Journal

of Leukocyte

Biology

50:444-452

(1991)

Activation of Human Neutrophils by Phorbol Ester Decreases the Cytoplasm Compactness and the Lactoferrin Content of the Granulocytes Nair Center

for

Esaguy,

Experimental

Artur

Cytology Salazar

P. Aguas,

Manuel

Vilanova,

(INIC) (N.E., A.P.A., M.T.S.) and Departments Institute for the Biomedical Sciences, University

and

Manuel

T. Silva

of Anatomy (A.P.A.) and Immunology of Porto, Porto, Portugal

(MV.),

Abel

Human neutrophils isolated from the blood were incubated in vitro for 30 mm with 25 ng/ml of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, to study its effect on the fine structure of granulocytes and on the subcellular distribution of lactoferrin (Lf). Flow cytometry analysis of the human neutrophils showed that PMA induced a decrease In size and granularity of the cells. By electron microscopy, the PMA-treated cells showed numerous empty vesicles, smoothing of the cell surface, and a marked decrease in the compactness of the cytoplasmic ground substance. High. resolution immunogold ultracytochemistry showed that neutrophils lost most of their content in Lf after the PMA Incubation. In thin sections, the cytoplasmic compartment of PMA-treated neutrophils contained an average of 1 .03 ± 0.3 Lf-positive vesicles per p.m2, whereas control granulocytes showed an average of 5.72 ± 1 .49 Lf vesicles per p.m2 of cytoplasm. Most of PMA-treated neutrophils kept some Lf-positive vesicles; a significant minority (10-15%) of the granulocytes showed cytological features of cell death. We conclude that the PMA-lnduced neutrophil activation is associated with microanatomical changes that include I) exocytosis of most, but not all, of the Lf-bearing vesicles; Ii) rounding up of the cell outline; and iii) decrease in the compactness of the cytoplasmic ground substance. Key

words:

secretion, munogold,

protein electron

kinase C, microscopy

INTRODUCTION The

neutrophilic

granulocyte

is a dormant

acetate agent

or microorganisms induced in vitro (PMA), [26,28,37].

endogenous cellular vation radicals through so-called

a

circulating

kinase

[9,44]. Neutrophil activation by phorbol 1 2-myristate 13-

C

(PKC)

,

1 5 ,3 1 ] . The

changes due to PMA-triggered neutrophil actiinclude the generation of microbicidal oxygen (e.g. , superoxide and hydrogen peroxide) the activation respiratory

of burst

NADPH-oxidase during the [4,36,42], extensive degran-

ulation [25,40,41], down-regulation receptors, mnhibmtmon of free Ca2 ulation of arachidonic acid release of the binding of leukotriene B4 factor We

[11

for neutrophils studied here

the

[35,48]. fine structure

neutrophils, and used immunogold

of fMet-Leu-Phe transients [19], stim[32,38], and inhibition and platelet-activating

1991 Wiley-Liss,

Inc.

the

cytoplasmic

modification it was not

PMA-treated neutrophils and a general rounding

ground

MATERIALS

Isolation

vesicles granulocytes

were in

Reprint

University

substance

of

AND

neutrophils.

This

to granulocytes leukocytes.

as

METHODS

of Neutrophils

Blood

Received

to

Lf-positive of the

appeared to be restricted found in other PMA-treated

from healthy volunteers was heparinized and to dextran sedimentation as described before procedure results in neutrophil-enriched cell

suspensions.

define the alterations in content and location of lactofer©

im-

PMA; total degranulation was not, however, observed even in neutrophils showing cytological features of cell death. PMA provoked a decrease in the compactness of

submitted [3]. This

of PMA-stimulated ultracytochemistry

cytochemistry,

up ofthe cell shape. Most ofthe lost after a 30 mm incubation

tumor promoter and inflammatory The cellular receptor for PMA is the

protein

proteins,

rin (LI). We document that show cytoplasmic vacuolization

phagocyte that has the ability of enhancing its metabolism (neutrophil activation) in response to several physiological stimuli, such as the engulfment of foreign particles can be

iron-binding

These

November requests:

21. Manuel

of Porto.

preparations

contained

1990:

March

T.

accepted

Silva.

Rua do Campo

Center

Alegre

8.

a few eryth1991.

for Experimental 823.

4100

Cytology. Porto.

Portugal.

Cytoplasm

and

Lactoferrin

in Activated

A

w

445

Neutrophils

B

Ui

I-

0

0

U)

U)

w

LU

C,)

U) cc.,

0

0

-J

LOG

FORWARD

LOG

SCATTER

FORWARD

SCATTER

Fig. 1 . Contour diagrams represented in a logarithmic scale of the cytometric analysis of the granularity (vertical axis) and size (horizontal axis) of control (left side [A]) and PMA-treated (right side [BJ) human neutrophils. The data show the forward and side scattered light distribution measured in an EPICS profile flow cytometer (Coulter) and reveal that PMA induces a decrease in the granularity and size of the neutrophils.

rocytes

and

leukocytes

ultrastructural

and

other

than

immunogold

neutrophils. studies,

For we

the

collected

some samples at this stage of the purification because the “contaminant” cells (erythrocytes cytes other than neutrophils) are useful

procedure and leukoas internal

(additional)

PMA

controls

of the

specificity

of the

effect

processed labeling.

Cytometric

size

et al. [14]. This samples containing

collected IBM (PS

samples tochemical

were

used

both

for

cytometric

and

ultracy-

analysis.

of Neutrophils

by PMA

The neutrophil-enriched cell suspensions were washed three times in Hanks’ balanced salt solution (HBSS, Gibco, Pasley, Scotland) by pelleting and resuspension, and divided in two aliquots of i106 cells. One aliquot was incubated for 30 mm at 37#{176}C in HBSS containing 25

and

granularity

ware

immunogold

profile

forward light.

total events computer

and using

flow

(narrow List mode then the

poputo their

cytometer angle) data

analyzed cytologic

and was in an soft-

of Coulter.

Microscopy

The cells were 0.3% glutaraldehyde

fixed

in a solution of 2% formaldehyde, in HBSS during 2 h at 4#{176}C, washed

in phosphate-buffered saline (PBS), resuspended in 5% bovine serum albumin (BSA; used as a protein matrix to protect the cells during further processing), and micro-

the PMA vehicle, washed in HBSS

aldehyde immunogold

cells were analysis or

in an EPICS

in 20,000 8530-021)

buffy coats were buffy coats were above (1 h, room in PBS containing

DMSO). The to cytometric

and

Analysis

ng/ml of PMA (Sigma Chemical Co., St. Louis, MO). The other aliquot was used to control the PMA effect, and was incubated for 30 mm at 37#{176}C in HBSS alone (plus 0.02% v/v and submitted

microscopy

(Coulter) by measuring their side (right angle) scattered

Electron

In Vitro Stimulation

electron

Single cell suspensions of the Percoll purified lations of neutrophils were analyzed according

and of the immunogold Lf labeling of neutrophils. The cell suspensions were further enriched in neutrophils by a Percoll density gradient method as described by Brattig results in the harvesting of leukocyte 94% or more of neutrophils; the

for

sites

that, marker

made as described before [2,33]. The fixed in the same aldehyde mixture as temperature), and washed several times 1 mM glycyl-glycine to quench free otherwise, [1]. The

could specimens

react later with the were dehydrated

446

Esaguy

et al.

hagen, Denmark (Cat. No. A186). The reactivity and specificity for Lf of the sera was checked by immunoblotting of transfers from SDS-PAGE gels [45].

lmmunogold

Ultracytochemistry

Thin-sectioned leukocytes in dilutions of the anti-Lf gold

coated

Cat. No. previously

by goat

IgG

according in detail

experiments as described

cells were microscope

used

occupied PMA-treated

(Sigma

Chemical

Co.,

to a multistep procedure [2 1]. We performed three

to control the specificity of before [21]. The thin-sectioned

viewed in a Siemens after uranyl acetate

Morphometric We

antirabbit

G-3766) described

types of labeling,

were sequentially incubated serum and of 10 nm colloidal

the

Elmiskop lA electron and lead citrate staining.

Quantification

a picture by

weight

method

Lf-containing and control

to compare

vesicles neutrophils

in

the areas

thin [46].

sectioned First, we

photographed at random 10 different neutrophils in each sample at X 18,000 magnification. The electron micrographs were enlarged in photographic paper and immunogold labeled vesicles were counted and traced in the paper. The labeled granules and the remaining cytoplasm were cut out with a scissors; the paper cuttings were weighted in an analytical balance. The weight values were converted into area values considering the micrograph magnification. of cytoplasm in each

At least neutrophil

the number of Lf-positive by Lf-positive vesicles the average values eters was calculated

5 m2

vesicles and

(and up to 8.5 iim2) quantified regarding

was by

and the areas occupied remainder cytoplasm;

the

for im2 of each for the individual

of PMA and control samples by the Student t-test.

were

of the cells. compared

three These

paramvalues

statistically

RESULTS Fig. 2. Low magnification electron micrograph of thin-sectioned neutrophil-enriched blood cells incubated for 30 mm in PMA. The two neutrophils shown present vacuolization of the cytoplasm and smoothening of the cell surface. The red blood cell seen at the bottom of the figure keeps its characteristic shape, indicating that the rounding up alteration of the neighbouring neutrophils is not due to a putative hypotonic change of the incubating medium. xlO,500.

We used immunogold 12-myristate

in ethanol and embedded

in LR

White

resin (The London

collected

on parlodion-coated

We the

Antibodies Polyclonal in the rabbit)

anti-human lactoferrmn (Lf) IgG (produced was purchased from Dakopatts a’s, Copen-

of the

forward

a cell

activator,

on

the

samples.

Analysis

compared

PMA-treated

(PMA),

of neutrophils and on the subcellular of lactoferrmn (Lf). Our ultrastructural and results were complemented with cytometric

Cytometric

thin and

grids.

13-acetate

microanatomy distribution cytochemical analysis

Resin Co., Ltd., Basingstoke, Hampshire, England); sections were obtained in an LKB ultramicrotome,

here thin section electron microscopy and cytochemistry to study the effect of phorbol

the neutrophils

and

side

size

and

granularity

by cytometric scattered

light

of control

and

measurement in an EPICS

of profile

flow cytometer. We found that PMA induced a significant decrease in size and granular content in most of the treated human neutrophils as shown in Figure 1 (compare

Cytoplasm

and Lactoferrin

/ ,

-

in Activated

Neutrophils

....e...

0-

...

14:.

U..,

.

.

..t”

A

V4

-:

,,

0-

-

& S

/

V

-

-..

4.,.

H-

.

iri-4

1k0

Fig. 3. Thin section electron micrograph of control human neutrophil labeled by anti-lactoferrin antibodies using the immunogold method (black dots). The cell contains numerous vesicles that are marked by the immunogold probe (arrowheads). The lactoferrin-positive vesicles are rod shaped and depict a content of moderate electron density. The neutrophil surface shows cell processes (see inset) that contrast with the smoothness of the surface of PMA-treated granulocytes (compare with Fig. 2 and insets in Figs. 4 and 6). x 29,000 (inset, x 10,500).

447

448

Esaguy et al.

Fig. 4. High magnification view of thin-sectioned, PMA-treated neutrophil labeled by the immunogold method with anti-lactoferrin serum. The cell contains a decreased number of lactoferrin-positive vesicles (marked by the immunogold black dots). A moderate number of vacuoles is present in the cytoplasm (see low magnification in inset); a few of these vacuoles are lactoferrin-positive (arrowhead). x68,000 (inset, x 14,000).

Cytoplasm

and Lactoferrin

TABLE

in Activated

1. Quantitative

Comparison

Number of Lf-Positive Control Neutrophils0

*The

results

are

and

to

the

two

and

No. vesicles

± 0.016/pm2 ± 0.062/pm2

referred

between

of the Area

of cytoplasm

0.037 0.221

Differences (P< 0.01).

449

Vesicles Between PMA-Treated

Area/am2 PMA Control

Neutrophils

1.03 5.72

1 pm2

of

groups

are

± 0.30/pm2 ± 1.49/pm2

neutrophil

cytoplasm.

statistically

significant

iron-binding protein stored in the so-called specific or secondary granules of polymorphonuclear leukocytes [5,12,16,21]. LF was detected in situ on thin-sectioned cells by the positioning of small ( 10 nm) black dots of colloidal gold (Figs. 3-5). We found that PMA caused a marked decrease in the number of Lf-positive granules of neutrophils. The vesicles that showed positive labeling depicted secondary Fig. 5. Thin section electron micrograph of human lymphocyte present in the neutrophil-enriched samples incubated in PMA. The ultrastructure of the lymphocyte is not significantly altered by the PMA treatment. Notice, in particular, that no decrease In cytoplasmic compactness is observed. x 20,500.

the typical granules

profile and [21]. Lf was

of the cytoplasmic arrowhead). Our quantitative demonstrated that positive

granules

vacuoles

induced

analysis neutrophils

of

after

the PMA

found an average of p.m2 of thin-sectioned left side [A = control cells] with PMA-treated cells] ofthe figure). The neutrophils were statistically different and granularity.

Electron Low granulocytes cell

shape

of cytoplasm). (P