Elastase in the different primary granules of the human neutrophil

Vol.

132,

No. 3, 1985

November

15,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1985

Pages

1130-1136

ELASTASE IN THE DIFFERENT PRIMARY GRANULES OF THE HUMAN NEUTROPHIL Rodolfo

C.Garcia'

, Christer

G.B.Peterson2,

Anthony

W.Segall

2

and Per Venge

1Department of Haematology, Faculty of Clinical Sciences, University College London, Gower Street, London WClE 6HX, U.K. 2Department of Clinical Chemistry, University Hospital, S-75185 Uppsala, Sweden Received

September

18,

1985

Surmuary: Elastase in the human neutrophil is associated with various subpopulations of primary granules of different density. The proportion of this enzyme that is extracted with acetate pH 4.2 and cetyltrimethylammonium bromide varies in the different subpopulations. Nevertheless, the electrophoretic mobility and relative proportions of elastase isoenzymes is the same in both extracts from On stimulation of neutrophils with N-formylmethithe different subpopulations. onylleucylphenylalanine, elastase is not released from the least dense subpopulation, whereas other two subpopulations do undergo degranulation to approximately the same extent. However, the release of elastase from these two denser granules differs after they are isolated and treated with calcium. D 1985 Academic Press,

Inc.

Elastase with

is

perhaps

inflmatory

sible

for

the most

tissue

and rheumatoid

Elastase

the neutrophil

subpopulations 10).

It

is

have

tent. of the

been described

three

The present isoenzymes

glycoprotein (2)

that

elastase study

have

(5).

or primary (8,9),

belongs addresses

which

the question

and their

the

weight

conditions

cytochemistry in

of which

two

specific

29-31,000

with

proteinases.

in their

granules

on neutrophil

There

Copyright All rights

$1.50

0 1985 by Academic Press. IJ~ reproduction in any form

Inc. reserved.

1130

are con-

distribution stimulation.

Abbreviations: FMLP, N-formylmethionylleucylphenylalanine; Boc-Ala-ONp, butyloxycarbonylalanine o-nitrophenyl ester; CTAB, cetyltrimethylauunonium SDS, sodium dodecyl sulphate; PAGE, polyacrylamide gel electrobromide; phoresis; ACIE, agarose crossed immunoelectrophoresis. 0006-291X/85

(7,

up to 25%

carbohydrate

of the subcellular

degranulation

prevail-

is present

(6,7),

of serine differ

among

proteoglycans,

elastase

in the

inmnrne

components

Ultrastructural

granules

and not

to the group (11)

under

with

degranulation

collagen,

shown that

of molecular

isoenzymes

of elastase

(2-4)

substrate

studies

in the azurophil

a basic

of carbohydrates at least

and the

e.g.

and subsequent

elastin,

responarteritis

surfaces,

is one of the major

and fibronectin

distribution

associated agents

glomerulonephritis, coated

attachment

of degrading

ing

mostly

the

capable

immunoglobulins

neutrophils

emphysema,

Appropriately for

proteinases

to be one of the main

phagocytes.

fibrinogen, between

in lung

a substrate

released,

and subcellular

of the neutral

appears

(1,2).

of polymorphonuclear the products

It

destruction arthritis

p rovide

complexes,

prominent

conditions,

Vol.

132,

No. 3, 1985

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METHODS Purification and stimulation of cells: Neutrophils were obtained from normal donor blood as already described (12). Stimulation was carried out by the addition of FMLP (2x10-7M) and cytochalasin B (5 pg/ml> or only cytochalasin B (controls) to cell suspensions (1.6x108/ml) in RPM1 1640 medium containing at 37'C. After incubation the cells were pelleted and heparin (5 units/ml), washed with cold 0.15M NaCl, before homogenization. Subcellular fractionation: All the procedures were carried out at 0-4'C. Cell disruption was performed as previously described (131, in the presence of proteinase inhibitors (14). Organelle-containing extracts devoid of nuclei were fractionated on continuous sucrose gradients as reported (14). Aliquots of the fractions collected from the gradients were subjected to radioimmunoassays, enzyme activity assays and extraction of components, as described below. Density measurement, by refractometry, had a sensitivity of 0.002 g/ml. In some experiments the granule bands were aspirated as one fraction each. In that way, two azurophil granule fractions were obtained, a light one of density between 1.19 and 1.22 g/ml and a dense one of density 1.23-1.27 g/ml. They were diluted with 1mM EDTA (pH 7.4) to a sucrose concentration of 0.3-0.4M, centrifuged at 100,OOOxg (rmax) for 15 min, and the pellets resuspended in 0.26M sucrose and stored at -200C. Extraction of granule components: Aliquots from gradient fractions containing 0.3 mg protein were extracted with 0.3 ml of 0.13M sodium acetate (pH 4.2) at 4oC for 20 hours and centrifuged at 75,000xg (rmax> for 15 min at 4oC. The supernatants were separated (acetate extracts> and the pellets were resuspended in 0.3 ml of O.~%(W/V) CTAB in 0.15M NaCl, kept at 4OC for 30 min and centrifuged as before. The supernatants were separated (CTAB extracts) and the residues resuspended in 0.3 ml of 0.26M sucrose (non-extractable material). An average 3.6% (range O-8%) of elastase appears to remain unextracted as measured by radioimmunoassay. Treatments of isolated granules: Aliquots of the light and dense azurophil granule fractions, isolated as described above, were incubated at 370C for 5 min with different salts. The incubation mixtures contained, in a total volume of 0.2 ml, either a) granules (0.11-0.18 mg protein), 0.2M sucrose, 0.75mM EDTA, O.Ol%(v/v) Triton X-100, and CaC12 or MgC12 (pH 7.2) as stated, or b) granules (0.37 mg protein), 0.2M sucrose, 25mM Tris-HCl (pH 7.11, 0.005% (v/v) Triton X-100, and sodium or ammonium salts as stated. After incubation the tubes were cooled on ice and centrifuged at 75,000xg (rmax) for 15 min, at 4OC. Elastase in the supernatants was measured by radioixmnunoassay. Enzyme assays: 5'-nucleotidase activity was measured by the method of Douglas et al (15), as modified in our laboratory (13). Cyanocobalamin-binding protein was determined as described by Kane et al (161, and cX-galactosidase activity according to Rest et al (8). Elastase was measured by its esterase activity towards Boc-Ala-ONp (17) and myeloperoxidase by oxidation of guaiacol (18), both in the presence of O.l%(v/v) Triton X-100. Radioimmunoassays: Elastase was determined by a solid phase radioinununoassay (19). Rabbit anti-elastase antibody was coupled to microcrystalline cellulose particles according to Wide (20). Elastase was labelled with 1251 as previously described (21). The incubation mixture contained 0.1 ml of antibody-coupled particles, 0.1 ml of sample suitably diluted in assay buffer (0.05M sodium phosphate, pH 7.4, O.OlM EDTA, 0,2%(w/v) albumin, 0.02M sodium azide, 0.08M NaCl, O.~%(W/V) Tween R20 and O.~%(W/V) CTAB) and 0.1 ml of radiolabelled elastase. After overnight incubation at room temperature, the particles were washed four times with 0.15M NaCl containing O.~%(W/V) CTAB. The sensitivity of the assay is below 2 pg/ml and the variability of the duplicates was 5.8%. Electrophoresis: SDS-PAGE was carried out according to Laemmli (22). Crossed immunoelectrophoresis was performed according to Laurel1 (23), utilizing QAESephadex-purified agarose (24). Elastase was purified from normal neutrophil granulocytes as described previously (11). The three isoenzymes were separated and showed one band each on SDS-PAGE, after reduction. Antibodies were raised in rabbits against the major, most basic isoenzyme of elastase. The three iso1131

Vol.

132,

No. 3, 1985

BIOCHEMICAL

enzymes showed complete double immunodiffusion Protein determination: Rad).

AND

BIOPHYSICAL

RESEARCH

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identity towards these antibodies as evaluated by and crossed immunoelectrophoresis. Protein was measured by the Coomasie Blue method (Bio

RESULTS Subcellular

distribution

and characterization

of elastase

in unstimulated

neutrophils On subcellular dual

distribution

corresponding another

(Fig.11 to the

component

but different tually

g/ml.

of unstimulated with

light

of the

relative

no elastase

structures, 1.19

fractionation

peaks

azurophil

in the region

of 1.212

showed

and 1.244

(8).

Myeloperoxidase,

a similar

distribution

in the two granule

subtypes.

There

of the gradients

occupied

granules,

distribution

i.e.

was vir-

by membranous

at densities

of elastase

was quantitatively

of 1.12 the

m 1

Elastasc

15-:_: z-lo-

.._-

: ..

5-.

2 z s

'-

j

EIEI

Ezl

15 Myelopcroxidase n

5

10

1s

20

Fraction

25

30

35

40

number

Fig. 1 : Subcellular distribution of neutrophil elastase Elastase and myeloperoxidase enzymatic activities in each fraction are expressed as a percentage of the total in the gradient. The number of cells fractionated was 1.2x10g. Recoveries were 136 and 84%, respectively. The density proThe position of the peak acfile is shown in the upper frame (dotted lines). tivity of 5'-nucleotidase (A), cyanocobalsmin-binding protein (B) and CY-galactosidase (Cl, markers for plasma membrane, specific granules and dense is indicated. The activity of these markers azurophil granules respectively, within the density intervals shown were 61, 68 and 60% of the total for each respectively. 1132

a

g/ml,

presents

and specific

The subcellular

elastase

granules

granules,

proportions

mitochondria

at densities

and dense azurophil

neutrophils,

to

vol.

132,

No. 3. 1985

same whether

assayed

establish

the

elastase

present

quots

for

proportion

within

to ACIE,

g/ml

(fraction

that

content

in

22) and 1.24

g/ml

shows maxima

g/ml

26-27)

cide

for

(fractions both

profiles

suggests

was expected, These

tinguishable

if

total

elastase

extractable,

is considered

(Fig.2

ent,

g/ml

a greater

different

i.e.

The pro-

whereas 20-21)

at 1.21 the profile

and 1.24-

of the primary

the

granules

frac-

are not

sum of acetate

case only

two peaks

coingra-

in 8 subcellular

of primary in which

However,

the maxima do not

observed

content,

of elastase

two peaks

a),

heterogeneity

subpopulations g),

that

ali-

and by CTAB was

(fractions

The fact

of the

and CTAB. The

fractions.

presents (Fig.2

TO g/ml,

extracts.

of the

26)

and was consistently

tionations.

the

d).

these

by acetate

extracts

at 1.20

(Fig.2

acetate

proportions

same in all

(fraction

1.18-1.28

with

to the density

1.25

than

extracted

the acetate

COMMUNICATIONS

composition

interval

was extractable

of the CTAB extracts

nules

and isoenzyme

was the

according

RESEARCH

or by radioiunnunoassay.

the density

sequentially

of elastase

of elastase

activity

in SDS-PACE and the relative

different

file

BIOPHYSICAL

characteristics

were

according

substantially

AND

esterase

in granules

of elastase

isoenzymes,

its

innnunological

of each fraction

mobility the

BIOCHEMICAL

dis-

and CTAB are appar-

as in Fig.1.

Acetate

extracts

CTAB

extracts

r

Total

l-l

so- a u)3020 10 go

Ai

16

16 Fraction

Fig,

20 II. number

2 :

20

20’1428 16

Extraction of elastase from granules of resting cells and after stimulation with N-formylmethionylleucylphenylalanine Radioimunoassayable elastase contained in acetate extracts (a, b, c), CTAB extracts (d, e, f) or the sum of both (g, h, if, from gradient fractions from unstimulated cells (a, d, g) and 5 min (b, e, h) and 20 min (c, f, i) after stimulation. The number of cells fractionated was 1.6x10g in each case. Protein recoveries were 90, 93 and 94% respectively.

1133

Vol.

132,

No. 3, 1985

Degranulation

BIOCHEMICAL

whether

portion

elastase

of the

gered

with

dary

granules

(25).

and Fig.3

2).

tractable

elastase 21-23)

3 B).

The densest d,e,f

the ratio

of acetate-

progresses.

(fractions

26-28,

1.245-1.27

of the

different

shown

to CTAB-extractable

three

granule

by CTAB, also

could

be detected

isoenzymes

before

fractions,

and after

largely

(Fig.2 g/ml),

decreases

g/ml

which with

of the acetate

a,b,c

(frac-

contains

stimulation that

as degranulation

the mobility

or relative

and CTAB extracts

stimulation,

ex-

and Fig.

2 and 3 indicate

is altered

acetate

d,e,f

degranulation

in Figs. in

when the

of the g/ml),

(Fig.2

1.205-1.220

elastase

sub-

in the preportion

the

of density stimulation

The results

were

1.180-1.200

substantial

trig-

and secon-

by stimulation

after

C).

lightest

In contrast,

in the granules

No differences

portions

f).

primary stimulation

16-20,

depleted

after

(Fig.2

extractable

and Fig.3

in the

(fractions

was not

or prowere

as described

substantially

region

distribution

of both

analysed

present

gradient

place

present

decreased

predominantly

(Fig.2

COMMUNICATIONS

neutrophils

the contents

is more apparent

has taken

the relative

stimulation,

granules

CTAB extractable,

tions elastase

RESEARCH

and 5 and 20 min after

The elastase of the

A) and hence

granules

before

in

after

and their

region

is mainly

changes

secrete

Cells

(Fig.

granule

which

are

isoenzymes

fractionated section

other

there

FMLP to partially

cellularly primary

BIOPHYSICAL

studies

To investigate

ceding

AND

pro-

from

the

were

analysed

by

were

used to study

SDS-PAGE and ACIE. Release Light

from

isolated

and dense

the release valent

granules

of elastase

to fractions

They showed analysed

primary

granules

isolated

from resting

in different

conditions.

A+B and the dense

an identical

by SDS-PAGE.

ones

composition Treatment

neutrophils The light

to fraction

and relative

with

proportion

B

100 mM sodium

lOO-

granules

C as shown

are equiin Fig.3.

of proteins

or ammonium chloride

when or

C

%I-

0020-

a40-

-

20IO

OStimulation

20

10 o-

time

( min )

1 0

J

20

Characteristics of azurophil granule subtypes %&&l granule fractions of density 1.18-1.20 g/ml (A), 1.20-1.22 g/ml (B) and 1.24-1.27 g/ml (C) from unstimulated and FMLP-stimulated cells. Acetateextractable (solid bars) and CTAB-extractable (open bars) elastase was measured by radioimmunoassay. 1134

Vol.

132,

BIOCHEMICAL

No. 3, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

I

i 1.5

Fig. 4 : Effect of calcium on the release of elastase from isolated granules released from light (open bars) and Elastase, measured by radioimmunoassay, dense (solid bars) azurophil granule fractions as a function of the concentration of free calcium.

acetate, from

at pH 7.1,

the dense

tive

effect:

with

increasing

40-50%

granule

calcium

Calcium

granules

(Fig.4).

whereas

The same results cells.

up to 45% (average

from the chloride

was dissociated

concentrations,

from FMLP-stimulated

producing

of the elastase

fractions.

up to 40% of the elastase

the dense lated

liberated

primary

were

Magnesium

from

less

only

than

and 20-35%

the

light

granules

8% was released

obtained

with

partially

30%) of the release

light

had a more selec-

granules

replaced

obtained

from

with

iso-

calcium,

calcium.

DISCUSSION Our studies

show that

the

same in the

different

subgroups

and FMLP-stimulated of the isoenzymes granules. ferent

(26,27). tase with ences tial

neutrophils. of elastase

Elastase forms

work primary

of calcium of calcium

points

These

of the primary

granules

also nules

be heterogeneity as well

with

on the release is not studies

specific

on the grounds in the packaging

as in the release

acetate of this for

of their

present

respect,

in the

as the

dif-

selectively

in the association as indicated

of elasby differ-

and CTAB and by the differenenzyme from

elastase, concomitantly

that

the

in the release

to be exocytosed

subpopulations,

is released indicate

as were

in this

is

from unstimulated

FMLP resulted

to a variability with

isoenzymes

both

same proportion

granule

extracted

to 55% of the myeloperoxidase unpublished).

Stimulation

have been reported

the various

effect

of elastase granules,

from myeloperoxidase

of the latter

proportions

proportion of primary

in the

differs

The present

in the

The effect

relative

in addition

of isopycnic of the contents components

isolated

as we have

granules.

found

(R.C.Garcia

that

up

& P.Venge,

to the heterogeneity centrifugation, of the different on cell

stimulation.

there

may gra-

Vol.

132,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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ACKNOWLEDGEMENTS: This research has been funded by the Medical Research Councils of Great Britain and Sweden. We wish to thank K.Lindblad and L.Bj&kman for their excellent technical assistance and the European Molecular Biology Organisation for the award of a short-term fellowship to R.C.Garcia.

REFERENCES A., Sloan, B., Weinbaum, G., Damiano, V., Sandhaus, R.A., Elias, 1. Janoff, J. and Kimbel, P. (1977) Am.Rev.Resp.Dis. 115, 461-478. J., Giles, P.J., Porcelli, L., Reim, C.F., Baugh, R. and 2. Travis, J. (1980) Ciba Foundation Symposium 75, 51-68. Powers, A.J. (1978) Agents Actions S, 11-18. 3. Barrett, J.A. and Kelley, D.G. (1980) J.Biol.Chem. 255, 8848-8858. 4. McDonald, S.J. and Regiani, S. (1984) J.Clin.Invest. 73, 1297-1303. 5. Weiss, J.M., Vaughan, D.W., Aiken, B.M. and Kagan, H.M. (1980) J.Cell 6. Clark, Biol. 86, 102-119. R., Bretz, U. and Baggiolini, M. (1975) B., Rindler-Ludwig, 7. Dewald, J.Exp.Med. 141, 709-723. J.K. (1978) Bi0chem.J. 174, 53-59. 8. Rest, R.F., Cooney, M.H. and Spitznagel, J., Coquin, Y. and Guichard, J. (1978) Lab. Invest. 38, 9. Breton-Gorius, 21-31. K., Olsson, I. and Spitznagel, J.K. (1977) Hoppe Seyler's Z. 10. Ohlsson, Physiol.Chem. 358, 361-366. K. and Olsson, I. (1974) Eur.J.Biochem. 42, 519-527. 11. Ohlsson, Biol. 85, 42-59. 12. Segal, A.W., Dorling, J. and Coade, S.B. (1980) J.Cell R.C., Harper, A.M. and Banga, J.P. (1983) Bi0chem.J. 13. Segal, A.W., Garcia, 210, 215-225. R.C. and Segal, A.W. (1984) Bi0chem.J. 2, 233-242. 14. Garcia, A.P., Kerley, R. and Isselbacher, K.J. (1972) Bi0chem.J. 128, 15. Douglas, 1329-1338. A.V. and Neale, G. (1974) Gut l5, 953-959. 16. Kane, S.P., Hoffbrand, A., Salgam, P., Fehr, K. and BBni, A. (1981) Biochem.Pharmacol. 17. Baici, 30, 703-708. A.C. (1955) Methods Enzymol. 2, 764-775. 18. Chance, B. and Maehly, T., Olsson, I., Venge, P. and Elgefors, B. (1977) Scand.J. 19. Olofsson, Haematol. l8, 73-80. Suppl. 142, 207-221. 20. Wide, L. (1969) Acta Endocrinol., W.M. and Greenwood, F.C. (1962) Nature (London) 194, 495-496. 21. Hunter, U.K. (1970) Nature (London) 227, 680-685. 22. Laemmli, C.B. (1966) Anal.Biochem. l5, 45-52. 23. Laurell, I. and Venge, P. (1974) Blood 44, 235-246. 24. Olsson, W. and Iden, S.S. (1980) Inflammation 5, 73-88. 25. Smith, R.J., Wierenga, J.M., Pember, S.O., Barnes, K.C., Shapira, R., Spitznagel, J.K. 26. Kinkade, and Martin, L.E. (1983) Biochem.Biophys.Res.Commun. 114, 296-303. J.M. (1983) Blood 1, 1116-1124. 27. Pember, S.O. and Kinkade,

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