Specific activation by concanavalin A of the superoxide anion generation capacity during U937 differentiation

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vo1.151, No. 2 , 1 9 8 8

Pages 802-808

March 15,1988

SPECIFIC ACTIVATION

BY CONCANAVALIN A OF THE SUPEROXIDE ANION

GENERATION CAPACITY DURING U937 DIFFERENTIATION

Jesfls Balsinde and Faustino Mollinedo*

Unidad de Biomembranas Centro de Investigaciones Biol6gicas, C.S.I.C. Vel~zquez 144, 28006 Madrid, Spain Received January 21, 1988

SUMMARY: Phorbol myristate acetate (PMA) induces changes in the human monocyte-macrophage-like cell line U937 which reflect cellular differentiation. PMA prompted the expression of the superoxide anion (02 ) generating capacity in U937 upon appropriate stimulation. A highly specific stimulation by Concanavalin A (Con A) of 0 2 release was observed in PMA-differentiated U937 cells, which exceeded in 10-20 times that obtained with Con A-stimulated monocytes and neutrophils. These results indicate that a highly specific machinery required for Con A stimulation, practically absent in mature monocytes and neutrophils, is synthesized during PMA-induced U937 differentiation. A novel cytochrome b putatively involved in 02 generation was detected in U937 cells. This cytochrome b content was increased during PMA-induced cell differentiation, although no linear correlation was found between capability to produce 02 by macrophage-like U937 cells and their content of cytocbrome b. © 1988 Acid.... Press,

Inc.

The mononuclear fense

against

of this

phagocyte

invading

system

system

pathogens.

are in part

plays The

mediated

an essential

antimicrobial by

the generation

(0 2 ), from which a series of highly reactive named "respiratory system, tion

burst".

has been implicated to be present cell

line

phagocytes.

U937,

which

as a model

for studying that

shown be

induced

is

believed

human monocyte

by ~ - i n t e r f e r o n acetate

To whom correspondence

be

system

of monocytic

maturation

(5) and reported

origin,

to more

(ii),

l~,25-dihydroxyvitamin

(PMA)

cytochrome

histiocytic

of U937 by

(i), a process

b-type

and function mature

anion

for the 02 genera-

an unique

The human

de-

that a NADPH oxidase

is responsible

(6-8).

host

functions

superoxide

species originate

part of the oxidase

to

in the

antitumor of

(2-4) indicates

context,

phagocytes

differentiation

by phorbol myristste

In this

as an integral

in mononuclear

been can

Recent evidence

similar to that found in neutrophils,

in mononuclear

role and

has

lymphoma been

(9,10).

used

It has

monocytes-macrophages D3

(12),

and

(13).

should be addressed.

Abbreviations: Con A, concanavalin A: FMLP, N-formyl-methionyl-leucyl-phenylalanine; 02, superoxide anion; PMA, 4 ~-phorbol, 12-myristate, 13-acetate.

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

802

Vol. 151, No. 2, 1988

In the present capacity agents.

during

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

work, U937

Furthermore,

studied

differentiation

the development

by

PMA,

upon

of the O2-generating

stimulation

with

various

we have examined the content of the above mentioned b-type

cytochrome

in U937

PMA,

particular

with

we have

cells

as they differentiate

reference

to

its

in culture

relationship

with

in the presence the

generation

of

of O 2

by these cells.

MATERIALS AND METHODS Materials. RPMI 1640, fetal calf serum, and L-glutamine were purchased from Flow Lab. (Irvine, U.K.). Antibiotics were from Llorente Lab. (Madrid, Spain). PMA, FMLP, and Con A were from Sigma (St. Louis, MO, U.S.A.). PMA was dissolved in dimethylsulfoxide at a concentration of 2 mg/ml. This stock solution was stored in aliquots at -20QC. Appropriate controls were performed to exclude an effect of dimethylsulfoxide on cells. Horse heart cytochrome c was from Merck (Darmstadt, F.R.G.). Lymphoprep was from Nyegaard Co. (Oslo, Norway). Cells. U937 cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM L-glutamine, i00 units/ml penicillin and 50 ~g/ml gentamicin. Cells were incubated at 37eC in a humidified atmosphere at 5% CO 2 and 95% air. Medium was changed on alternate days until cell harvesting. Cell differentiation was induced by adding PMA to a final concentration of 35 ~g/ml. Human peripheral blood monocytes and neutrophils were obtained from adult donors by Lymphoprep centrifugation, after previous erythrocyte sedimentation with 1.3% (v/v) dextran (14, 15). Remaining erythrocyte contamination of the neutrophil-enriched pellet was eliminated by hypotonic lysis, resulting in a final cell population of more than 98% neutrophils, as assessed by Giemsa-Wright's stain. The mononuclear cells were washed twice with phosphate-buffered saline, and the cell suspension was added to Petri dishes to allow cells to adhere. After incubation at 37~C for 2 h, the Petri dishes were washed 6 times with phosphate-buffered saline. The nonadherent cells were washed away and monocytes were collected with a rubber policeman. Superoxide anion and protein assays. Release of 0 2 was measured by the superoxide dismutase-inhibitable reduction of ferricytochrome c in a discontinuous assay (2), Neutrophils, monocytes and U937 cells were assayed immediately after cell harvesting in a final volumen of 1 ml of a glucose-containing buffer (I0 mM Hepes, 150 mM NaCI, 1.2 mM MgCI2, 1.3 mM CaCI2, 5.5 mM glucose, pH 7.5). 5x10 5 cells were preincubated for 5 min at 37~C with cytochalasin B (5 ~g/ml), and then challenged with the appropriate stimulus for i0 min at 37QC.-At the end of this period, reaction was stopped by placing the tubes on ice. After centrifugation at 400 x g at 4QC, supernatants were collected and read at 550 nm. Controls with superoxide dismutase (i0 ~g/ml) were run in parallel to assure that cytochrome c reduction was due only to the superoxide anion generated during cell stimulation. Protein was measured as described by Bradford (16) with bovine serum albumin as standard. Cytochrome b determination. Cells were homogenized as described by Mollinedo and Schneider (14). The postnuclear supernatant obtained was diluted with TNC buffer (50 mM Tris-HCl, I00 mM NaCI, pH 8.0) and centrifuged at 29000 rpm (i00,000 x g) for 90 min at 4~C in a Beckman L8-70 ultracentrifuge, using a 30-type rotor. The pellets, representing the membranous fractions, were resuspended in approximately 2 ml of TNC buffer, sonicated and solubilized by adding 0.1% sodium deoxycholate (17). Two-milliliter samples were divided in two cuvettes, and reduced versus oxidized difference spectra from 600 to 400 nm were recorded in a Beckman DU-SB spectrophotometer after addition of sodium dithionite and potassium ferricyanide respectively (18). The concentration of

803

Vol. 151, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cytochrome b was calculated from the absorbance maximum at a millimolar extinction coefficient of 21.6 mM -I c m - I (19).

559

nm

by

using

RESULTS U937 35

cells

ng/ml

for

adherent of

were

either

different

grown

periods

(more than 80%),

macrophages,

(results

not

phase

of

exposed

Cell

the

undifferentiated

different

periods

triggering

U937

of time,

with different

the cells stimuli

A,

was

a dramatic

as compared

increase

cells

when

for 40 h, monocytes

different

stimuli:

a significant

about

incubated

PMA at

U937

became

properties

surface

blue

antigens

exclusion,

was

cells.

with

PMA

the ability

I). The maximum

at

to produce

for

O 2 upon

capacity of O2 genera-

with PMA. capacity

FMLP or PNA were

35 ng/ml

Interestingly,

triggered

by Con

used as stimulants,

in

U937.

to elicit being

Trypan

in the 0 ~ generation

Fig. 2 shows the O 2 generation ed U937

by

acquired

(Fig.

PMA,

macrophage

assessed

were

to

with

the phenotypic

after 40 h incubation

to that obtained

PMA-differentiated

of

or treated

and differentiated

cells

tion by U937 cells was achieved there

When

expression

viability,

higher than 90% in undifferentiated When

time.

formed clumps and expressed

including

shown).

to exponential

capacity of undifferentiated and neutrophils

upon

PMA, FMLP and Con A. Undifferentiated

2-fold

production

higher

than

of O2. Monocytes that

of

U937, PMA-treat-

stimulation

with

U937 cells were unable

also gave a weak response,

undifferentiated

U937

cells.

2O

E

O

16 O

~ 12 ' ~~ O

8

o

4

E

0 I

I

I

I

I

0

I0

20

50

40

50

Time in culture (hours) ~ . O; production by U937 cells after PMA preincubation. Cells were preincubated~with 35 ng/ml PMA for the periods of time indicated, and then stimulated with 50 ~g/ml Con A (~--~), 10 -7 FMLP (O .... O), or I ~g/ml PMA (O--Q) as described in Materials and Methods. O; production was monitored by the cytochrome c reduction assay (see MateriaIs and Methods). Data are mean Z standard error of five different determinations.

804

three

In

V o l . 151, N o . 2, 1 9 8 8

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

E

Neutrophlls

U937 (PMA)

5C 0

x

o

~

25 20

o 15 ~,,0

~L

Monocytes

U957

¢-

PFC

PFC

PFC

PFC Stimulus

Figure 2. O2 production by undifferentiated U937, PMA-differentiated U937 for 40 h, monocytes, and neutrophils. O2 generation was determined as described_$n Materials and Methods after cell stimulation with 1 ~g/ml PMA (P), i0 "M FMLP (F) or 50 ~g/ml Con A (C). Data are mean ± standard error of at least three independent determinations.

contrast,

PMA-differentiated

19.5

cytochrome

nmol

a value which

20

are

triggered respiratory

times cells the

c

higher

achieved

reduced/5xl05 than

specialized

generation

U937

of

that

cells/lO

obtained

in r e s p i r a t o r y O~

in

a

respiratory min

Con

activity

stimulation,

with Con A - s t i m u l a t e d

neutrophils,

activity.

differentiated

U937,

PMA

and

although

FMLP

this

burst was not as high as in neutrophils.

0 02C 0 016 w r,.) z <

133 rr 0 nn

0 012 0008 0 004 0

- 0.004 -0 008 400

I

I

450

,500

I

550

600

WAVELENGTH (nm)

Figure 3. Dithionite difference spectrum of total membranes prepared from PMA-differentiated U937 cells. Reduced versus oxidized spectra were obtained after addition of sodium dithionite and potassium ferricyanide, respectively, to samples in matched euvettes. The spectrum shows peaks of a b~type cytochrome at 428, 530 and 559 nm.

805

of

A

burst

after

burst

also

latter

Vol. 151, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1 Cytochrome b content as a function of time in culture of PNA-treated U937 cells Time (h) I

nmol cytochrome b/mg membrane protein 2

0

0.104 t 0.011

24

0.176 t 0.020

40

0.248 3 0.030

iCells were incubated for the times indicated in the presence of 35 ng/ml as described in Materials and Methods. 2Cytochrome b was calculated in the membrane pellets obtained from the postnuclear fractions as described in Materials and Methods. Values are mean standard error of four different preparations.

A

novel

previously

b-type

cytochrome

reported

the total membranes dithionite

in

human

with

an

alpha

neutrophils

band

and

spectroscopy

(Fig.

559

monocytes

isolated from the postnuclear

difference

at

3).

nm

(6),

which was

has

been

observed

in

fraction of U937 cells using

Most

of

this

cytochrome

b

was

localized in a region distinct from mitochondria upon subcellular fractionation in

a

sucrose

cytochrome content

gradient

measured

increased

cytochrome presence

is

not

with

content

of PNA,

(results of

U937

rose

more

whereas

the

not

shown).

This

mitochondrial

origin.

differentiation than rate

2-fold of Con

indicates

(Table after

The

i).

40

h

A-stimulated

increased about 20-fold after differentiation

that

the

b-type

b-type

cytochrome

Cell-membrane cell

culture

superoxide

~-type in

the

generation

(Fig. 2).

DISCUSSION U937

are

immature

human

monocyte-like

cells

characteristics of functionally mature monocytes

which

exhibit

only

some

(20). This is to our knowledge

the first report demonstrating the induction of the superoxide anion generating capacity induce

in the human

maturation

of

cell line U937 by PMA. U937

pendent

and nonspecific

et al.

(21) were unable

of U937 cells, U937

assessed

tumor killing

by

Incubation with PMA is known Fc

enhancement

receptors

~-interferon

antibody-de-

(13). However,

Roux-Lombard

and l ~ , 2 5 - d i h y d r o x y v i t a m i n

for 0 2 production.

This

to

and

to detect PMA induction of the 02 generating

despite of

to be stimulated

cells,

discrepancy

capacity

D3 enabled

could be explained

by the lower PMA concentration used during U937 differentiation in the previous study,

namely I ng/ml

(21), as compared to that employed in this work,

35 ng/ml.

806

namely

Vol. 151, No. 2, 1988

The

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

appearance

followed

of

monoeyte

the

distinct

generating

maturation,

of monoeyte-macrophage a

02

PMA-differentiated of differentiated

as evidenced

surface antigens.

respiratory

burst

cells. U937

response

This

cells,

is

the

which

upon specific Con A stimulation. burst in mature phagocytes,

capacity by

in

U937

herein

cell morphology

reported

and expression

Furthermore,

it is worth to note that

is

by

achieved

first

report

are highly

different

showing

responding

stimuli

the

in

preparation

for 02

production

Con A is a weak stimulant of the respiratory

such as monocytes

and neutrophils

(Fig. 2). Thus,

the fact that Con A is able to trigger the 02 generation in U937 cells treated with PMA for 40 h at a rate 20 times higher than in human neutrophils that a highly

specific machinery

during PMA-induced

needed

for Con A stimulation

is synthesized

U937 differentiation.

This latter machinery

coupled to Con

A stimulation would include: processes

suggest

involved

a) receptors for Con A; b) transduction signalling

in Con

A

stimulus-response

coupling;

and

c)

increase

in

the expression of the components of the O2 generating system. We report here for the first time the presence of a novel ~-type cytochrome similar

to that

previously

reported

22, 23).

We found no direct

and

generation

O~

indicates

that

ability

(5, 6) and monocytes

(6,

correlation between increase of b-type eytochrome

capacity

the

in neutrophils

during

PMA-induced

to generate

02

U937

during

differentiation,

stimulation

is

which

controlled

by other factors in addition to the amount of cytochrome b present. As a matter of fact, the different capabilities of PMA-differentiated U937 cells to produce 02

upon

stimulation

by

distinct

agents

clearly

reflect

events

occurring

at

the level of signal transduction processes.

The

results

reported

here

respiratory burst responses U937 cells.

This observation

signalling

processes

that

different

in human monocytes, indicates

mechanisms of cell activation, tion

show

stimuli

neutrophils

that different

elicit

distinct

and differentiated

stimuli

follow distinct

and different activation mechanisms or transduc-

are predominant

in each

cell

type.

Concerning

the

PMA-treated U937 cells, it is interesting to note that prolonged cell exposure to phorbol

esters has been reported

C activity

(24, 25).

neutrophils,

to induce disappearance

In this context,

PMA-treated

U937

cells

of protein kinase

we have found that,unlike monocytes are much

less

responsive

and

to PMA or FMLP

than to Con A. It is well known that PMA and FMLP act through protein kinase C

activation

trigger

and

different

PMA-differentiated differentiated

subeellular signal U937

cells

redistribution

transducing cells

constitute

for a

processes Con

A

good

model

(26),

whereas

(27).

seems

to

The high specificity

of

stimulation to

study

Con

A

indicates signal

that

transduction

proceses which putatively do not involve protein kinase C activity, those elicited by the leetin Con A.

807

these

especially

Vol. 151, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENTS This work was supported by a grant from the "Fondo Nacional para la Investigaci6n Cientifica"

(CSIC 424). J.B. is

recipient of a predoctoral fellowship

from the Spanish Ministry of Science and Education.

We thank Mrs. Ana Chao for

typing the manuscript.

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