Secretion by mononuclear phagocytes of lysosomal hydrolases bearing ligands for the mannose-6-phosphate receptor system of fibroblasts: Evidence for a second mechanism of spontaneous secretion?

Vol. 105, No. 3, 1982 April 14, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 922-927

SECRETION BY MONONUCLEAR PHAGOCYTES OF LYSOSOMAL HYDROLASES BEARING LIGANDS FOR THE MANNOSE-6-PHOSPHATE

RECEPTOR SYSTEM OF FIBROBLASTS:

EVIDENCE FOR A SECOND MECHANISM OF SPONTANEOUS SECRETION? Wendy Jessup Cell

Received

and Roger

T. Dean,

Biology Research Group, Department of Applied Biology, Brunel University, Uxbridge, Middlesex, U.K.

January

27,

1982

8-Hexosaminidase secreted by peritoneal macrophages in response to stimulation by zymosan or NH4C1, or spontaneously by a macrophage-like cell line (p388D1), is susceptible to receptor-mediated endocytosis by human fibroThis endocytosis is almost completely blocked by exogenous mannose-6blasts. phosphate and therefore seems to depend on a mannose-6-phosphate ligand on the enzyme. It is suggested that macrophage lysosomal enzyme packaging may involve mannose-6-phosphate recognition markers, and that a continuous hypersecretion mechanism may exist which does not depend on a defect in this ligand. Considerable lysosomal from

evidence

enzymes may be important

Golgi

to lysosomes

6-phosphate receptors

recognition either

markers

A dysfunction

the

of this

fibroblasts secretion,

and thence

Such a defect

in which absent

Secretion

normal

fibroblasts,

that

the elevation

of lysosomal

enzyme molecules

into

Mononuclear range

as I-cell

insertion

(9),

to complementary complex

leads

to a failure

products

destined

accumulation disease

by this

weak bases treatment

to the Golgi in

the diversion

can be stimulated most

of which

minimum essential buffered saline.

0 1982 b-v Academic Pre.rs. of reproduction in any f‘orm

II) ligand

It

(6-8).

prevents

seems

recycling of

of newly-synthesized

vesicles.

are

to secrete ineffective

NEAA, non-essential

medium;

0006-291X/82/070922-06$01.00/0 Copyrighi .A It rizhrs

for

intervention

lysosomal

enzymes

on fibroblasts.

Inc. reserved.

922

amino

by a

The

Abbreviations EMEM, Eagle's PBS, phosphate

of

of these

The absence

(1,8).

(3).

(Mucolipidosis

by pharmacological with

to

route

of the mannose-6-phosphate

be induced

to result

secretory

phagocytes

of agents

from other

extracellular

mannose-

intracellular

mechanism

rapid

back

is believed

to bind

genetically

pH caused

receptors

receptors

sorting

for

and routing contain

of the receptor-enzyme

hydrolases

can also

retention

(2) or by a direct

such as by incubation

of mannose-6-phosphate

wide

occurs

on fibroblast

enzyme molecules

the transfer

to abnormally

ligands

intracellular

are believed

surface

an enzyme required

(5).

with

unoccupied

cell

lysosomal

enzymes. (41,

which

receptor-mediated

to segregate

carbohydrate

in their

allowing

via

that

Newly-synthesised

(1).

in the Golgi,

lysosomes,

is

suggests

acids;

Vol. 105, No. 3, 1982 correlation and

between

to

have

cause

an

the

hydrolase

normal

for their

We have

be

line

secrete

lysosomal

unstimulated in

l-cell

fibroblasts

small

(4)

(12) amounts

of

and

specific

per

24h.

We present

and

P388Dl

cells

with

that

culture

these

cells

which

(9).

lysosomal

little

is

Knowledge

packaging

may

lesions

under

release.

from medium

studies

have

may

that

(ll), (11)

of be

known

the

useful

in

and

that

may

in

10%

B-hexosaminidase

have

mechanistic such

contrast

to

normal

of

the

only

defined

cellular by

as activity

macrophages

markers, discuss

as

release is

secreted

and

the

types

recognition (ll),

cells

cell

spontaneously

than

cells

P388Dl

possible

and

of

macrophage-

that

hypersecretion

greater

other

secretions murine

cells

which

mannose-6-phosphate (13)

is

P388Dl

discussion, of

the

hypersecreting

cells

release

of

It

by

cells

this

induced

demonstrated

(10). enzymes

evidence

this

the

muscle In

contains

that

relatively

enzyme

spontaneously

smooth

fibroblasts of

such

inflammation

inflammatory

conditions

prepared

endothelial

extracellular

of

from

other or

suggests

chronic

the

lysosomal

induce

function.

enzyme.

the

implications

precede

lysosomal

with

to

macrophages,

spontaneously

of

common

fibroblasts

which

Previous

release

features

on in

and

enzymes

materials

of

B-hexosaminidase

P388Dl.

RESEARCH COMMUNICATIONS

secretion

exist

macrophage

macrophages, cell

enzyme maintenance

secretory

examined

certain

initiated

events

mechanisms

peritoneal

the

data

can

intracellular

understanding

like

in

considerable

secretion

the

of

lysosomal

role

while

AND BIOPHYSICAL

capacities

macrophage

important

However,

of

BIOCHEMICAL

in the

common

mechanistic

observation.

Methods The collection and purification of mouse peritoneal macrophages and the cultivation of the cell line ~388Dl were as described previously (10). Secreted enzymes were collected from adherent cultures (0.5 x 106 cells/cm2)following incubation in serum-free EMEM supplemented with NEAA, 100 I-U./ml penicillin and 100 pg/ml streptomycin, plus either 50mM NH4Cl or 50ug/ml zymosan (from Saccharomyces cerevisae) in peritoneal macrophage cultures. After 24h (or 5h for zymosan-treated cultures) the media were harvested, concentrated in an Amicon filtration unit with a PM-10 membrane, and dialysed against PBS. Stock cultures of normal and B-hexosaminidase-deficient (Sandhoff disease) fibroblasts were maintained in EMEM containing 10% (v/v) heat-inactivated foetal calf serum plus antibiotics. All cultures were maintained at 31°C and gassed with 5% (v/v) CO2 in air. B-hexosaminidase (N-acetylB-D-glucosaminidase, EC3.2.1.30) was assayed at pH 4.5 using 2mM 4-methylumbelliferyl-2-acetamido-2-deoxy-B -D-glucopyranoside One unit of activity is the amount of enzyme which catalyses the release (14). of 1 nmol of 4-methylumbelliferone per hour. Lactate dehydrogenase (EC 1.1.1.27) was assayed as previously (15). Protein was measured according to Lowry (16). Endocytosis of secreted enzyme was measured in confluent cultures of Sandhoff fibroblasts pre-incubated for 24h in serum-free EMBM plus NEAA, then presented with the concentrated, dialysed enzyme preparations diluted in fresh medium to 500 units of @-hexosaminidase /ml, with additions as indicated. After incubation for 24h the medium was aspirated, the monolayers washed four times with cold PBS and cell lysates prepared by adding 0.1% Triton X-100 in PBS. Results Lysosomal to

either

and Discussion hydrolases zymosan

were or

NH4C1,

obtained and

from from

the

the 923

media

medium

of of

macrophage

unstimulated

cultures p388Dl

exposed cells.

Vol. 105, No. 3, 1982

BIOCHEMICAL

AN6, BIOPHYSICAL TABLE

Lysosomal

Cell

type

enzyme

1

secretion

by mononuclear

phagocytes

Extracellular

enzyme

Stimulus

Macrophage

NH4Cl

Macrophage

Zymosan

P388Dl

None

RESEARCH COMMUNICATIONS

(% total)

@Hexosaminidase (50m.l) (5Wdml)

Lactate

Dehydrogenase

81.6

_f 1.1

26.7

+ 6.0

46.1

f 3.5

9.8

+ 8.4

30.1

2 1.4

3.7

+ 1.3

Cells biotics

were cultured in serum-free EMHM supplemented with NEAA and antiplus the secretory stimuli indicated for 24h (5h for zymosan). Medium and cells were harvested and assayed separately. Secretion is expressed as the percentage of the total (cellular + medium) activity which is present extracellularly at the end of the incubation, and is mean ? S.D. of triplicate measurements.

In

all

instances

no

equivalent

secretion

was

release

accompanied

of

secretion

of

B-hexosaminidase Sandhoff

whereas that

by

an

equivalent

recognition of

is

enzyme

3),

are

by

uptake

also

giving

shown

in

enzymes,

P388Dl

Sandhoff

fibroblasts.

cultures

is

Table

therefore

of

6-phosphate

(17) Perhaps

existence enzymes

cells. enzymes

(although some

these

which

cells

disprove types

by

also

that ligand

and

does

not that

have cells this represent

924

mechanism

a defect

reported

(18,19)

) this

and category

which

by P388Dl

the

in

the

hypermannose-

mannose-6-phosphate to

cystic

by

therefore

spontaneous

represent

(ll),

uptake

in cells of

require

been

molecule.

enzymes

fibroblasts,

these

the

fibroblast

lysosomal

p388Dl

a second

on

enzyme

dependent

of

rate fibro-

@-hexosaminidase

macrophage

I-cell

subsequent Sandhoff macrophage

p388Dl

(4,5).

is

endothelial

reports cell

that

of

the

mannose-6-phosphate

inserted

possibility Other

including recent

of

not

suggests

Endoglycosidase-H

by

proposal

the

This

carbohydrate

reduced

secretion

from

the

One

with

the

incubation,

(13).

secreted

shows

is

lysosomal

the

by

3% of

effect.

a carbohydrate

common

distinct

ligand.

to

spontaneous

ligand for

receptor-negative lysosomal

In

The

evidence

secretion

2.

little

greatly

via

to the

&hexosaminidase

spontaneously

@-hexosaminidase

mannose-6-phosphate provide

of

had

fibroblasts

of

support

fibroblasts

properties

of

uptake

further

human

those

since

endocytosed

depressed

phosphorylated

preparations

sensitive

internalised The

with

was

was

during

contain

lysis,

1).

Uptake

mannose

may

cell

dehyrogenase

secretions

2.

of

common

macrophage

(Table

macrophage

to

lactate

2mM mannose-6-phosphate

enzymes

in

due

enzyme (Table

Table

concentration

mannose-6-phosphate

blasts

in

of

lysosomal markers

digestion of

inclusion

macrophage

from

shown

than

marker

@-hexosaminidase

as

rate

rather

cytosolic

derived

fibroblasts

control

the

selective

hypersecrete

fibrosis tumour

fibroblasts cells

of

(20).

hypersecretion.

E VI

0.16

7.92

mannose-6-phosphate

mannose

2mM

2mM

+ 0.21

2 0.24

+_ 0.31 3

95

100

(5OmM NHE) % control rate

from

stimulus)

secretions

(+ secretory

phagocytc

4.59

0.13

5.31 0.12

0.24

+ 0.09

+

+

87

100 3

Macrophage (50)1g/ml Zymosan) Uptake rate 8 control

Enzyme source

mononuclear

23.8

2.7

104

f_ 0.11

* Uptake

rate

= units

internalised/mg

cell

protein/h.

rate protein/h

12

100

B control

release)

+ 0.12

f_ 0.13

rate

Uptake

23.0

(spontaneous

~388Dl

by human fibroblasts

Measurement of the endocytosis of B-hexosaminidase (500 units/ml) presented to confluent cultures of f%hexosaminidase-deficient fibroblasts was as described in Methods. In parallel experiments, the uptake of normal fibroblast B-hexosaminidase (prepared from NH4Cl-stimulated secretions) was 29.8 unitsjmg cell and inhibited to 8% of this control rate by 2mM mannose-6-phosphate.

8.38

*Uptake

Macrophage

bhexosaminidase

(enzyme only)

during period

of

None

Additions endocytosis

Endocytosis

TABLE 2

Vol. 105, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL TABLE

Effect of Prior

of prior macrophage

3

Endoglycosidase-H phexosaminidase

treatment

treatment by human

Addition endocytosis

None

+Endoglycosidase-H

RESEARCH COMMUNICATIONS

on endocytosis fibroblasts.

during

Uptake rate (% control)

None

loo

+ 2mM mannose-6-phosphate

19.4

None

23.0

+ 2mM mannose-&phosphate

9.6

Macrophage Bhexosaminidase was prepared from NH4Cl-stimulated cells as described in Methods. 10,000 units of B- hexosaminidase were incubated with 4OmU Endoglycosidase-H in O.lM citrate-phosphate buffer (pH 5.5) containing 0.01% bovine serum albumin for 120h at 37OC (total volume, 2.2ml.J. Control enzyme was incubated under identical conditions, except that Endoglycosidase-H was omitted. Endocytosis of the enzyme preparations by Sandhoff fibroblasts was determined as described in Methods.

The

rate

of

concentration units/mg cell of

cell

rates

compared

for

not

the to

of

internalised

macrophage

internalised

macrophage

is

believed

lysosomal

lysosomal

enzyme

sorting,

although which

can

enzymes. logical lysosomal

for

the

they

do

As

the

from

those

enzyme

during

the

possess

of

enzyme

is

interaction

between work

in

mannose-6-phosphate

of

or Further

mannose-6-phosphate extracellular

in

and

activity

specific

is

involved

fundamental

events

should

be

receptors

in

macrophages

926

of

is

lysosomal

by

factors is

plasma in

yet

macrophages, (21,22). lysosomal

the

physio-

including

directed

lysosomal

therefore

for

receptor

some

research

disposition

It

evidence in

and

phagocyte

system

No clear

including

more

ligand

controlled

receptors glucosamine

this

half-life

mononuclear markers.

initiated

glycoproteins,

notkaownif

the

newly-synthesised

a common

fibroblasts.

a mannose/N-acetyl

degradation

a mannose-6-phosphate

be

of

uptake

shown).

of

that

units/mg secretions

in

not

packaging

indicates

operative

macrophages

and

the

29.8

since

(data

5.3-8.4

from

rapid

period, 16h

fibroblasts

may

of

difference

recognition

with

secretion

packaging. of

types

present

the

uptake was

cell

share

of

a

was

(prepared

a particularly

The

endocytosis

function

intracellular

reflect

several

expression

it

cause

to

at

uptake)

measurement enzyme

The

).

for

parallel

unlikely

also

though

yet

our

"high-uptake" (8)

(presented

saturating

mannose-6-phosphate

cells

even

mediate

identification in

contain

these

different

available

in

involves

enzymes that

be

B-hexosaminidase

receptor.

possible

quite

is

that

enzymes

a complementary

with

fibroblast

It

known.

fi-hexosaminidase to

1OmM NH4Cl

of

It

macrophage

protein/h,

exposed is

of

established

protein/h cells

uptake

previously

towards and

their

enzymes.

role

Vol. 105, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Acknowledgements This work was supported by the Medical Research Council, the Arthritis Rheumatism Council and the Cystic Fibrosis Research Trust.

and

References M., Gonzalez-Noriega, A., Grubb, J.H., and Fischer, H-D 1. Sly, W.S., Natowicz, (1981) in Lysosomes and Lysosomal Storage Diseases. (Callahan, J-W., and eds.) pp 131-146, Raven Press, New York. Lowden, J.A., 2. von Figura, K., and Weber, E. (1978) Bi0chem.J. 176, 943-956. 3. sly, w-s., and Stahl, P. (1978) in Transport of Macromolecules in Cellular Systems (Silverstein, S., ed.) pp. 229-244, Dahlem-Konferenzen, Berlin. 4. Hickman S., and Neufeld, E.F. (1972) Biochem.Biophys.Res.Commun. 49, 992-999. 5. Kornfeld, S., Tabas, I., Varki, A., and Reitman, M. (1981) Biochem.Soc. Trans. 9, 11P. 6. Weismann, U., DiDonato, S., and Herschkowitz, N.N. (1975) Biochem.Biophys. Res.Commun. 66, 1338-1343. 7. Wilcox, P., and Rattray, P. (1979) Biochim.Biophys.Acta 586, 442-452. 8. Gonzalez-Noriega, A., Grubb, J.H. Talkad, V., and Sly, W.S. (1980) J.Cell Biol. 85, 839-852. 9. Davies, P., and Allison, A.C. (1976) in Immunobiology of the Macrophage (Nelson D.S., ed.) pp. 428-461, Academic Press, New York. 10. Jessup, W., and Dean, R.T. (1980) Bi0chem.J. 190, 847-850. 11. Hasilik, A., Voss, B., and von Figura, K. (1981) Exp.CellRes. 133, 23-30. 12. von Figura, K. (1978) Exp.Cell Res. 111, 15-21. 13. Kaplan, A., Fischer, H.D., Achord, D., and Sly, W.S. (1977) J.Clin.Invest. 60, 1088-1093. 14. Barrett, A.J., and Heath, M.F. (1977) in Lysosomes : a laboratory handbook (Dingle, J.T., ed.) pp. 19-145, North-Holland, Amsterdam. 15. Dean, R.T., Hylton, W., and Allison, A.C. (1979) Biochim.Biophys.Acta 584, 57-65. 16. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J.Biol.Chem. 193, 265-275. 17. Hosli, P., and Vogt, E. (1977) BiochemBicphys.Res.Commun. 79, 741-748. 18. Jessup, W. (1982) Biochem.Soc.Trans (in press). 19. Harris, A. (1981) Clin.Genet 20, (in press). 20. Allison, A.C. (1979) J.Clin.Path. 27, suppl. 7, 43-50. 21. Stahl, P., Schlesinger, P.H., Sigardson, E., Rodman, J.S., and Lee, Y.C. (1980) Cell 19, 207-215. 22. Faghihi-Shirazi, M., and Dean, R.T. (1982) Biochem.Soc.Trans (in press).

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