Eosinophils do respond to FMLP

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1987 70: 379-383

Eosinophils do respond to fMLP M Yazdanbakhsh, CM Eckmann, L Koenderman, AJ Verhoeven and D Roos

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From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.

Eosinophils By

Maria

Yazdanbakhsh,

Carel

M.

Do Respond

Eckmann,

Leo

to fMLP

Koenderman,

Arthur

Eosinophils were isolated from normal human blood by separation over Percoll gradients, which resulted in eosinophil suspensions of a purity higher than 95% and recoveries of about 65%. Normal human eosinophils were found to respond to formyl-methionyl-leucyl-phenylalanine (fMLP)

fMLP

as was

tions

(iO8

of

another

at

treatment

of

concentrations

>iO

mol/L

with

an

increase

in

the

concentration of intracellular free calcium, oxygen consumption, nitroblue tetrazolium reduction, and chemiluminescence. The maximal response of eosinophils to fMLP was lower than that of neutrophils isolated from the same blood samples and required at least ten times as much

D

IFFICULTIES human

Most

studies

tial

have

these

eosinophils

hampered

so far have

eosinophilia; normal isolation

in obtaining

blood

used

are

responsiveness

of eosinophils

and

to this to fMLP

mol/L. patients

However, previous studies with eosinophilia have shown

these

report, in the

as well

cells.

we

with

In addition,

and

have

dismutase

Molecular

Biochemicals,

ries,

Alderley

sulfoxide

incubations (10

and stored

.tg/mL)

had

erythrocytes,

City,

of

lucigenin

and Fura-2

OR).

cytochalasin

NBT were was bought

Digitonin

B (ICI

UK)

1,000

was

times

were

the

Research

dissolved

final

for

cell

of DMSO

on the cells.

Percoll. Percoll suspensions (Pharmacia Fine Chemicals, Uppsala, Sweden) were prepared with specific gravities of 1 .077, 1.082, and 1.084 g/mL at room temperature. The specific gravities were measured with a density meter (Anton Paar, Graz, Austria, model DMA 46). The osmotic value of all Percoll suspensions was 290 mosm

The NaCI,

(measured

with

incubation 2.7 mmol/L

I .0 mmol/L

MgC12,

a semi-Osmometer,

medium KCI,

Knauer,

for the cells 8.1 mmol/L

5.5 mmol/L

consisted

Na2HPO4, glucose,

and

albumin.

Blood,

Berlin,

FRG).

of I 38 mmol/L 0.6 mmol/L

0.5%

(wt/vol)

S

1987

by Grune

of

fMLP

ability

to

eosinophils

eosinophils

showed respond

be

used

to to

to

after

that

do respond

not

stimulate neutrophils. concentrathese

fMLP.

We

fMLP

and

isolate

eosino-

& Stratton,

Inc.

preparation. Blood was obtained from healthy volunteers 3% to 8% eosinophils in their peripheral blood. Granulocytes were purified from the buffy coat of 500 mL of blood anticoagulated with 0.4% (wt/vol) trisodium citrate (pH 7.4). The buffy coat was diluted three times with phosphate-buffered saline (PBS) containing 13 mmol/L trisodium citrate (PBS-citrate) and centrifuged (20 minutes, 1,000 g, room temperature) over Percoll with a specific gravity of 1.077 g/mL. The erythrocytes in the pellet fraction were lysed with isotonic ammonium chloride at 4#{176}C as described.’ The granulocytes were subsequently incubated for 30 minutes at 37#{176}C in Hanks’

buffered

(vol/vol)

fetal

I .084

salt calf

solution

serum

with

(pH

7.0

20 at

mmol/L

g/mL.

This

suspension

was

HEPES

37#{176}C),to restore

gravity of the cells. Thereafter, in Percoll with a specific gravity layered

and the

5%

original

the granulocytes were at room temperature of on

top

of Percoll

with

a

of 1.1 g/mL and overlaid with 0.2 mL PBS. The tubes were centrifuged for 20 minutes at I .000 g at room temperature. Neutrophils (>98% pure) from the upper interface were collected, washed in PBS-citrate, and resuspended in incubation medium. The cells from the lower interface were also washed and gravity

resuspended

in the

incubation

medium;

the

resulting

cell

suspension

represented a 50% to 70% recovery of the eosinophils present in the buffy coat, at more than 95% purity. The contaminating cells were neutrophils. Eosinophils were also isolated as described by Roberts and Gallin.3 Briefly, whole blood was anticoagulated with EDTA (final concentration, 0.3%, wt/vol). Prewarmed blood was incubated with IMLP (l06 mol/L) for 15 minutes at 37#{176}C.Thereafter, it was layered over Percoll with a specific gravity of I .082 g/mL and centrifuged (25 minutes, 400 g, room temperature). Erythrocytes and eosinophils constituted the layer below the Percoll. The erythro-

From Blood

the

CaCl2, human

Central

Transfusion

Clinical

in dimethyl

concentration

eosinophils or to a suboptimal with

should

concentra-

not

pur-

Laborato-

at -70#{176}C.The final concentration

no effect

reduc-

FRG). Phorbol myristate acetate Corp. Katonah, NY), fMLP (Vega

Cheshire, at

to

METHODS

bovine

(Junction

and

Park,

(DMSO)

(NBT)

dinitrate), Co (St Louis).

(Darmstadt, Midland

Tucson),

report, able

properties.

AND from

Probes

from Merck Consolidated

individ-

also

their

fMLP

fMLP did

Purification

blood

lost

therefore

specific

free calcium,

tetrazolium

altered

(l0,lO’-dimethyl-9,9’-biacridinium obtained from Sigma Chemical from

are

by

opsonized of the

In the present

either respond

normal

Low

phils.

suspended

In addition, we demonstrate with fMLP by the method

MATERIALS

chased (PMA;

normal

in intracellular

nitroblue

Gallin3

Superoxide

from

that

Roos

in themselves

stimulus. whole

had

conclude

specific

metab-

generation

response to by pretreatment

eosinophils

by an increase

consumption,

oxygen

(LTC4)

of fMLP.7 human

and chemiluminescence. eosinophils isolated

Roberts

C4

in eosinophils

normal

to fMLP

oxygen tion, that

that

Acdo not to l06

on eosinophils from that fMLP enhances

stimulates

of LTC4 in to be augmented

low concentrations

show

respond

and

as leukotriene

uals, the generation particles was shown cells

of

to formyl-

normal human eosinophils concentration range of l0

of Fe receptors

release

with

those

was described.

eosinophils

neutrophils.

which

by cells to

Dirk

Cell

patients from

consumption primed these

that

for

and

with

cells.

neutrophils

(fMLP)

cording respond

olite

from

different

normal

these

In a recent report,3 a method for the human eosinophils based on the differen-

methionyl-leucyl-phenylalanine

the expression

from with

eosinophils

eosinophils

individuals.”2 of normal

studies

needed mol/L).

#{176}2

tion

J. Verhoeven,

Immunology,

Submitted

July

Supported

by

Address

Netherlands for

to

March

Dirk

Laboratory Service,

Cross and

of Amsterdam. 900-512-057

requests

Red

Experimental

20. 1987. of

the

(FUNGO). which is subsidized the Advancement ofPure

Central

Transfusion

No.

the

Laboratory

10. 1 986; accepted Grant

reprint

Secretariat,

of

and

University

Medical Research lands Organizationfor

Blood

Laboratory Service

P0

of Box

Roos. the

Foundation

PhD.

do

Publication

Netherlands

9406.

for

by the NetherResearch (ZWO).

1006

Red

AK

Cross

Amsterdam,

The Netherlands. The publication charge

payment.

“advertisement” indicate

costs

ofthis

article

This

article

must

in accordance

with

were defrayed therefore /8

U.S.C.

in part

be hereby §1734

by page marked solely

to

this fact.

1 987 by Grune

& Stratton.

Inc.

0006-4971-87-7002-0006$3.OO/O

Vol 70, No 2 (August),

1987:

pp 379-383

379

From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.

YAZDANBAKHSH

380

cytes were lysed with the previous paragraph. 90%,

with

recoveries

Metabolic an

isotonic ammonium chloride The purity of eosinophils of 70%

oxygen

to 90%.

Oxygen

reactions. electrode

as

as described in was higher than

consumption

described

was measured

before.9

NBT

with

reduction

was

measured by incubating cells for 30 minutes at 37#{176}C with NBT (500 sg/mL) in the presence or absence of stimuli. At the end of the incubation, the cells were fixed with paraformaldehyde (final concentration, 1%, vol/vol) and spun on a glass slide with a cytospin centrifuge. Thereafter, the slides were stained with nuclear fast red (0.1%, wt/vol), and the percentage of NBT-positive cells was scored as described before.’#{176}One cell sample was stained with MayGrtinwald/Giemsa

and

used

for

the

determination

of

the

Lucigenin-enhanced chemiluminescence was determined as follows: Minivials were filled with 1 mL of cell suspensions (10 cells/mL). The vials were placed in polyethylene counting vials in a scintillation spectrometer with a controlled temperature of 37#{176}C (Packard

Tricarb

started

by the

3310,

addition

Downers

Grove,

1 mL

of prewarmed

of

lucigenin (640 zmol/L) continuously for ten minutes of the apparatus. Where indicated, cytochalasin B (10 sg/mL) for three counted

of cytosolic

neutrophils.

pended mmol/L

Mg504,

mmol/L

potassium

(wt/vol)

by incubation

warm 40

albumin

1 zmol/L

HEPES

DMSO. pended

After

The

were

with

the loading

and

in HEPES

continued

as

that

found

in

analysis was performed test for paired observations.

with

Statistical t

RESULTS Oxygen

consumption.

sponded

to fMLP

The

response

about

l0

for

the

Normal

by a rapid

was

dose

mol/L

reaction

(Fig

cytochalasin fMLP.’5 response

1).

It

with

that was

optimal

was has

been

the

a maximum

shown

these

cells

cytochalasin

B. This

on the 02 consumption

of either

and

that

shown

previously

response

effect

mol/L,

twice of

the 02 consumption also augmented

the by that

neutrophils

to

by eosinophils by pretreatment

agent

by

cell type.

itself

had

in of no

Cytochalasin

B augmented the fMLP-stimulated neutrophil respiration 100% to 200%, whereas that of eosinophils was enhanced

02

at

concentration

106 was

re-

consumption.

fMLP

about

response

B enhances We found to fMLP

in oxygen reaching

The

of the maximal

eosinophils

human

increase

dependent,

fMLP.

neutrophil

magnitude

by by

to 60%. at

a concentration

consumption

priming in

same

of

I0_8

by eosinophils;

these

cells

fMLP

(10_8

tion

in response

mol/L)

(Table for

1 ). two

mol/L

however, Eosinophils

minutes

to a suboptimal

showed

concentration

did

not

it was

stimulate

capable

pretreated an

of with

02 consumpof PMA (0.5

(vol/vol)

use.

oxygen

resus-

Prior

consumption

to each

( 10 cells/ concentration, 106

or neutrophils

medium

at 37#{176}C for

analysis.

test or Student’s

t

the

cells.’4

for a further 0.1%

on ice before

incu-

with the

the cells were washed,

kept

of

stock

five times

with

eosinophils

incubated

cuvette. Fluorescence

was

procedure,

medium,

diluted and

l-mmol/L

incubated

Statistical

fMLP

ester

is approximately

ie, 0.35 jzL/106

Student’s

30%

0.5%

were

of eosinophils

neutrophils,

Fura-2”

methyl

cells/mL) a

1.2

and

loaded

(l0

(from

were

Fura-2-loaded

were

cells

incubation

cells

in HEPES

cells/mL)

Cells

content

eosinophils

and

HEPES,

glucose,

at 37#{176}C and then diluted

Control

measurement,

mL)

7.4)).

eosinophils

mmol/L

mmol/L

Fura-2/AM

medium.

minutes.

medium

were washed and susNaCl, 6 mmol/L KCI, I 20

5.5

Prewarmed

for 20 minutes

was

incubation

in human

CaCl2,

(pH

reaction

of the cells at 37#{176}C with the acetoxy

with

DMSO)

(132

phosphate,

(Fura-2/AM).

bated

Ca2

neutrophils mmol/L

and

medium 1 mmol/L

human

Fura-2

free

Eosinophils

in HEPES

The

and a stimulus. Each vial was in the out-of-coincidence mode cells were preincubated with minutes at 37#{176}C.

containing

Measurement

IL).

the fluorescence after digitonin addition, and Fmj. 5 the fluorescence after Mn2 quenching. Agonists used gave no autofluorescence changes in unloaded control cells. Fura-2 contents in the cells were determined by adding Fura2-free acid to the cell suspensions. On average, the Fura-2 content of the cells was 1 10 smol/L, under the assumption that the free water

cell

composition.

ET AL

(final

1 5 minutes

before

performed

with

transfer

to a U)

measurements

were

a spectrofluo-

5) U

rometer

(model

contents

of the

holder was wavelengths Fura-2

RF-540, cuvette

Shimadzu were

Corp.

magnetically

Kyoto, stirred,

iapan). and

thermostated at 37#{176}C. The excitation were 335 nm and 505 nm, respectively.”

fluorescence

as a function

of [Ca2]i

was

The

the

)00

cuvette

and emission Calibration of

determined

0

0

essen-

E C

as described for Quin-2 fluorescence’2”3 with the following modifications. To saturate all trapped Fura-2 with Ca2, 5 ,mol/L digitonin was added to the cell suspension. At this digitonin concentration, the cell membrane was found to be permeable for small solutes without causing lysis of the cells, thereby avoiding a dramatic tially

shift

in

autofluorescence.

quenched

by adding

MnCl2)

Subsequently, 0.5

mmol/L

to the cell suspension.

Ca2-independent

fluorescence

Mn2t

The percentage

found

to be 36%

Cytosolic

the Ca2-dependent

where

224

224

nmol/L

were

Fura-2

Fura-2

was

mmol/L

and

the

by addition

fluorescence quenched

of

by Mn2.

the

Fura-2/Ca2

Fig 1 . Oxygen consumption by eosinophils (--) and neutrophils in response to various fMLP concentrations. The results show the maximum amount of oxygen consumed by cells stimulated with fMLP (usually three to four minutes after stimulation). The amount of oxygen consumed by cells at rest has already been subtracted. Each point represents the mean ± SEM of (n) experiments. (-)

+0.36[F F

[fMLrjM

was

as follows:

nmol/L

K4 for

of0.5

quenched

fluorescence

was calculated

is the

a stock

signal

Both signals

F-(F. [Ca2]

Fura-2

(from

ofCa2-independent

of the total

free Ca2

the

Mn2

-F.]) -F complex,”

Fma. is

From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.

EOSINOPHIL

RESPONSE

381

TO FMLP

Table

1 . Priming

Effect

chemiluminescence

of fMLP C.,

Oxygen Consumption (nmol 02/10’ cells/mm)

Test Condition PBS

Eosinophils

Lag time

12

(Mini

Neutrophils

Eosinophils

Neutrophils

0.6

8.4

6.5

5)

pretreat-

ment fMLP

+

PMA

(10’

1.6

0.3

±

±

0.2

2.0

±

±

1

0.3

E

mol/L)

pretreatment

+

PMA

8.6

1.4

±

3.0

±

0.4

5.4

1.3

±

3.3

±

0.3

Oxygen consumption by eosinophils and neutrophils in response to PMA (0.5 for eosinophils and 1 .0 ng/mL for neutrophils) is shown. The cells were preincubated with either PBS fMLP (10’ mol/L) for two minutes before the addition of PMA. fMLP by itself did not stimulate either cell type. The lag time is the time after the addition of PMA. before maximum velocity is reached, as defined by Cohen and Chovafliec.” All results are expressed as means ± SEM of three experiments. The differences between PBS- and fMLP- (10’ mol/L)-treated cells were all significant at P < .05 using Student’s t test for paired observations.

ng/mL

8

4

ng/mL)

with

a shorter

velocity

than

control

results

were

tions tion

obtained

of PMA

compared used

more

reduced

neutrophils.

NBT.

hence had

When 95% The

the

tg/mL)

before

increased the percentage NBT from 1% to 19% NBT

Cytochalasin by either

with

fMLP

82%.

and

was

no priming

I ng/mL.

effect.

with

PMA and

(0.1 eosinophils

of fMLP cytochalasin

l0

mol/L

of eosinophils capable that of neutrophils

is B

fMLP

of reducing from 21% to

B alone did not stimulate the reduction of cell type. When the eosinophils were primed

(l0

mol/L)

of formazan-positive ng/mL) increased

from

for three

minutes,

the

eosinophils in response 50% to 90%.

Lucigenin-enhanced

percentage to PMA

chemiluminescence.

to fMLP by a rapid increase in chemiluminespeaked at about one to two minutes and reached

value

8.9

±

1.4

x

I0

cpm

(25

(mean

±

SEM

of

experiments). second

slide

peak

that

reached

(mean

minutes.

a nine

eosinophil

small

peak

cpm

±

SEM

Figure

3A

with

to fMLP

fMLP

was enhanced is an oxygen-dependent

not

necessarily

dase

activity,

correlate evident

affect

itself.

with from

and a reaction

Cytosolicfree

4)

U,

0

a N

0

io

io

io

[fMLP] NBT reduction by eosinophils (--) to various concentrations of fMLP. ± SEM of three experiments

and neutrophils (-I Each point represents

in

metabo-

effect

on

levels

in eosinophils

free

and neutrophils Fura-2. The

fMLP, rose

the intracellular

to values

10_6 mol/L intracellular nmol/L (mean (Fig 4). When

neutrophils

93 ± 6 nmol/L of n experiments).

fMLP Ca2

of more

calcium

concen-

were change

also induced concentration,

± SEM, eosinophils

n

was (n = Upon

Ca2 than

the

or neutrophils. measured in Fura-2

and

phils

(M)

oxygen no

of

cytochalasin

Ca2

mol/L

icc8

the

had

oxi-

kinetics

after the addition of fMLP change in Quin-2 fluorescence by Pozzan et al.’3 The resting

(n = 9) and (mean ± SEM

0

the

fluorescence in neutrophils was similar to the neutrophils as observed

4)

E

of the

in

from

Intracellular

Ca2.

the

cytochalasin B. process but does

Thus,

eosinophils

trations in both eosinophils with the fluorescent indicator

unlike

parameters

B alone

of either

decreased

assay,

difference

distinct

l0 ten

of eosinophil one

by

02 consumption.

Cytochalasin

x

around

was inhibited by cytoneutrophil chemilumi-

other

the

peak

in this

with Chemiluminescence

2.3

±

at

second

biphas-

increasing

14.1

first

NBT reduction, In contrast, the

‘I)

C.)

the

and the contamination.

response

chemiluminescence

Fig 2. response the mean

that

and 3B).

lism

4)

of

experiments)

02 consumption chalasin B (Fig

chemiluminescence

test

shows

was

by a slowly

a maximum

neutrophil

eosinophil

to fMLP

followed

nine

increased

increasing

The

response

was of

chemiluminescence

B could NBT

The

ic; an initial

nescence

N eutrophils

responded cence that of

j.tg/

Fig 3. Lucigenin-enhanced chemiluminescence. (A) fMLP (2 mol/L) was added at time 0 to eosinophils (--). neutrophils (-) and a 1 :1 mixture of eosinophils and neutrophils (. . ). (B) fMLP (2 smol/L) was added to the cells preincubated with cytochalasin B (10 sg/mL). The purity of the eosinophils and neutrophils was 95% and 96%, respectively.

neutrophils

human

concentrations of the cells with with

as

concentra-

neutrophils

of normal

stimulation

Similar concentra-

of neutrophils

eosinophils

response

maximal

PBS.

Higher

stimulated

of

and neutrophils to increasing shown in Fig 2. Pretreatment (10

with

the suboptimal

with

of fMLP)

than

as a higher

for the stimulation

experiments

solvent

reduction.

NBT mL),

with

eosinophils:

in our (the

as well

pretreated

are needed

with

DMSO

lag time cells

75

±

(Fig in free

6 nmol/L

7), respectively addition of l0

concentration I mol/L.

a very rapid to a value

in neutroIn eosinophils, increase of 297

in the ± 23

= 9). This response was transient were stimulated with I0 mol/L

From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.

YAZDANBAKHSH

382

two

50C

A

aC a 0

methods

response was

to ionomycin

assessed

(Fig

or

platelet-

SB, C).

DISCUSSION report

we have

shown

that

normal

phils

respond to the chemotactic increase in free intracellular calcium,

100

NBT IMLP

(10’M)

30s

reduction,

shown

Fig 4. A representative experiment showing the effect of fMLP (10 mol/L) concentrations of (A) neutrophils and values, see the text).

from a series of nine on intracellular free Ca’ (B) eosinophils (for Ca2

that

and these

with

fMLP

augmented

the

response

to fMLP Despite

burst

these

fMLP,

cells

intracellular Comparison

with

no

eosinophils eosinophils

Normal

compared

purified described

with

the

in the

level

of free

than

free

calcium

the response free

Ca2

and

1 13

was

levels

addition

were

79

fMLP

to

experiments,

addition normal

of fMLP, eosinophils

nmol/L

above

ments).

In contrast,

the

The

our

method

same

donors

parameter

resting

blood

±

SEM

significant).

(mean

±

of After

concentration by 205

SEM

isolated

for

after

after

contaminating

five

arations

the

fMLP

of five

experi-

fMLP

treat-

significant

An example difference

is shown

between

A

in Fig 5A.

the eosinophils

There isolated

B

was

20%

of

neutrophils.

the

However, the large

evidence

the

that

the

neutrophils with

that

a purity was

These

eosinophils

results

were

could

found

to

be

from the priming experiments majority of the eosinophils can

intracellular

to I08

intracellular

free

seen

exhibited

of the response

shown mol/L

Ca2,

maximally

responses is as follows:

98%

50%

of the experiments respond to 108

in 48

for

to fMLP.

The

±

that

mol/L),

respond

ment ofblood had lost their ability to respond to fMLP (I0_6 mol/L): the increase in the intracellular free Ca2 was only 27 ± I 3 nmol/L above resting values (mean ± SEM of five experiments).

with

formazan-positive. we conclude that

eosinophils

purified

(mean

not

(l0

as

intracellular

in normal

whole

levels

response

from

eosinophils

eosinophils

concentrations

eosinophil

suggest the presence of lower numbers of fMLP receptors on the eosinophil surface. In the NBT reduction test with fMLP

the intracellular Ca2 increased significantly

resting

higher

optimal

of

lower

by

in the difference

as that was

isolated

6 nmol/L

±

for an

as high to fMLP

In addition,

required

reduction tests. to exhibit an

compared

as a sensitive

of stimuli.

12 nmol/L

of

of neutrophils. were

is twice

response

B

neutrophils

by two different

of whole blood with fMLP Gallin.3 The change in intracellu-

measured

to a number ±

that

and

of NBT eosinophils

that

after

Cytochalasin

isolated

eosinophils

after treatment by Roberts and

paired

increase

PMA maximal

an

we have

enzymes

eosinophils

consumption of normal

their

of fMLP

between

procedures.

were

showed

Ca2t

granule mol/L).’7

of both

eosino-

fMLP by consumption,

Recently,

release x l0_6

(2

in the 02 the capacity

oxidative

also

human

peptide oxygen

chemiluminescence.

cells

treatment

neutrophils,’7

lar

the

(PAF)

200

In this 0

when factor

activating

ET AL

whereas mol/L Ca2

in Fig fMLP

concentrations

not

to

prep-

seen

(one did not in free

responded no further was

to

by neutrophils

I ). (b) Eosinophils with an increase (ie,

due

Eosinophil

an 02 consumption seen

neutrophils fMLP

were

(a)

already increase upon

in

stimu-

lation with I0_6 mol/L fMLP). Thus, the lack ofresponse of the eosinophil suspensions to 10_8 mol/L fMLP proves that the contamination with neutrophils was negligible. (c) Cyto-

no

chalasin

by the

B inhibited

the

eosinophil

chemiluminescence

in

1mm

CJ\%

1000

500

100

fMLP

ionomycm

(1pM)

(1pM)

PAF

(1pM)

C + C’1

0

0

0 Cl) 0

C

B

A

C.)

1000 500

>.

0 100

tt

IMLP

ionomycin

(1pM)

(1pM)

lonomycin

(1pM)

PAF

(1pM)

Fig 5. Intracellular free Ca’ concentrations in eosinophils isolated by our method (top panel) and by the method described by Roberts and Gallin3 (bottom panel) (see Materials and Methods). Effects are shown of the addition of (A) fMLP (10’ mol/L). representative of five experiments (for Ca’ values, see the text); (B) ionomycin (10-’ mol/L). representative of five experiments (in normal eosinophils. the intracellular free Ca’ level increased to 590 ± 40 nmol/L as compared with 670 ± 160 nmol/L in eosinophils purified by the method described by Roberts and GaIlin3; mean ± SEM of five experiments); and (C) PAF (10’ mol/L). representative of two experiments (the free intracellular Ca2 increased to 354 nmol/L and 352 nmol/L in normal eosinophils and eosinophils isolated with fMLP,3 respectively; mean of two experiments).

From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.

EOSINOPHIL

RESPONSE

response

to

Thus,

fMLP

the

while

(Fig

3).

(d)

The

kinetics

response

kinetics

of

the

reduction

test

examined,

eosinophils

neutrophil

where

were

seen

In addition,

laboratory

has

that

were

able

completely

in the

ruled

out.

induces

an increase

without

an appreciable

very

pure

respond

separation presume

of

therefore,

after

phils

still

can

that

free

as our

of

eosinophils

and

Ca2

possibility Gallin3

eosinophils treatment be called

from do not

a

neutrophils. respond with Our

The to fMLP fMLP,

results

the

of

not

fMLP

stimulate

the

to a subsequent et al7 and this

we

be accounted

PAF

was

by the

normal.

of Roberts

(with

inability

to

latter

to purify

to ionomycine

we have

isolated

Gallin3

from

and

had

due of the

response

In addition,

method donors purified

donors

their

was

eosinoblood

response

for by our

because

This

normal

presumably

altered

of

human

blood.

fMLP-treated

fMLP,

donor

method that

unlike

from This

same

the

to whole

that, to

the by

evidence

added

respond

successfully

or

obtained

obtained to

from

method

observation

cannot

cells

nonselected eosinophils

eosinorandom,

with similar results to those obtained with by the same method from our selected

3%

to

8%

eosinophils

in

their

peripheral

blood).

authors

In conclusion,

that,

fMLP,

eosino-

contradict

do

can

An impor-

concentrations

eosinophils our

desensitization.’8

phils

and

low

to fMLP

the

ability

receptor

and

the

very

where either

respond

the

still

facilitating

at

eosinophils

mol/L.

cells are primed to respond of another stimulus (Shaw

the eosinophils

lost

these

purification

human l0

in themselves

Gallin,3

from

eosinophils

fMLP (lO_6 mol/L) induced a decrease thus

of blood normal.

can

even

by

and

deduced

that

unlikely.

reported

of the neutrophils,

eosinophils

fMLP

findings

isolated

phils,

be

normal

study).

eosinophils

in eosinophils

highly

eosinophils in which blood. This treatment

gravity

that

observation

as well

Roberts

in the specific

cannot

that

which these dose

Roberts

that

stimulated

is that

In experiments

P. Venge,

neutrophils

show

at concentrations

mol/L),

were

gradient

possibility

lag time this

in

of eosinophils the

preparations

eosinophils, suboptimal

in a granulo-

in intracellular

render

method for human was added to whole

The

NBT

in another

H#{226}kansson and 1986).

activation

However,

(>98%)

to fMLP

Recently,

(L.

March

point

(I0’

are

and

to fMLP

tant

NBT

out

opinion

respond

from cells

to reduce

present

by contaminating

result

the

in a chemotatic

to fMLP

secreted fMLP

In

carried

eosinophils

communication,

products

(e) identifiable

work

latter to

fMLP-induced is different

to be able

visualized

to respond

personal with

and

is due

response.

to fMLP. preparation

neutrophils.

of the

morphologically

shown

of

response

of eosinophils

response cyte

that

B-inhibitable

chemiluminescence the

augmenting

cytochalasin

eosinophils

383

TO FMLP

and

normal

therefore

use of fMLP

human

any

will yield

eosinophils

isolation

altered

method

do that

respond involves

to the

eosinophils.

the REFERENCES

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N-formylmethio-