0022-1554/91/$3.

30 Histochemistry

The Journal of ©

Copyright

1991

and

Vol.

Cytochemistry

The Histochemical

by

Society.

Original

D.

and

SNOW,2

ROBERT

Department Labs,

RACHEL

Received

BRAMSON,

University

ofWashington,

Kingston,

Ontario,

November

1. 1990

University,

for publication

Relationship Between and AA Amyloid in

HENDERSON

Seattle, Canada

and amyloid

deposition

AA amyloidosis. brane-derived

Antibodies HSPG (either

demonstrated

a

and

Washington

in revised

form

sites ofHSPG

in a mouse

against

the

model

basement

of

rnernchains)

deposition

in amyloid

sites,

whereas

and

occurs

in familial ofamyloid

acid

polypeptide

amyloid

is present

Mediterranean from

protein

1972).

The

amino

terminal

76 amino two

as serum

is known

in

and

other

types

protein)

acids

of the

thirds

amyloid

various

fever,

a molecular

(AA

response appears

amyloid.

of 8500, et al.,

AA protein

whose

and The

76 amino-

designated

1976; correspond

occurring

protein

malignancy

is a unique

(Hoffman

in-

biochemically

weight

A (SAA)

of

ofsystemic

(Eriksen

of a naturally

to be an acute-phase

to underlying forms

and identified

isolated

with

A protein

known

as a secondary

distinguishable

type

27,

1991

WIGHT,

the

Em et al.,

serum

et al.,

andDepartment

ofPathology,

Richardson

and

May

16,

tive immunostaining

1991;

accepted

May

was observed

23,

1991

in these

(0A2146).

locales

with

a

polydonal antibody to the protein core of a dermatan sulfate proteoglycan (known as “decorin”). Imrnunogold labeling ofHSPGs (either protein core or GAG chains) in amyloidotic mouse spleen or liver revealed specific localization of HSPGS to amyloid fibrils. In the liver, heparan sulfate GAGS were also immunolocalized to the lysosomal compartment

of hepatocytes and/or Kupifer cells adjacent to sites of amyloid deposition, suggesting that these cells are involved in HSPG production and/or degradation. The dose temporal and ultrastructural relationship between HSPGS and AA amyloid further implies an important role for HSPGS during the initial stages of AA amyloidosis. (J Histochem Cytochem WORDS:

KEY

39:1321-1330, AA amyloid;

aminoglycans;

blood

disorders,

clinically

N.

1991)

Heparan

sulfate;

Proteoglycans;

Glycos-

P component.

no posi-

Introduction AA Amyloidosis

THOMAS

(ADS,RB,HM,TNW),

February

protein core or GAG virtually concurrent deposition of HSPGS and amyloid in specific tissue sites regardless of the organ involved (spleen or liver) or the induction protocol used (arnyloid enhancing factor + silver nitrate, or daily azocasein injections). Polydonal antibodies to AA amyloid protein and amyloid P component also demonstrated co-localization to

flammatory

MAR,

(RK).

Previous histochemical studies have suggested a dose ternporal relationship between the deposition ofhighly sulfated glycosaminoglycans (GAGs) and amyloid during experimental AA arnyloidosis. In the present investigation, we extended these initial observations by using specific iminunocytochernical probes to analyze the temporal and ultrastructural idationship between heparan sulfate proteoglycan (HSPG) accumulation

1321-1330, 1991 Printed in USA.

KISILEVSKY

ofPathology,

Queens

10, pp.

Article

A Temporal and Ultrastructural Heparan Sulfate Proteoglycans Experimental Amyloidosis’ ALAN

No.

39,

Inc.

1984).

concentration

to the protein

SAA in the

ing

increases any

Although made

about

can

be induced

such

In these

animals, liver,

function

and

kidney

(AEF)

and

rad et al., within

24-48

hr in spleen,

Sipe,

1982).

AA

by giving

repeated

(reviewed

in Kisilevsky,

within

7-10

occurs days

stimulus and

1983).

primarily

of repeated

as silver

AA amyloid

liver,

injections

in the injections

AA amyloid deposition amyloid enhancing factor

(such

animals,

to be amyloid-

models

deposition

1978).

it is believed

et a].,

1985). In addition, in animals using

In these

24 hr, dur-

1978;

or azocasein

an inflammatory 1982).

known,

(McAdam

AA amyloid

(Snow and Kisilevsky, can be rapidly induced

within

et al.,

is not

in animal

as casein

usually

(McAdam

by the liver

ofantigens spleen,

process

its normal primarily

osis

a thousandfold,

inflammatory

kidney

nitrate)

(Axel.

deposition

(Snow

and

occurs Kisilevsky,

1985). 1 Supported

by the

Alzheimer’s

ican Health Assistance Foundation, Research, and NIH grant AGO5 2

Correspondence

of Pathology,

Disease

the French

Program

Foundation

of the

Amer-

for Alzheimer’s

136.

to: Dr. Alan

Neuropathology

Research

Labs,

D. Snow, Univ. RJ-05,

Seattle,

of Washington, WA

98195.

Dept.

Previous

histochemical

studies

experimental AA amyloidogenesis poral relationship exists between can (GAG) (i.e.

,

amyloid

accumulation, enhancing

have

regardless factor

demonstrated

in a mouse initial amyloid and

ofthe silver

induction nitrate,

that,

during

model, a close ternand glycosaminoglyprotocol or daily

used

azocasein

1321

Downloaded from jhc.sagepub.com by guest on March 22, 2015

1322

SNOW,

injections),

the

the

length

alcian

tissue

(Snow

and

Kisilevsky,

ical investigations tween late

amyloid

sulfate

al.,

1987a).

In addition,

been

et al.,

and GAG(s)

and

Wight,

1989).

P component deposits

AA amyloidosis, dyes

1988;

other

than

common al.,

A close

amyloid

temporal

spleen

and

liver.

The

Materials Rapid three

and Amyloid

CBA/J

of both pared rupted

spleen

administered tion

of silver

and

0.5

mice

from -

weeks

into

each

AA

between

was prepared

using

animal, embedded

initial fixed

of each

The

ethanol and

injection was pre-

form

of a dis-

protein)

spleen and

sectioned

10%

was

A 2%

deionized

Animals

of

AEF

animal.

double-distilled

induction. in 90%

in the

ml (1 mg/mI

injection.

in paraffin,

a single

nitrate.

1982)

ofO.5

tail vein

for subcutaneous after

received silver

et al.,

injection the

each and

groups

solu-

for

The

1.5 hr at room

and processed

7.0),

tissue

either

and

0.25%

was removed,

glutaraldehyde

immersion-fixed

temperature,

washed

three

for paraffin

embedding

in

times

with

or for electron

Amyloid

received

Induction.

daily

A group

subcutaneous

of 12 CBA/J

injections

female

mice

of azocasein

(0.5

(6-8 ml of

solution) as prepared by the technique ofJanigan and Druet (1966). Groups of three animals were sacrificed at Day 5, 7, 10, 11, or 14, and the spleen and liver were removed from each animal and processed (as described above) for both paraffin embedding and electron microscopy. For both the

rapid

and

treated

traditional

amyloid

mice served

induction

Immunohistochemistry. sell et a!.,

1980)

experiments,

a group

of five

un-

as controls. An affinity-purified

and

a monoclonal

polyclonal

antibody

(MAb)

antibody

(known

(Has-

as HK-102)

(Kato et al., 1988) (gift of Dr. Koji Kimata, Aichi, Japan) to the protein core of the basement membrane-derived HSPG were used. In addition, an MAb (known as HK-249) (gift of Dr. Koji Kimata) against a glucosamine sulfate alpha 1-’4 glucuronic acid-containing determinant in heparan sulfate chains of a basement membrane-derived HSPG (Snow et al., et al.,

Koike

1987)

was employed.

1988).

Immunostaining

The

basement

membrane-derived

of these antibodies were isolated sarcoma as previously described of tissue

sections

was

from the (Kato et

accomplished

using

the

peroxidase-anti-peroxidase technique (Sternberger, 1986). The polyclonal HSPG core protein antibody was used at dilutions of 1:10, 1:30, and 1:50, whereas the two HS mAb (against either the protein core or GAG chains) were used either undiluted (as hybridoma supernatant) or at a 1:10 diluA polyclonal

Chemical

and

antibody Scientific;

against Westbury,

the

amyloid

NY)

(used

employed for immunohistochemistry To analyze nonspecific binding, controls Tris-buffered

saline

clonal

antibody

to the small

as “decorin”)

(Gloss!

was also used

to determine

AA

amyloid

using

tissue

either

protein

gen.

Isolation

periments

(TBS)

et al.,

deposits.

The

sections

1984)

(gift

of Dr. Hans

specificity

ofthe

HSPG

each

of the

with chains)

HSPG

was described

antigen

previously

primary

antibody.

proteoglycan

DSPGs

or GAG

of the

of the sulfate

whether

incubated

core

instead

dermatan

(Accurate of 1:100)

was

at the light microscopic level. consisted of sections incubated

also

with

P component at a dilution

Kresse,

accumulate HSPG

FRG)

in association

with

for the

(Kato

et al.

was confirmed

antibodies

with

used

(known

Munster,

antibodies

pre-absorbed

A poly-

(DSPG)

excess

(against HSPG

anti-

immunoabsorption

cx-

1988).

,

water,

were

sacrificed

and

liver

were

formaldehyde

at 6 tm.

KISILEVSKY

a 7%

tion.

an important role AA amyloidosis.

of amyloid,

paraformaldehyde

buffer.

fixative

old)

weeks

al.,

in both

association

induction

old)

(AEF)

(Axelrad

intravenously

20’C,

intimate

rapid

factor An

ml was used

for 24 hr at

(6-8

described

at 1, 2, 3, 4, or 7 days removed

between

WIGHT,

microscopy.

1990a;

heparan sulfate proteoglyGAG chains) and amyloid in experimental amyloidwas observed

of 3%

HSPGs used for the production mouse Engelbreth-Holm-Swarm

techniques

further suggests of experimental

For

homogenate. nitrate

(Coria 1976).

relationship

and

amyloid

in amyloid

Methods

enhancing

as previously

the

Cohen,

accumulation

fibrils stages

play

in Snow

examined

and

ultrastructural

Induction.

female

amyloid

sulfate,

protein

Skinner

temporal

may

identified

immunohistochemical

HSPG

HSPGs and AA amyloid for HSPGs in the early

they

(Snow

(reviewed

heparan

ofamyloid

1979;

and and

that

constituent

investigation,

deposition

amyloids

ofamyloidosis

type

et

of different

were used to identify and immunolocalize cans (HSPGs) (both protein core and P component to AA amyloid deposits osis.

et

(Snow et al., 1987b). as heparan sulfate, have

suggesting

To date,

ofthe

In the present

(Snow

befibrils,

pathogenesis

Holck

(i.e.,

a solution

M phosphate

Tra&tional

a pro-

alone

with

PBS (pH

accumu-

red

is the only

regardless

al.,

identified

that

inflammation

in a number

in the

be-

ruthenium

1987c,1988,1989a,1990b), role

have

tricle

the above

biochem-

MAR,

non, after 4 days ofAEF ± silver nitrate treatment, a group ofthree animals under anesthesia were fixed for 10 mm by perfusion through the left yenin 0.05

suggested a close anatomic relationship proteoglycans (PGs) and AA amyloid

demonstrated

a common

et

deposition,

in situ and in isolated fibril preparations Highly sulfated PGs and/or GAGS, such

now

and

involved

relationship

as the major

use of cationic

blue) sulfated

were

quantitative

temporal

or

of the

(Scott

GAGS

and

acute

Use

technique

experimental

during

or kidney),

1985).

sulfated

close

GAG

during

not occur

and cuprolinic tween highly

the

heparin

spleen

did

staining

Qualitative

and

and/or

in mouse

cess that

1985).

liver,

Kisilevsky,

highly

confirmed

initial

heparan

that

spleen,

,

and

chloride

) suggested

1965

(i.e.

(Snow

blue-magnesium

Dorling,

both

concerned

of induction

BRAMSON,

In addi-

Detection munostained were ing

stained to the

of Amyloid with

the

Deposits. HSPG,

for the presence method

Figure splenic

ofPuchtler

DSPG, of tissue

Adjacent

serial

or amyloid amyloid

et a!. (1962)

sections

to those

P component by either

or a polyclonal

im-

antibodies

congo

red accord-

antibody

(1:10

1. lmmunolocalization of heparan sulfate proteoglycans and amyloid P component to AA amyloid protein deposition sites in spleen. Figures represent tissue taken 4 days after mice were given AEF and silver nitrate. (A) Splenic amyloid (arrows) in the perifollicular area and white pulp (arrowhead), as shown by Congo red staining under polarized light. (B) Serial section demonstrating HSPG core protein immunostaining (polyclonal, 1:50 dilution) in the same areas as the amyloid deposits (compare with A). (C) Lack of HSPG core protein immunostaining (polyclonal, 1:30 dilution) in perifollicular area of spleen taken from untreated normal animal. A similar lack of HSPG immunostaining (for either protein core or GAG chains) was observed on Day 1 after mice were treated with AEF + silver nitrate, when no amyloid deposition was observed. (0) HSPG core protein immunostaining (MAb HK-102, undiluted supernatant) in perifollicular area of spleen (arrows) corresponding to sites of amyloid deposition (not shown). (E) Serial section from D demonstrating lack of immunostaining in perifollicular area of spleen (arrows) with a polyclonal antibodytothe DSPG core protein (1:10 dilution). (F) Sametissue section as E immunostained with the polyclonal DSPG core protein antibody (1:10 dilution) demonstrated positive immunostaining of collagen fibrils (arrowheads) associated with blood vessels outside the spleen. (G) AA amyloid protein(polyclonal, 1:10 dilution) immunostaining ofperifollicular area in spleen (arrows). (H)Serial section from G demonstrating positive immunostaining with an MAb to HSPG core protein (HK-102, 1:5) in exact areas (arrowheads) of AA amyloid protein deposition. (I) Serial section from H demonstrating positive immunostaining in the perifollicular area of the spleen (arrowheads) with a polyclonal antibody (1:100 dilution) to the amyloid P component. (J) Serial section from H demonstrating lack of immunostaining with the monoclonal HSPG core protein antibody (HK-102, 1 :5) pre-absorbed with excess HSPG antigen. Bars: A-E - 25 pm; F - 5 pm; G-J - 13 pm.

Downloaded from jhc.sagepub.com by guest on March 22, 2015

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.j

1324

SNOW,

dilution)

against

red staining,

the AA amyloid

a specific

birefringence

protein

indicator

as viewed

(Kisilevsky,

ofamyloid,

under

1987).

Positive

was demonstrated

polarized

light

congo

1A) and

by a red/green

(Puchtler

et al.,

1962).

Temporal Relationship Between Initial Amyloid and HSPG Accumulanon. Adjacent serial sections stained with either congo red to detect amyloid deposits

or antibodies

chains

(HK-249,

mine

the

and

HSPG

tional

undiluted

temporal

protein

deposition

(polyclonal,

supernatant)

of the

or time-sequence in both

1:10 dilution)or HSPG

relationship the

were

between

rapid-induction

GAG

used

to deter-

initial

amyloid

model

and

the

tradi-

immunogold

labeling

Wight,

1988).

of 3%

hydrogen

drops

mAb

core

(1:10

were

then

and

floated

on

1988).

then

rinsed

on

goat anti-rat

dilution)

gold

in TBS.

bOB

uranyl

electron

TBS.

dilution)

by rinsing

with

with

for

microscope

the

were

and

nor-

either

(a)

the

antibodies,

rinsed

citrate,

and

with

were

filtered

dried,

viewed

using

taken

perifollicular

jacent

couna JEOL

to the

Co-accumulation Amyloid Spleens

P Component taken

as expected, addition,

HSPG

ofAmyloid,

from

in

untreated

showed spleens

inability

this

no evidence (Figure

and

of the animals

1C)or

GAG

stained

with

presence

congo

red,

of amyloid.

immunostained

chain

with

antibodies

in the

2, AEF

+

deposition

perifollicular silver (by

areas

nitrate-treated positive

spleen

mice

congo

red

areas of the spleen, as previously 1985). HSPG immunostaining demonstrated in the exact

ofthe

staining)

described on adjacent

HSPG (core protein or GAG locales where amyloid was being

a close temporal relationship and HSPG accumulation.

DSPG

DSPG

was observed

antibody

control

1981)

areas

the

or due

DSPG

anti-

of collagen fibrils and vessels in the capsule of the studies

with

demonstrating

ad-

(Figure

artifact

to detect

iF). Previous

associated Orford,

antibody

ofpreparation

immunostaining with blood

(Figure

on

lack of posi-

in the perifollicular

was not an effect

of the

whereas

showed

polyclonal

(after

demonstrated

1D),

locales

immunostaining

positive DSPG tissue associated

positive

(Figure

tissue

the

animals

protein

have

demonstrated

collagen fibrils (Scott and and therefore serve as an inthat

the antibody

is effective

tissue.

serial

sections

of spleen

immunostained

core protein, demonstrated

AA amyloid, and amyloid that all three components

tissue

The

sites.

specificity

of the

HSPG

antigen

jacent

serial

failed

core

HSPG

(Figures

are localized

HSPG

to demonstrate

with

P component

1G-1I)

to the same

protein

(Figure

tissue sections pre-absorbed

any immunostaining

1J) imwith

on ad-

sections.

In

1C). At Day early

in a few

(Snow serial

areas HSPG areas

(not shown)

(see Figure

demonstrated

congo red immuno-

the

demonstrated weak to no staining in the perifollicular areas. At Day 1, the group ofrnice treated with AEF + silver nitrate showed no evidence of amyloid (by congo red staining) or HSPG deposition

same

with

lack of DSPG mice

core

or GAG chain antibodies was confirmed when munostained with the heparan sulfate antibodies

and

Spleen

animals

of untreated

core protein

HSPGs,

miusing

and concurrent all perifollicular

nitrate-treated

HSPG of HSPG

these

that DSPGS are closely Haigh, 1985; Scott and ternal

1D) areas

in a few perifollicular

silver

+

for

sections

Adjacent

Results

AEF

tive immunostaining

in

at 60 kV.

from

accumulation

serial

spleen

con-

lB and

all the perifollicular

was observed

immunostained

1 hr in

both

10 times

Samples

4 days)

or goat

for

antibodies

(Figures

throughout

spleen.

gen, since connective

were

KISILEVSKY

injections. Initial amyloid (by positive HSPG (by core protein or GAG antibody

in amyloidotic

et al., 1988; Sections

by incubation

then

HK-249

immunostaining

intensities

accumulation

1E). The

polyclonal

(Kato

at 4’C.

monoclonal

water.

lead

of 10%

(b) the

antigen

polyclonal

distilled

acetate

separate

containing

overnight

the

Grids

under

on drops

or (c) either

HSPG

and

floated

on drops

drops

followed

for

(Mar

WIGHT,

at Day 7. By Days 10, 11, and 14, amyloid immunostaining increased, now involving Spleens

on four

(1:10 dilution),

incubated

of filtered

(1:100

to 10-nm

followed

were

were

incubated 50-tl

chains,

pre-absorbed

drops

IgG

terstained

daily azocasein staining) and

microscopic

described

by rinsing

antibody

HS GAG

Sections

IgG (1:100

anti-rabbit TBS,

polyclonal against

Electron

sections

followed

Grids

antibody

et al.,

containing

5 mm,

water. protein

Snow

grids for

Level.

as previously

(20 mm)

dilution)

or monoclonal

jugated

nickel

peroxide

serum

the HSPG

was accomplished

Briefly,

of distilled goat

at the Ultrastructural

MAR,

of the spleen. A similar time-sequence relationship between tial amyloid deposition and HSPG accumulation was observed

of the Labeling

HSPG

increased

staining)

model. Immunogold

mal

to the core

concurrent

showed

BRAMSON,

amyloid

perifollicular

and Kisilevsky, sections clearly

chain) accumulation deposited, indicating

between initial amyloid deposition By Days 3, 4, and 7, amyloid (Figure

Co-accumulation ofAmyloid, HSPGs, Amyloid P Component in Liver Time-sequence ing) and HSPG

analysis ofamyloid (determined (determined by immunostaining

and

by congo red stainwith HSPG anti-

bodies) accumulation in the liver revealed initial deposition in the walls of a few central veins at Day 2 after AEF + silver nitrate and at Day 9 using the regimen ofdaily azocasein injections. With time, amyloid tral

and

veins and In addition

amyloid

concurrent throughout to the

P component

HSPG

accumulation

occurred

the parenchyma (Figures co-localization of amyloid (Figure

2D)

was also

in most

cen-

2A and 2B). and HSPGs,

immunolocalized

to

Figure 2. Immunolocalization of heparan sulfate proteoglycans and amyloid P component to AA amyloid protein deposition sites in liver. Figures represent liver tissue take on Day 4 after mice were given AEF and silver nitrate. (A) Amyloid accumulation in the wall (arrowheads) of a central vein in the liver as demonstrated by congo red staining under polarized light. (B) Serial section from A immunostained with a polyclonal antibody (1:10 dilution) to the HSPG core protein. HSPG core protein is primarily localized to sites of amyloid deposition (compare with A). (C) AA amyloid protein immunostaining (polyclonal, 1:10 dilution) in walls of central veins (arrowheads) and parenchyma in liver. (D) Serial section from C demonstrating amyloid P component immunostaining (polyclonal, 1 :100 dilution)

walls of central veins and in exact areas of AA amyloid protein deposition (compare with C). (E) Heparan sulfate GAG undiluted supernatant) within hepatocytes and Kupffer cells in liver (arrows), in areas that do not contain amyloid. (F) Higher HS GAG chain immunostaining within hepatocytes andr Kupffer cells in liver (arrows). (0) Serial section from E demonstrating cytes and/or Kupffer cells usingthe HK-249 monoclonal heparan sulfate GAG chain antibody pre-absorbed with excess HSPG staining (HK-249, undiluted supernatant) within hepatocytes andkr Kupffer cells (arrowheads) in areas adjacent to positive (arrows). Bars: A-E.G = 25 pm; F,H - 6.3 pm. in

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chain immunostaining magnification

from

(MAb HK-249, E demonstrating

lack of immunostaining

in hepato-

antigen. (H) HS GAG chain immunostaining of amyloid

immunodeposits

AA

AMYLOED

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AA amyloid heparan HK-249)

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amyloid

the

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in the liver (Figure

2C).

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(Figures

deposits

HS GAG

HSPG

2E

(Figure

chain

antigen

and

2H).

2F)

Serial

antibody

and

(HK-249)

demonstrated

in

sections

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areas

adjacent

deposition

in Alzheimer’s

that

was observed

excess

years

in these

areas,

tamed

heparan

lation

in conjunction

with

study,

histochemical

staining

(compare

Fig-

and

older.

These

Sulfate Amyloid

Immunolocalization

Proteoglycan Core Protein to AA Fibrils in Spleen and Liver

Ultrastructural silver

analysis

nitrate

tein antibody

of amyloid Immunogold

HSPG

(not

core

amyloid fibrils (Figures core protein antibody absorbed (Figure

body

with 4D).

excess

Immunogold revealed

liver (not

(not

AEF

and liver(Figure The

antigen the

sulfate

In the of central core pro-

to amyloid 3B). In many

decorated

individual

specificity when this

failed

+

in between

shown).

localized

antibody

3B and 4C). was confirmed

Heparan

fibrils

areas

specifically

shown)

labeling with immunolocalization

shown).

initial

accumulated in the walls labeling with the HSPG

protein

HSPG

4 after

amyloid

HSPGS

the spleen

the

at Day

in perifollicular

demonstrated

in both

instances

spleen

demonstrated

cells

large amounts (Figure 3A).

fibrils

of the

treatment

reticuloendothelial

liver, veins

of Heparan

to label

of the antibody

HSPG pre-

amyloid

fibrils

HK-249 HS GAG chain antito amyloid fibrils in spleen and GAG

chain

immunostaining

was

also observed within the cytoplasm of hepatocytes or Kupffer cells adjacent to amyloid (Figures 4A and 4B). The gold particles were primanly located within vesicles within these cells (Figures 4A and 4B).

present

investigation

ultrastructural tein

in a mouse

chemical

has demonstrated

relationship model

observations

core

and

and

and

by Snow

demonstrated amyloid

Analogous

studies

HSPGS

Down’s

titative

assessment

present

study

of the amyloid

this

and

syndrome of events

accumulation study have

other

and

may

not

demonstrated

relied

HSPG

amyloid

proteins.

localized

to AA amyloid

with

amyloidosis.

AA

ofthe specific protein sites. Although

This

HSPG

The

DSPGs

The

as a model

beta-amyloid

protein

tionship

to study

amyloidotic

and

mouse

tissue

spleen (Snow

chains

with intact

the AA HSPG.

to chondroitin

sul-

present

antibody

did,

Scott

be due

with

no deposicontaining however,

and

Ordford,

1981;

was capable

of de-

ofthe

in AA

DSPG

epitopes

(i.e.

, amyloid

present

to lack

deprevi-

immunostaining

to masking components

simply

liver, sites

an observation

the antibody

lack of DSPG

be due

investigation to AA amyloid

of DSPGs

in AA

sites.

localization specifically

employing between

The

GAG

in conjunction

fibrils, 1985;

that

or other

deposition

the

protein

Haigh,

The

or may

ous studies

patients

(BAP)

and

antigen.

chains

PG

or quan-

be made.

and

core protein

collagen

indicated

ultrastructural

GAG

with

occurred

core

protein

in

GAGs AA

of the

could core

in both spleen and was detected in tissue

with

(Scott which

P component), amyloid

agrees ofHSPG

DSPG

associated

amyloid

ages were used

digestion

protein

im-

demon-

no qualitative

antibodies

accumulation

by the

involving

chains and

previously

on

the protein

used

co-localization

sites may

at various

GAG

HSPGs to show that only HSPGs were protein in the kidneys ofhuman patients

DSPG

In one study,

and

(Snow

and

amyloid

relationship

core

both

recently

sulfate,

mouse

a temporal

suspected

biochemical

(1987a)

analysis,

that

et al. (1988) dermatan

and

deposits containwith Alzheimer’s

HSPG accumulate in the tissue concurrent protein, and are most likely in the form ofan

Norling

deposition

et al.

study

tecting

to AA amyloid-

In a similar demonstrated

core protein

GAG

in experimental

AA amyloid

HS accumu-

blue)

previous

latter

1980),

be unique

that event.

was previously

with

Snow

of the

shows

35 con-

1989). These studies, in suggest that heparan

,

before

described

HSPG

sequence

Since

by papain

Scott,

in the present

the

amyloidosis.

ously

initial

(Verga et al. investigation,

aged also

a 16-fold increase in heparan sulfate and/or heparin mouse spleen during the early stages of experimental

histo-

between

alcian

ofHSPG

Kisilevsky (1985). that HSPGs (both

deposition

In

sections

implying

(using

agrees

studies.

core proteins

fate,

patients

serial

is an early

PGs than

deposits

munocytochemical

tect

observed

with

to AA amyloid

AA amyloid.

previous

1990a).

is an early event in the pathogenesis of amywould confer even greater significance for

the role ofthese particular and Wight, 1989). The specific localization

and

occur concurrently regardless ofthe organ involved or the induction protocol used (i.e. , AEF + silver azocasein injections). This temporal relationship

between

This

accumulation generally.

pro-

AA amyloidosis (spleen or liver) nitrate or daily

osis.

sulfate loidosis

strated in the

on

GAGs localized to pre-amyloid cerebral gray matter of patients syndrome the present

temporal

extending

made

techniques GAGs)

and

KISILEYSKY

et al.,

syndrome

deposits

the BAP

disease or Down’s conjunction with

AA amyloid

a close

HSPGS

of amyloidosis, initially

Immunohistochemical protein

between

WIGHT,

(Snow

immunoreactivity,

increased amyloid deposition tion of DSPG core protein

Discussion The

cortical

sulfate

highly sulfated ing BAP in the

Ultrastructural

disease

in Down’s

with

HS antibody

MAR,

such patients the earliest deposition ofBAP was observed as “amorphous” or “diffuse” cortical deposits in the brains of patients aged 18 and 24 years. This occurred before the accumulation of fibrillar amyloid

with

pre-absorbed

of the

to

immunostained

no immunostaining

again indicating the specificity ure 2G with Figure 2E).

HSPG

Furthermore,

sulfate GAG chains were immunolocalized to the cytoplasm ofhepatocytes and Kupffer

BRAMSON,

cationic

sulfated

and et al.,

of heparan

to amyloid dyes

PGs

liver,

and

to analyze AA

in isolated

1987b),

fibrils

and

sulfate agrees

core

the structural

amyloid

fibrils

fibril

preparations

in human

protein

with in

amyloidotic

previrelamouse

from liver

Figure a Ultrastructural immunolocalization of heparan sulfate proteoglycan core protein to AA amyloid fibrils in liver. (A) Large quantities of amyloid (a) accumulation in the wall of a central vein in the liver, Day 4 after AEF + silver nitrate treatment. (B) Higher magnification of area of amyloid accumulation from A, demonstrating HSPG core protein immunostaining (polyclonal, 1:10 dilution). In many instances, 10-nm gold particle decorate individual amyloid fibrils (arrowheads), demonstrating a close relationship between HSPG core protein and amyloid fibrils. Bars: A - 2 pm; B = 8 pm.

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Figure 4. Ultrastructural

of HSPGs (core protein and GAG chains) in liver amyloid and in cells adjacent to amyloid accumulation. (A) lmundiluted supernatant) to heparan sulfate GAG chains demonstrates large amounts of amyloid accumulation within containing HS GAG chain immunostaining are located in the vicinity of amyloid deposits. (B) Heparan sulfate GAG supernatant) of amyloid fibrils (a) and within vesicles (arrowheads) of adjacent cells. (C) Immunodetection of HSPG core protein (polyclonal, 1:10 dilution) localized to amyloid fibrils in liver, Day 4 after AEF + silver nitrate treatment. (D) Lack of immunostaining of amyloid fibrils using the HSPG core protein antibody (polyclonal, 1:10 dilution) pre-absorbed with excess HSPG antigen. This demonstrates the specificity of the HSPG core protein antibody. Bars: A,B = 0.22 pm; C 0.6 pm; D 0.3 pm. immunolocalization munogold labeling with the MAb (HK-249, the cytoplasm of hepatocyte. Hepatocytes chain immunostaining (HK-249, undiluted

(Young

et al.

(ruthenium tural

,

1989).

relationship

amyloid

In

red and fibrils,

these

exists both

in situ

suggests

sociated

with

The

presence

cells and/or level

that amyloid

for the synthesis

and

HSPGs

suggests

two

indicated highly

or parts

cationic

that

sulfated

in isolated

with specific anatomic and

fibril

heparan structural thereof

PGs

or

reagents

a close

struc-

and

preparations.

intimately

cells,

which

either

of HSPGs

in vesicles

was observed that

that

in the cytoplasm

these

accumulate

may

asof liver

in liver

latter

made

study,

degraded

ofthe

servations

be responsible

in AA amyloid

are

is part

Use

at the ultrastructural

cells

HSPGs

in a cell compartment

sulfate antibodies relationship and are

that

observed

AA

fibrils.

of HS GAGs

Kupffer

in liver,

studies,

blue)

between

of immunogold labeling has now confirmed this further

latter

cuprolinic

by these

degradative within GAG

and

brain

chain

(Snow

antibody

level, to neuronal to be age-related

sions

lysosomes

(Tsuchida

present

investigation

from

accumulation

of HS GAGS

et al.

of recent ,

1990a).

(HK-249)

GAGS, at the ultrastructural These granules are believed derived

the

The finding

liver cells is reminiscent

in Alzheimer’s

the same

cells

process.

localized

lipofuscin intracellular

et al., 1987; Sohal

ob-

In this

HS

granules. incluand Wolfe,

1986). Evidence

localization (Duong

in the

ofamyloid et al., 1989;

Downloaded from jhc.sagepub.com by guest on March 22, 2015

P component, Coria

also

agreeing

et al., 1988;

Holck

demonstrates

with et al.,

the

co-

previous

studies

1979;

Skinner

AA

AMYLOID

and

Cohen,

AND

1976).

P component in the present licular

deposition 1979;

tion

ofP

heimer’s

Cohen,

deposits

Down’s

sulfated suggests

splenic

1976).

In a recent

in the cerebral

syndrome

patients

demonstrate

recent

studies

of amyloid P component for and therefore the accumulation deposits

may

be

The full significance ation between Nevertheless, of(a)

tein

into

a beta-pleated

tant

ingredient

and/or

of Alz-

to contain

both

due

to

the

of

and

ultrastructural

a role

of these

time.

Nevertheless,

may

be an important

1987),

in AA

processing ofthe

(b)

HSPGs

ofthe amyloid as an

the site of ultimate

amyloid

AA proimpor-

depo-

stabilizing amyloid once formed and to occur (reviewed in Snow and Wight,

possibilities

is correct

the present factor

investigation

in the early

is not

known

implies

that

sequence

ofevents

at this HSPGs leading

deposition.

We would/ike

to thank

Dr Koji

for

the

MA, Kisilevsky

R, WillmerJ,

Ericksen N, Ericsson arnyloid protein-AAman and monkey

cultured

Beck

(decorin)

5), Skinner

factor.

disease

and other

EC, Scheibel in Alzheimer’s

antibody

Lab

M (1982):

Invest

H (1984):

fibroblasts. heparan

amyloidosis.

AB (1989): Immunodetection disease. Acta Neuropathol(Berl)

of the 78:429

Glenner GG (1972): Amino acid of unknown origin. J Biol Chem

ofproteodermatan 2 59:14144

association

and

EP (1976): Mouse protein of huSci USA 73:964

of human

dermatan

sulfate

serum

sulfate.

amyloid

) Biol Chem

262:1456

LH, Eriksen

protein

one of two serum

amyloid

H, WilczekJ,

Rennard

Isolation of a heparan sulfate-containing proteoglycan brane. Proc Nat! Acad Sci USA 77:4494

5, Martin from

GR (1980):

basement

mem-

N, Walsh

KA, Benditt

NH2-terminal

protein

sequence

(apoSAA)

EP (1984): identity

Muonly

with

gene products.

Holck M, Husby G, Sletten K, Natvig)B (1979): nent (protein AP): an integral part of the amyloid munol 10:55 )anigan

D’r,

ity and

rapid

RL(1966):

Druet

Experimental

induction.

Am

) Exp Med

) Pathol

The

amyloid

P compo-

substance?

amyloidosis.

Scand ) Im-

Role

of antigenic-

48:1013

Kato M, Koike Y, Suzuki 5, Kimata K (1988): Basement membrane proteoglycans in various tissues: characterization using monoclonal antibodies to the Engelbreth-HoIm-Swarm mouse tumor low density heparan sulfate proteoglycan. J Cell Biol 106:2203 Kisilevsky

R (1987):

From

arthritis

to Alzheimer’s

disease:

on the pathogenesis of amyloidosis. Can J Physiol Kisilevsky R (1983): Amyloidosis: a familiar problem pathogenetic developments. Lab Invest 49:381

current

Glycoconjugates,

65:1805

in the light

of current

B8

Mar H, Wight

TN (1988): Colloidal gold immunostaining sections. J Histochem Cytochem 36:1387

ultra-thin

concepts

Pharmacol

M, Suzuki 5, Kimata K(1987): A monodonal antibody against sulfate of EHS-tumor proteoglycan. Proc IX Int Symp on

Y, Kato

the heparan

on deplasticized

McAdam

KPWJ, Elm RJ, Sipe )D, Wolff SM (1978): Changes amyloid-A and C-reactive protein after etiocholanolene-induced flammation. ) Clin Invest 61:390

in human in-

serum

McAdam

Selinger

KPW), Li), Knowles), Foss NT, Dinarello CA, Rosenwasser U, MJ, Kaplan MM, Goodman R (1982): The biology ofSAA: iden-

tification ofthe with monoclonal

inducer, in vitro synthesis, antibodies. Ann NY

and Acad

heterogeneity Sci 389:126

demonstrated

Norling B, Westermark GT, Westermark P (1988): Immunohistochemical identification ofheparan sulphate proteoglycan in secondary systemic yloidosis. Clin Exp Immunol 73:333

Scott

H, Sweat F, Levine M (1962): On the binding J Histochem Cytochem 10:355

)E (1980):

Collagen-proteoglycan

in tendon

Haigh M (1985): and non-calcifying

Scott

Orford

of the

study.

Snow AD, Kisilevsky

Localization J 187:887

proteoglycan

serum

protein

Br ) Exp Pathol

59:305

Aspects of the amyloid Amyloidosis. New York,

Lab

Invest

R, Stephens

red by of pro-

fibril interactions Rep 5:71

sulphate-rich

acute-phase

am-

glycosaminoglycans Hiswchemistry 5:221

collagen Biosci

R(1985): Temporal relationship and amyloid deposition during

A histochemical

of Congo

at the d band in the gap region.

synthesis.

M, Cohen AS (1976): 0, Pasternack A, eds.

Snow AD, Kisilevsky glycan accumulation

staining ofacid in salt solutions.

Dermatan

collagen

SipeJD (1978): Induction both RNA and protein

Biochem

Proteoglycan-type-I connective tissues.

CR (1981):

ates with rat tail-tendon J 197:213

Skinner Wegelius

microscopy.

(1965): Differential by alcian blue

ScottJE, in bone JE,

interactions.

by electron

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associ-

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In 339

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53:37

C, Anastassiades

ization of tissue and plasma glycosaminoglycans amyloidosis and acute inflammation. Qualitative sis. Lab Invest 56:665

T (1987a): during and

Characterexperimental AA quantitative analy-

Snow, AD, Kisilevsky R, Willmer), Prusiner SB, DeArmond 5) (1989a): Sulfated glycosaminoglycans in amyloid plaques in prion diseases. Acta Neuropathol (Berl) 77:337 Snow AD, Mar H, Nochlin

Robey PG. Barrach

AA:

159:641

osis.

Biosynthesis

sulfate

47:139

types of cerebral

J Biol Chem

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Further

Duinen 5, Shelanski of amyloid P corn-

LH, Pearshall N, LagunoffD, Benditt homology with nonimmunoglobulin amyloid substance. Proc Nat! Acad

M, Kresse

human

Hamazaki H (1987): P component with HassellJR,

amyloid

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Chen

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in

DSPG

E, Prelli F, Larrondo-Lillo M, Van B (1988): Isolation and characterization

T, Pommier P component

Gloss!),

Ericsson

tissue

ScottJE, DorlingJ (mucopolysaccharides)

ML, Frangione ponent from Alzheimer’s Lab Invest 58:454 amyloid

the

antibodies.

of amyloid-enhancing

F, Castano

Duong

Kressefor

HSPG

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Dr Hans

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the

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(c) HSPGs its degradation

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playing

latin

amyloid proteins at a later ofthe amyloid fibril. Fura specific

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HSPGS

SAA,

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precursor

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thermore,

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