Differences

in arachidonic

myelomonocytic Center

for

S. Becker,S

Environmental

Chapel

Hill,-

tCell

Chapel

Hill,

North

H. S. Koren,t

Medicine

and

and

Molecular

Biology

Lung

and

Biology

Section,

US.

Words: arachidonic #{149}dfferentiation

acid

eicosanoids

HL6O

U937

INTRODUCTION Permanent myeloid leukemic and histiocytic lymphoma cell lines are being used as models for normal phagocyte biology I 18]. The ease of culture of homogeneous cell lines such as HL6O, U937, and ML3 has facilitated studies of monocytic and granulocytic cell development [11, 14], as well as studies of phagocyte interactions with other cell types, such as adherence to endothelial and ML3 cell lines

have

cells [12]. The been isolated

promyelocytic from human

eral blood leukocytes [8, 15] and the U937 isolated from a histiocytic lymphoma [34] a suspension culture [22]. The phenotypic

118

29].

Arachidonic

Journal

acid

of Leukocyte

‘Department

of Medicine,

Environmental

Protection

University Agency;

of North

and

Carolina,

SABB..Environmental,

is released

Biology

from

cell line has and converted expression,

membrane

Volume

5!,

and

to func-

research

Effects

Research

approved

for

sarily

reflect

names

or

for

!992

form

can

described

be

in

reesterified

publication. the

article

U.S. Approval

views

and

commercial

back

has

been

into

lipid

reviewed

Environmental does

policies

products

pools.

not

of the

signify

Agency

constitute

by

Protection that nor

the

does

endorsement

the

contents

and neces-

mention

or

Health

Agency

of trade

recommendation

use. AA,

arachidonic

hydroxyeicosatetraenoic acid;

HLA,

acid; human

chromatography; LTB4,

leukotriene

saline;

PG,

prostaglandin;

medium;

TxB2,

Reprint Lung

antigen; NS, PGE2,

thromboxane

requests:

M. CB# 4,

1991;

FCS, HPLC,

IgG, not

fetal

calf

serum;

HETE,

l2-hydroxy-5,8,i0-heptadecatrienoic

interferon--y; B4;

Biology, June

acid; H HT,

leukocyte IFN--y,

triene;

and

this

Laboratory,

Abbreviations:

Received

lipids

free

concentrations [26] in HL6O cells and alterations in adenosine 3,5-cyclic monophosphate cellular concentrations [17] and interleukin 1 activity [21] in U937 cells. Alternatively, the inhibition of arachidonate metabolism by 5-lipoxygenase inhibitors (cirsilol and AA861) can inhibit HL6O cell proliferation [35]. Thus the eicosanoids synthesized and released by these cell lines can have profound effects on their cellular functions. In addition, the release of arachidonate metabolites by these cell lines may regulate immune and inflammatory functions of other co-cultured cell types; for example, human macrophage-derived PGE2 can induce inhibition of lymphocyte proliferation and interleukin 1 production [25]. Because arachidonate metabolites have been shown to be important lipid mediators of the cellular biology of many cultured cell types, the profile of metabolites produced by cultured histiocytic and promyelocytic cells may be critical in modulating their own functions as well as the biology of other co-cultured cell types. Therefore, we compared the arachidonic acid metabolite profile of three human cell lines (HL6O, ML3, U937) commonly used as models of phagocyte development. The cell lines were studied after induction of differentiation with interferon-’y (INF-y). IFN-y has been shown to induce maturation changes in HL6O, U937, and ML3 cells including an increase in Fe receptors [19] and human leukocyte antigen HLA-DR receptors [1].

The

been

February

the

Alternatively, free arachidonic acid can be converted by either cyclooxygenase or lipoxygenases into potent autacoids prostaglandins (PGs), leukotrienes (LTs), and hydroxyeicosatetraenoic acid (HETEs) [10, 321. Leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) have been shown to affect important histiocytic and promyelocytic cell biochemical pathways and functions, including intracellular inositol trisphosphate formation [2] and increased intracellular calcium

HL6O periph-

tions, and biochemical activities of these cell lines (including surface antigens [11], lysozyme secretion [4], cytokine production [31], phagocytosis [9], generation of toxic oxygen species [19], and signal transduction pathways [36]) have been characterized in response to stimulation by a variety of agents after induction of differentiation into monocytic lineage. Monocytes and macrophages can mediate immune processes and inflammation by the production and release of soluble mediators, including arachidonic acid metabolites [7, 28,

M. Friedman*,l

and

Carolina

Abstract: The production of arachidonic acid metabolites by the HL6O, ML3, and U937 human phagocyte cell lines was determined after incubation with interferon-’y (IFN-y, 500 U/ml) or vehicle for 4 days. Cells were prelabeled with tritiated arachidonic acid, [3H]AA, for 4 h, and media supernatants were analyzed by high-performance liquid chromatography. None of the cell lines produced [3H]AA metabolites in large amounts during an unstimulated, basal release period (30 or 60 mm). In response to 10 tM calcium ionophore A23187 incubation (30 mm), undifferentiated and IFN-y-differentiated HL6O cells formed both cyclooxygenase products (thromboxane and prostaglandins) and lipoxygenase products (leukotrienes and hydroxyeicosatetraenoic acids). In contrast to the HL6O cells, IFN-y-differentiated U937 cells formed primarily cyclooxygenase products and undifferentiated and IFN-y-differentiated ML3 cells did not form any [3H]AA metabolites in response to A23187. These results indicate the need to be careful in selecting a cell line for use in a phagocyte assay system when cyclooxygenase and/or lipoxygenase products could influence the assay results.J. Leukoc. Biol. 51: 118-123; 1992. Key ML3

by human

cell lines

M. C. Madden,* *

acid metabolism

high-performance

immunoglobulin

significant;

PBS,

prostaglandin

E2;

liquid G;

LT,

leuko-

phosphate-buffered SFM,

serum-free

B2. C.

Madden,

7310,

UNC,

accepted

Center

for

Chapel

Hill,

June

24,

1991.

Environmental NC

27599.

Medicine

MATERIALS Tritiated cis form),

AND

METHODS

arachidonic thromboxane

acid

Fia (6-keto-[3H]PGF1a), [3H]LTE4, [3H]-15-HETE, were purchased from Other eicosanoids pliers: [14C]PGF2 and [3H]PGE2

([3H]AA; 60-100 Ci/mmol; B2 ([3HJTxB2), 6-keto-prostaglandin

>98%

[3H]PGD2, [3H]L’1C4, [3H]LTD4, [3H]-12-HETE, and [3H]-5-HETE New England Nuclear (Boston, MA).

were obtained from Amersham and [3H]LTB4

from the following sup(Arlington Heights, IL) from Advanced Magnetics

(Cambridge, MA). 12-Hydroxy-5,8,10-heptadecatrienoic ([3H]HHT) was prepared by incubation of platelet somes with [3H]AA as previously described [13]. HL6O and U937 cells (purchased from American

acid microType

Culture Collection, Rockville, MD) and ML3 cells (generously donated by Dr. Stephen Haskill, Lineberger Cancer Center, University of North Carolina, Chapel Hill) were maintained at 37#{176}Cin a 5% C02, 95% room air environment with RPMI medium UR Scientific, Woodland, CA) supplemented with 10% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT) and gentamicin (40 sg/ml; Valley Biologicals, State College, PA) and cultured in 75-cm2 flasks. Cells were incubated with or without 500 U/rn! INF-’y (Biogen, Cambridge, MA) for 4 days. Cell viability was assessed by trypan blue dye exclusion. The number of Fe receptors was determined using rosette formation with sheep erythro-

1 ml for determined

30

mm at 4#{176} or 37#{176}C. Rosette by standard light microscopy.

formation Expres-

sion of cell surface receptors (CR1, CR3, CR4, Leu-M3, HLA-DR) were monitored by flow cytometry as previously described [5]. Cells were transferred into a 50-ml polypropylene tube, centrifuged (500 g, 10 mm, 4#{176}C),and washed with phosphatebuffered saline (PBS). The cells were dispersed by agitation, and cultures were incubated for 4 h in 10 ml serum-free RPMI medium (SFM) containing 50 mM HEPES and 5 Ci [3HJAA while turning (0.5 rpm) in a roller bottle incubator (Bellco, Vineland, NJ) maintained at 37#{176}C.Following incorporation of [3H]AA, cells were centrifuged, the supernatant was decanted, and the cells were washed with PBS (10 ml) to remove unincorporated label. Cultures were then incubated with 10 ml SFM for 30 or 60 mm (termed the basal release period in this paper), after which the media were removed and centrifuged and aliquots of supernatants were counted for tritium release. The remaining media supernatants were processed for analysis by high-performance liquid chromatography (HPLC) Following the basal release resuspended

for eicosanoid removal of media

period, in 10 ml

cells SFM

formation. supernatants

during

were dispersed by agitation containing 10 sM calcium

the and iono-

phore A23187 (dissolved in dimethyl sulfoxide at a final concentration of 0.13% v/v; Calbiochem Boehring, La Jolla, CA) for 30 mm (referred to in this paper as the stimulated release period). Media were removed and centrifuged and an aliquot of supernatant was counted for tritium; the remainder was saved for HPLC analysis. One molar NaOH was added to cell cultures, an aliquot was removed 20 h later, and radioactivity associated with the cells was determined. Cellular tritium release, expressed as a percentage, was determined as the amount of radioactivity in the medium divided by the amount in the medium plus the amount associated with the cells. Media were analyzed by HPLC using a modification of a previously

published

HPLC

procedure

that

recovers

4#{176}Cunder N2 until HPLC analysis. Just prior to analysis, samples were brought to room temperature, mixed, and centrifuged at 2000 g for 6 mm. An aliquot of the supernatant was then injected into an HPLC system, which has been described previously [24]. Separation of [3H]AA metabolites was accomplished with a reverse-phase method using a methanol gradient from 58 to 100% for 100 mm at a flow rate of 1.1 ml/min through a 5-tm ODS Ultrasphere column (Beckman, Fullerton, CA). Metabolite peaks were identified as eicosanoids based on the retention time compared to externally applied authentic standards. [3H]PGE2 and [3H]PGD2 coelute in this method and are reported here as the combined products [3H]PGE2/D2. Retention time variability of arachidonate metabolite standards was ± 0.3 mm (n = 12). Data are reported as mean ± SEM (n). Statistical analysis was done using t-tests for unpaired variates with P < .05 considered significant [33].

RESULTS

cytes coated with immunoglobulin G (IgG) (OrganonTeknika-Cappel, Malvern, PA). Cells (106) were incubated with 0.2% red blood cells (Becton-Dickinson, Cockeysville, MD) in was then

of tested eicosanoid standards [20]. Media supernatants were added to ethanol to a final alcohol concentration of 80% and stored at - 70#{176}C.Samples were thawed, proteins precipitated by centrifugation (500 g, 20 mm, 10#{176}C),and supernatants dried by rotary evaporation (30#{176}C). The samples were then redissolved in 1.0 ml 30% methanol and stored at

>80%

No statistical differences (P = NS) in the viability as assessed by trypan blue dye exclusion was observed in HL6O and U937 cell lines treated with IFN-’y or incubated with vehicle (medium) for 4 days. The viabilities of IFN-y-treated HL6O and U937 cells were 84 ± 5% (n = 7) and 89 ± 5% (n = 9), respectively, and ofuntreated HL6O and U937 cells were 89 ± 3% (n = 4) and 91 ± 4% (n = 7), respectively. There was a slight but significant (P = .016) decrease in the viability of ML3 cells incubated with IFN-’y (79 ± 2%, n = 7) compared to control cultures (88 ± 3%, n = 4). Seventy seven percent of the IFN--y-treated HL6O cells and 60% of the ML3 cells formed rosettes with sheep red blood cells, whereas only 8 and 9% ofthe undifferentiated (vehicletreated) HL6O and HL3 cells, respectively, formed rosettes. We have previously shown increased Fe receptor-mediated binding by IFN--y-treated U937 cells [22]. Treatment of HL6O cells with IFN-y for 4 days induced an increase in the number of other differentiation markers, including a 1.0- to 4.1-fold increase in CR1, CR3, CR4, and Leu-M3 and an 11.7-fold increase in the number of HLA-DR receptors cornpared to control cell cultures (Table 1). ML3 cells incubated with IFN-’y had 1.0- to 4.5-fold increases in CR1, CR3, CR4, and HLA-DR receptors compared to control cultures (Table 1). A slight decrease (40%) in Leu-M3 receptors was observed in IFN-’y-treated ML3 cells. These data therefore suggest that the incubation with IFN-’y (500 U/mI) for 4 days induced differentiation of the HL6O and ML3 cells. There was no change in the number of differentiation markers (CR1, CR3, CR4, Leu-M3, HLA-DR) in U937 cells incubated with IFN-y compared to control cultures (data not shown). Similar experiments using other clones of U937 cells (obtained from the Tissue Culture Facility, Lineberger Cancer Center, University of North Carolina, Chapel Hill) and/or other sources of human recombinant IFN-y (Boehringer Mannheim, Indianapolis, IN) also failed to induce alterations in the presence of these differentiation markers. Following incubation with INF-’y or vehicle alone for 4 days, the cells ence of [3H]AA Journal

of Leukocyte

were washed for 4 h. With Biology

and then cultured HL6O cells, there Volume

51,

February

in the preswas similar 1992

119

TABLE

1.

Expression

of and

Differentiation ML3

Markers

Fluorescen Cell line

Antigen

HL6O

ML3

‘Cells IFN--y

were (500

surface fluorescence

Control

Fold change

IFN-y

6.3

12.9

2.0

8.6

34.9

4.1

CR4

18.5

18.7

1.0

Leu-M3

1.9

4.2

2.2

HLA-DR CR1 CR3 CR4

2.9 1.1 3.8 1.8

33.9 4.0 17.6 3.8

11.7 3.6 4.6 2.1

Leu-M3

2.2

1.4

HLA-DR

4.9

22.6

Cells and

incorporation undifferentiated 64 ± 11% (n (n = 5) when

e intensity

CR3

U/ml). was

the incorporated basal release

HL6O

CR1

incubated

markes

on

CeIlSa

for

3 days

were

then

analyzed

by

corrected

for

in

in

the

stained

with

flow

cytometry.

each

sample.

of =

presence

the [3H]AA cell cultures. 8) when treated incubated with

or

-0.6

4.6

absence

fluorescent

(control)

antibodies

Nonspecific

of to cell

background

into

both differentiated and HL6O cells incorporated with IFN-y and 62 ± 11% vehicle (P = NS). Following

the incorporation of [3H]AA, cells were incubated free media. Differentiated and undifferentiated released 13-18% (30 and 60 mm, respectively;

in serumHL6O cells P = NS) of

period.

radioactivity Analysis

into the media during the of the media from IFN-’y-

treated cells by HPLC showed that most of the released radioactivity was associated with either free [3H]AA or the solvent front, which includes released phospholipids (Fig. 1A). [3H]PGE2/D2 was found in small amounts in media supernatants of HL6O cells. Similar profiles of arachidonate metabolism by differentiated HL6O cells were observed for both 30 and 60 mm of basal release (data not shown). After the basal release period, HL6O cell cultures were washed and then incubated with 10 jsM calcium ionophore A23187 for 30 mm and the media processed for HPLC analysis. HL6O cells, differentiated with IFN-’y, released 20 ± 3% (n = 8) of the cell-associated tritium, and undifferentiated HL6O cells had a similar release of 19 ± 3% (n = 6; P = NS). Cultures of IFN-13-differentiated HL6O cells had the ability to synthesize and release several arachidonate metabolites derived from cyclooxygenase and lipoxygenases activities in response to A23187. Chromatograms showed the presence of 6-keto-[3H]PGF10, [3H]TxB2, [3H]PGE2/D2, [SH]HHT, [3H]L’1C4, [3H]LTB4, [3H]-15-HETE, and [3HJ-5-HETE in media supernatants (Fig. 1B). An unidentified product eluted with a retention time of approximately 54 mm, which is similar to the retention time of LTD4. Alternatively, based on its retention time, this product may be 5,12-diHETE, which has been reported to be formed by HL6O cells [31. Undifferentiated HL6O cells, when incubated with 10 tM A23187 ionophore, had a similar specFRONTIPHOSPHOUPIDS

B.

A.

0

6

,

#{243}Q.

w

2

C.. -J

444

4

44,4

4

x 0.

6

0. 0

2

I0

20

a’

6.s

C-

0.

C-

4

44

4

4 41

4

C.)

0

x 0.

Fig. 1. Reverse-phase HPLC analysis of [3HJAA HL6O cells during the basal release period (60 ionophore A23187 incubation. (A) Basal release (60 mm). (B) Stimulated release of IFN--y-incubated release of cells not incubated with IFN-y. 3.3 x are typical of vi - 6-7 experiments/per group.

120

Madden

ci aL

Arachidonate

metabolism

in human

cell

lines

metabolites

mm) of

produced

and

during

IFN-7-incubated

cells. 1O cells

were

by

calcium cells

(C) Stimulated used.

Results

trum

of products

compared

to cultures

of IFN-y-differentiated

cells (Fig. 1C), and the amounts of 6-keto-[3H]PGF1a, [3H]TxB2, [3H]PGE2/D2, [3H]HHT, [3H]LTC4, [3H]LTB4, and [3H]-15-HETE detected were similar in the two cultures. IFN-y-treated ML3 cells incorporated 57 ± 10% (n = 7) ofthe [3H]AA and 54 ± 17% (n = 4) without IFNC.y incubation (P = NS). IFN-y-differentiated ML3 cells released significantly less tritium (16 ± 4%, n = 4) in the 30-mm basal release period compared to undifferentiated ML3 cells (35 ± 4%, n entiated ML3

=

3; cells

P = 0.006). Differentiated released 25 and 23%,

and respectively,

A. C-O

4, 8

C.)

undifferof the

product derived from [3H]AA, acids, which are observed to be cell types via elongase activity

x 0.

\

2

0

20

30 RETENTION

40 TIME

50 60 (MINUTES)

70

80

90

B;

r 0a:

12

[10, 30]. Like the IFNC.ydifferentiated cultures, undifferentiated ML3 cells produced free [3H]AA, phospholipids, and the unidentified peak with a retention time of 89 mm as the major tritiated products (data not shown). IFN-’y-treated U937 cells incorporated 55 ± 7% (n = 12) ofthe [3H]AA during the labeling period, whereas untreated cultures incorporated 49 ± 4% (n = 7) of the [3H}AA. Differentiated and undifferentiated U937 cells released 15 ± 1% (n = 7) of the cell-associated tritium during the 30-mm basal release period (P = NS). The radioactivity released by IFN-y-treated cells was mainly associated with free AA and phospholipids, but a small amount of some cyclooxygenase products ([3H]PGE2/D2, [3H]TxB2, [3H]HHT) was detectable (Fig. 3A). When incubated with A23187, IFN-’y-differentiated U937 cells released 23 ± 3% (n = 12) of the incorporated tritium and cultures produced primarily cyclooxygenase products (6-keto-[3H]PGF10, [3H]TxB2, [3H]PGE2/D2, [3H]PGF2a, [3H]HHT) and [3H]-15-HETE as determined by HPLC (Fig. 3B). Undifferentiated U937 cells released primarily free AA, phospholipids, and a small amount of [3H]-15-HETE but, in contrast to IFN-’y-treated U937 cells, nondetectable amounts of cyclooxygenase prod-

-.

‘5

I0

cell-associated tritium in response to A23187 (P = NS). The major peaks observed in the chromatograms of the basal and A23187 release period of IFN-y-treated cells corresponded to free [3H]AA and phospholipids associated with the solvent front (Fig. 2A and B). ML3 cell cultures differentiated with IFN-’y released nondetectable amounts of [3H]AA metabolites with the exception of [3H]-15-HETE. The unidentified peak that eluted with a retention time of approximately 89 mm may be a longer-chain such as 22:4 to 26:4 fatty produced by other cultured

AA

I

ZI

z

U,

..JO 0’

‘5

COO.

4

8

4

0. 4

2

I

0

Fig.

2.

of n release

period

cells. (30

2 experiments

I

30 RETENTION

HPLC

ML3

release =

I

20

Reverse-phase

IFN-y-treated

sM)

I

K

50 TIME

analysis

(A)

Basal

mm).

2.8

the

basal

for

I

40

of

E’HIAA

release x

10

cells

release

I

60 (MINUTES)

period were

period

70

metabolites

produced

(60 mm). used. and

(B)

Results

n

=

by

A23187

7 for

(10

are

typical

the

A23l87

period.

ucts (Fig. 3C). Undifferentiated U937 cells released 12 ± 3% (n = 7) and 26 ± 5% (n = 7) of the incorporated tritium during the basal and stimulated release periods, respectively (P = NS compared to the same period for differentiated

Undifferentiated had similar profiles response to A23187, Profiles of arachidonate

cells). Undifferentiated produced nondetectable ucts, but some 15-HETE

myelomonocytic HL6O cells (i.e., formation of both lipoxygenase and cyclooxygenase metabolites) have been reported in human peripheral blood monocytes [29] and human alveolar and peritoneal macrophages [7, 28]. The results re-

U937 cells stimulated with amounts of cyclooxygenase was produced (Fig. 3D).

A23187 prod-

ported results

DISCUSSION We now report that the three human cell lines HL6O, ML3, and U937, after differentiation with INF-y (500 U/ml for 4 days), produced very different profiles or arachidonic acid metabolites in response to the calcium ionophore A23187. IFN-’y-differentiated HL6O cells produced relatively abundant amounts of both lipoxygenase products (LTB4, LTC4, 15-HETE) and cyclooxygenase products (PGE2, HHT, and TxB2) under the culture conditions employed. In contrast, differentiated U937 cells released primarily cyclooxygenase products, and undifferentiated and differentiated ML3 cells did not release detectable amounts of either lipoxygenase or cyclooxygenase

products

in

response

to A23187.

and IFN-y-differentiated HL6O cells of arachidonate metabolites produced in including the production of 15-HETE. metabolism similar to those of these

here for HL6O which showed

cells that

differ from undifferentiated

previously HL6O

published cells pro-

duced nondetectable amounts of 15-HETE in response to exogenously added arachidonic acid [23]. These present results and those of Lundberg et al. [23] suggest that the metabolism of endogenous arachidonate in undifferentiated HL6O cells is different from that of exogenously added, free arachidonate. As previously mentioned, ML3 cells did not produce detectable amounts of tritiated arachidonate metabolites except for a small amount of [3H]-15-HETE. The lack of production of arachidonate metabolites in the ML3 cell line in response to A23187 is in contrast to the production of several eicosanoids by HL6O cells and may be due to the ML3 cells being less

Journal

“mature”

of Leukocyte

than

the

Biology

HL6O

cells

Volume

[15].

5!,

February

1992

121

4-

SOLVENT

B.

FRONT!

PHOSPHOLIPIDS 0

6-

AA -.

“-F.

5.

I

4,4,

C.)

0 *

C.)

0

0.

x

0.

RETENTION

6

TIME

(MINUTES)

D.

(1)

6

C/) 0

0 0.

:1 0

5

0

5

I 0.

I 0. C/)

Cl)

0

0

I 0. I-.

4

I 0.

4

z

z m

0

0

Li.

3

Li.

3

>( 0.

0

0.

2

2

0

I 0

I 10

I 20

:jL

I 30

40

50

5-HETE

HHT

1#{149}

L

I

I

I

I

60

70

80

90

‘

0

I

I

I

10

20

30

Fig. 3.

A23187

Reverse-phase

(10 tM period

HPLC

A23187) release (30 mm) of

release

analysis period untreated

of [3HJAA

metabolites

produced

(30 mm) of IFN-y-treated cells. 5.7 x lO cells

cells. used.

were

by

I 60

I 70

I 80

I 90

cells.

(A)

Basal

release

period

of untreated cells n = 7 experiments

of IFN-y-treated (30 mm). for both

cells

(30

mm).

(B)

(D) Stimualted (10 iM the basal release period

StimuA23187) and the

period.

tively; P = NS) in response to A23187, the data suggest similar rates of release or reesterification of [3H]AA. Therefore, with similar amounts of substrate ([3H]AA) present in undifferentiated and differentiated U937 cells, but decreased

Madden

U937

(C) Basal release period Results are typical of

The results presented here show primarily cyclooxygenase product formation by U937 cells in response to A23187. Similarly IFN-’y-differentiated U937 cells have been reported to produce PGE2 and TxB2, measured by radioimmunoassay, in response to phorbol myristate acetate stimulation [31]. In addition, lack of detectable leukotriene (LTB4, LTC4, LTD4) production by A23187-stimulated cells has been reported [27]. Another report showed that IFN-’ydifferentiated cells (after [3H]AA incubation overnight, cornpared to 4 h in this report) formed lipoxygenase products as the major arachidonate metabolites during the basal release period [6], suggesting that the [3HJAA incorporation period or possibly use of a different U937 clone may be important in determining the eicosanoid profile of U937 cultures. Formation ofcyclooxygenase metabolites by U937 cells occurred only after differentiation with IFN-’y. Because undifferentiated and IFN-’y-differentiated U937 cells released similar amounts of the incorporated tritium (26 and 23%, respec-

122

I 50

RETEN11ON liME (MINUTES)

RETEN11ON liME (MINUTES)

lated release

I 40

ci al.

Arachidonate

metabolism

in human

cell

amounts of cyclooxygenase metabolites produced by undifferentiated cells, the data suggest that there is less cyclooxygenase activity in the undifferentiated cells and that IFN‘y-induced differentiation of U937 cells increases cyclooxygenase activity. As discussed previously, metabolites of arachidonic acid affect numerous cellular functions in vitro and consequently may mediate biological responses being measured in in vitro assays. For instance, LTB4 can increase the binding of phagocytes to endothelial cellsin vitro [16]. The differences in arachidonate products formed by the cell lines in assay systems using human histiocytic and promyelocytic cell types should therefore be carefully considered with regard to their contribution to the assay results. Furthermore, correlation of the arachidonate metabolites produced by these cell lines with other aspects ofcellular functions may provide further insight into the developmental biology of the phagocyte system.

ACKNOWLEDGMENTS Supported

lines

in part

by EPA

Cooperative

Agreement

CR812738.

REFERENCES 1. Amatruda, T.T., Bohman, R., Ranyard, J., and Koeffler, H.P. Pattern of expression of HLA-DR and HLA-DQ antigens and mRNA in myeloid differentiation. Blood 69, 1225, 1987. 2. Andersson, T., Schlegel, W., Monod, A., Krause, K. -H., Stendahl, 0., and Lew, D.P. Leukotriene B4 stimulation of phagocytes results in the formation of inositol 1,4,5-triphosphate. A second messenger for Ca2 mobilization. Biochem. j 240, 333, 1986. 3. Anthes, J.C., Bryant, R.W., Musch, MW., Ng, K., and Siegel, MI. Calcium ionophore and chemotactic peptide stimulation of peptidoleukotriene synthesis in DMSO-differentiated HL6O cells. Inflammation 10, 145, 1986. 4. Bar-Shavit, Z., Teitelbaum, S.L., Reitsma, P., Hall, A., Pegg, L.E., Trial,J., and Kahn, AR. Induction ofmonocyte differentiation and bone resorption by 1,25-dihydroxyvitamin D3. Proc. Nail. Acad. Sci. USA. 80, 5907-591!, 1983. 5. Becker, S., Warren, KM., and Haskill, S. CSF-1-induced monocyte survival and differentiation into macrophages in serum-free cultures. j Immunol. 139, 3703, 1987. 6. Bomalaski, J.S., Freunlich, B., Steiner, S., and Clark, M. The role of fatty acid metabolites in the differentiation of the human monocyte-like line U937. J. Leukoc. Biol. 44, 5!, 1988. 7. Brown, G.P., Monick, MM., and Hunninghake, G.W. Human alveolar macrophage arachidonic acid metabolism. Am. j PhysioL 254, C809, 1988. 8. Collins, Sj., Ruscetti, F.W., Gallagher, R. E., and Gallo, R.C. Normal functional characteristics of cultured human promyelocytic leukemia cells (HL6O) after induction of differentiation by dimethylsulfoxide. j Exp. Med. 149, 969, 1977. 9. Collins, S.J., Ruscetti, F.W., Gallagher, RE., and Gallo, R.C. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc. NaiL Acad. Sci. USA. 75, 2458, 1978. 10. Crawford, MA. Background to essential fatty acids and their prostanoid derivatives. Br Med. BulL 39, 210, 1983. 1!. Dayton, E.T., Perussia, B., and Trinchieri, G. Correlation between differentiation, expression of monocyte-specific antigens, and cytotoxic functions in human promyelocytic cell lines treated with leukocyte-conditioned medium. J. Immunol. 130, 1120, 1983. 12. DiCorleto, P.E., and de la Motte, CA. Characterization of the adhesion ofthe human monocytic cell line U937 to cultured endothelial cells. j Clin. Invesi. 75, 1153, 1985. 13. Eling, T.E., Tamer, B., Ally, A., and Warnock, R. Separation of arachidonic acid metabolites by high-pressure liquid chromatography. Methods Enzymol. 86, 51!, 1982. 14. Fischkoff, S.A., and Condon, ME. Switch in differentiative response to maturation inducers of human promyelocytic leukemia cells by prior exposure to alkaline conditions. Cancer Res. 45, 2065, 1985. 15. Fukuda, M., Koeffler, H.P., and Minowada, J. Membrane differentiation in human myeloid cells: expression of unique profiles of cell surface glycoproteins in myeloid leukemic cell lines blocked at different stages of differentiation and maturation. Proc. Nail. Acad. Sd. USA. 78, 6299-6303, 198!. 16. Gimbrone, MA., Brock, A.F., and Schafer, Al. Leukotriene B4 stimulates polymorphonuclear leukocyte adhesion to cultured vascular endothelial cells. j Clin. Invest. 74, 1552, 1984. 17. Goldring, S.R., Amento, E.P., Roelke, MS., and Krane, SM. The adenosine 3’ :5-cyclic monophosphate response to prostaglandin E2 is altered in U937 cells in association with maturational events induced by activated T lymphocytes and !,25dihydroxyvitamin D3. J. Immunol. 136, 346!, 1986. 18. Harris, P., and Ralph, P. Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell

lines. j Leuk. Biol. 37, 407, 1985. 19. Harris, P.E., Ralph, P., Gabrilove, J., Welte, K., Karmali, R., and Moore, M AS. Distinct differentiation-inducing activities of gamma-interferon and cytokine factors acting on the human promyelocytic leukemia cell line HL-60. Cancer Res. 45, 3090, 1985. 20. Henke, D., Danilowicz, R., and Eling, T.E. Arachidonic acid metabolism by isolated epidermal basal and differentiated keratinocytes from the hairless mouse. Biochim. Biophys. Acia 876, 271, 21.

1986.

Knudsen,

P.J.,

Dinarello,

posttranscriptionally 1

activity

by

CA.,

inhibit increasing

J. Irnmunol.

phosphate.

and

Strom,

monocyte intracellular

137,

3189,

Prostaglandins of interleukin

TB.

expression cyclic

adenosine

mono-

1986.

Larrick, J.W., Fischer, D.G., Anderson, S.J., and Koren, H.S. Characterization of a human macrophage-like cell line stimulated in vitro: a model of macrophage functions. J. Immunol. 125, 6, 1980. 23. Lundberg, U., Serhan, C.N., and Samuelsson, B. Appearance ofan arachidonic acid 15-lipoxygenase pathway upon differentiation of the human promyelocytic cell-line HL-60. FEBS Leit. 185, 14, 1985.

22.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33. 34.

35.

36.

Journal

Madden,

MC.,

Dailey,

L.A.,

Eling,

T.E.,

and

Friedman,

M.

The effect ofozone exposure on rat alveolar macrophage arachidonic acid metabolism. Exp. Lung Res. 17, 47-63, 199!. Monick, M., Glazier, J., and Hunninghake, G.W. Human alveolar macrophages suppress interleukin-! (IL-!) activity via the secretion of prostaglandin E2. Am. Rev. Respir. Dis. 135, 66, 1987. Naccache, PH., Molski, T.F.P., Spinelli, B., Borgeat, P., and Abboud, C.N. Development ofcalcium and secretory responses in the human promyelocytic leukemia cell line HL6O. j CelL Immunol. 119, 241, 1984. Nolfo,

R.,

and

Rankin,

J.A.

U937

and

THP-l

cells

do

not

release LTB4, LTC4, or LTD4 in response to A23l87. Prosiaglandins 39, 157, 1990. Ouwedijk, R.J.T., Zijlstra, F.J., van den Broek, A.M.W., Brouwer, A.J., Wilson, H.P., and Vincent, J.E. Comparison of the production ofeicosanoids by human and rat peritoneal macrophages and rat Kupifer cells. Prostaglandins 35, 437, 1988. Pawlowski, NA., Kaplan, G., Hamill, AL., Cohn, Z.A., and Scott, WA. Arachidonic acid metabolism by human monocytes.J. Exp. Med. 158, 393, 1983. Rosenthal, M.D., and Hill, JR. Human vascular endothelial cells synthesize and release 24- and 26-carbon polyunsaturated fatty acids. Biochim. Biophys. Acia 795, 17!, 1984. Roux-Lombard, P., Cruchaud, A., and Dayer,J.M. Effect of interferon ‘ and 10,25-dihydroxyvitamin D3 on superoxide anion, prostaglandin E2, and mononuclear cell factor production by U937 cells. CelL Immunol. 97, 286, 1986. Serhan, C.N., Wong, P.Y. -K., and Samuelsson, B. Nomenclature of lipoxins and related compounds derived from arachidonic acid and eicosapentaenoic acid. Prostaglandins 34, 20!, 1987. Snedecor, G.W., and Cochran, W.G. Statistical Methods. Ames: Iowa State University Press, 1978. Sundstrom, C. , and Nilsson, K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). mi. j Cancer 17, 565, 1976. Tsukada, T., Nakashima, K.,and Shirakawa, S. Arachidonate 5-lipoxygenase inhibitors show potent antiproliferative effects on human leukemia cell lines. Biochem. Biophys. Res. Commun. 140, 832, 1986. Verghese, M., Uhing, R.J., and Synderman, R. A pertussis/ cholera-sensitive N protein may mediate chemoattraction receptor signal transduction. Biochem. Biophys. Res. Commun. 138, 887, 1986.

of Leukocyte

Biology

Volume

5!,

February

1992

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