the hepatocyte

1

IL-1B

culture medium at 25 hr after

FIG. 4. RT-DCR amplification of &l/3 mRNA of the hepatocytes treated with dermatan sulfate. Lane l-3; RTpPCR products from nontreated hepatocytes. Lane 4-6; RTePCR products from DS-treated hepatocytes. Lane 1,4; RT-PCR using ILla primers, lane 2,5; RT-PCR using IL-la primers, Lane 3,6; RT. PCR using GADPH primer.

Effect of Dermatan

Sulfate

on Hepatocyte

1

Growth

2

3

35

4

5

6

7

8

9

10

11

12

13

14

15

16

FIG. 6. Effect of delayed addition of dermatan sulfate on RT-PCR amplification of IL-lfi mRNA. In Lane 1 to 4 or 5 to 8, WE medium containing dermatan sulfate and growth factors was replaced at 5, 15,20, or 25 hr after plating. In Lane 9 to 12 or 13 to 16, WE medium containing growth factors was replaced at 5, 15, 20 or 25 hr after plating. In Lane 1 to 4 and 9 to 12, RT-PCR amplification product of IL-l& In Lane 5 to 8 and 13 to 16, RT-PCR amplification product of GADPH. Total RNA was isolated at the end of the 300hr incubation period and the levels of IL-l/3 and GADPH mRNA were determined by RT-PCR amplification.

plating (lane 4), it was detected at 20 hr after plating (lane 3). Hence, more than 10 hr of exposure to DS, before DNA synthesis started at 30 hr after plating, was required to induce IL-l/3 mRNA expression in hepatocytes.

Gl phase of the cell cycle (11). The inhibitory effect of DS may be observed when IL-lp is induced in the Gl phase. Although the content of DS observed in the regenerating liver changes over time (I), their relationship to cell proliferation has not been clarified. Hepatocytes synthesize heparan sulfate as the major component (12). Therefore, DS secreted

DISCUSSION

We found that, among GAGS, DS was the most potent inhibitor of DNA synthesis of adult rat hepatocytes in primary culture stimulated with insulin and EGF. We thought that this inhibitory action fo DS might be mediated by stimulating the production of some factor which inhibits hepatocyte growth. The results of thymocyte costimulator assay adapted in the present study suggest that the hepatocyte culture medium in the presence of DS may have contained IL- la, II-l/3 or IL-6. Among these, IL-lp

and IL-6 are known inhibitors

synthesis in hepatocytes, specific primers for IL-lp

of DNA

whereas IL-la is not ( 11,7). Using or IL-6, RT-PCR of total RNA from

the cultured hepatocytes in the presence of DS revealed the presence of IL-l/3 but not IL-6 mRNA (data not shown). These results suggest that inhibition of DNA synthesis induced by DS might be mediated by the induction of IL-l/3 in hepatocytes. When DS or IL- l/3 were added to the hepatocyte culture medium at 5 hr after plating, the inhibitory effect on DNA synthesis by DS was equipotent to about 6 ng/ml of IL-lp (Fig. 5). This value was very similar to that estimated by the thymocyte co-stimulator assay (Table 2). It also can not be ruled out that DS may have act as an IL-l/3 inducer of slightly contaminated Kupffer cells or sinusoidal endothelial cells in the hepatocyte preparation, since these cells are known to produce IL-l/3 (15). Even if such contamination did occur, DS would still be a important factor in the regulation of liver growth. The inhibitory effect of DS on DNA synthesis seemed to require more than 10 hr of exposure to DS before the start of DNA synthesis (Fig. 5), and the time course of IL-lfl mRNA induction by DS showed a similar profile (Fig. 6). IL-l@ inhibits DNA synthesis in hepatocytes only when it is added in the

from neighboring cells in the contact with the hepatocytes may act as a signal for hepatocyte growth. Our sincere gratitude is extended to Dr. Jennette A. M. M&r, Imamura, Mr. T. Tanahashi and T. Ito.

Dr. T.

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