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From the Institutes of Pathology*
and
Immunology,
Otto-von-Guericke-University,
Magdeburg, Germany; and the Mt. Sinai Medical
School,
New York, New York
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophages (M
) are commonly found in many different amyloid
diseases, including AA and AL amyloidosis, and it has been proposed
that they are involved in amyloidogenesis. They synthesize a broad
range of proteases that may process the precursor protein to generate
the fibril protein, and they may also be involved in the degradation of
the deposits.2-4
Multinucleated giant cells (MGCs) are
considered to be a specific phenotype of M and they are also,
although less commonly, found in amyloid deposits and often show an
intimate spatial relationship to the deposits.5-9
Earlier
animal studies provided evidence that MGCs might be involved in the
resorption of amyloid
deposits.10
We have previously found that MGCs, like osteoclasts, selectively
express cathepsin K. The expression of cathepsin K in MGCs was not
restricted to a specific disease and was found in tuberculosis,
sarcoidosis, sarcoid-like lesions, and foreign body reactions
(Röcken C, Bühling F, unpublished observation).
Cathepsin K belongs to the papain family of cysteine proteases and has
unique properties.11,12
In adults it is expressed in
osteoclasts and their mononuclear precursors, in bronchial epithelium,
bile duct epithelium, M, and smooth muscle cells of atherosclerotic
arteries, in synoviocytes of patients suffering from rheumatoid
arthritis, and also in breast carcinoma cells.13-18
It is
not expressed in M under normal conditions.13
Cathepsin
K is the most potent mammalian elastase yet described and it has been
shown to possess unique collagenolytic activity. This activity does not
depend on destabilization of the triple helix, but rather it cleaves
type I and II collagen at the ends (telopeptide) and at multiple sites
within the native triple helix.12,19
Other proteinases
with collagenolytic activity cleave collagen at the telopeptides only
(eg, cathepsins L, B, and S) or within the molecule at a single site
(eg, MMP-1, -2, -8, and -13).12
Cathepsin K also shows
gelatinolytic activity in the pH range 4.0 to 7.0 and it may function
both intracellularly and extracellularly.19
The stability
of cathepsin K is modulated by glycosaminoglycans; in the presence of
chondroitin 4-sulfate there is an increase in the hydrolysis of soluble
and insoluble collagen by cathepsin K.20
The role of
cathepsin K in bone turnover is well established; mutations in the
cathepsin K gene lead to pycnodysostosis and cathepsin K knock-out mice
develop osteopetrosis.21-23
As yet there has been little
investigation into the putative role of cathepsin K in other diseases.
Our previous observations have shown that cathepsin K is expressed not only by osteoclasts, but also by MGCs in general. Therefore we performed a study to determine the presence of cathepsin K in MGCs adjacent to amyloid deposits and to investigate its ability to degrade fibril proteins in vitro, hence indicating a possible involvement in the pathology of amyloidoses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and heparan sulfate were purchased from Sigma (Deisenhofen, Germany). Serum amyloid A-rich high-density lipoprotein was obtained from Calbiochem-Novabiochem GmbH (Bad Soden, Germany). Recombinant human cathepsin K was produced as previously described.24
Case Selection
Fifty-three archived, formalin-fixed, and paraffin-embedded
autopsy and biopsy specimens from 23 patients with histologically
proven AA or AL amyloidosis were used in this study. Age, sex
distribution, and basic diseases are summarized in Table 1
. The classification of amyloid was
based on immunohistochemistry and clinical history as described
elsewhere.1,25
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Twelve patients had suffered from AL amyloidosis. The
immunohistochemical classification showed that the deposits consisted
in five patients of 
-light chain and in three of 
-light
chain.26,27
The amyloid deposits of four patients stained
for amyloid P component only; two of these patients had suffered from a
plasmacytoma whereas the other two had no disease commonly associated
with either AA or AL amyloidosis. These cases were classified as
myeloma-associated or primary AL amyloidosis, respectively, as
described in detail elsewhere.26
Specimens of both liver
and kidney were available from eight of these patients, from the spleen
in six patients, and from the tonsil, breast, lymph node, and lung each
in one patient. Large interstitial and occasional vascular deposits of
amyloid were found in every case.
Additionally, unfixed amyloid-containing tissue was available from a single patient with generalized AA amyloidosis and this was used for the preparation of amyloid fibril proteins and degradation experiments with recombinant cathepsin K as described below.
Two of the 31 patients have previously been published in detail as case reports.28,29
Light Microscopy
Deparaffinized serial sections from formalin-fixed tissue
were used to immunolocalize MGCs and cathepsin K. The presence of
amyloid was demonstrated by the appearance of green birefringence from
alkaline alcoholic Congo Red staining under polarized
light.30
Immunostaining was performed using monoclonal
antibodies directed against AA amyloid (monoclonal; dilution, 1:500),
and CD68 (1:5,000), as well as polyclonal antibodies directed against
-light chain (1:7,500), amyloid P component (1:1,600; all Dakopatts,
Hamburg, Germany), and cathepsin K (1:1,000).14
Before
immunostaining the specimens were pretreated with 10 mmol/L
ethylenediaminetetraacetic acid (EDTA) (2 x 10 minutes, 450 W
microwave oven; CD68, amyloid P component) and 0.5 U/ml protease I (16
minutes; -light chain; Ventana, Strasbourg, France).
Immunoreaction was visualized with the avidin biotin complex method
applying a Vectastain ABC alkaline phosphatase kit (distributed by
Camon, Wiesbaden, Germany) or UltraTech HRP streptavidin-biotin
universal detection system (Immunotech, France). Neufuchsin and
3,3-diaminobenzidine-tetrahydrochloride, respectively, served as
chromogens. The specimens were counterstained with hematoxylin.
The specificity of immunostaining was controlled using specimens containing known classes of amyloid (AA amyloid), using positive controls recommended by the manufacturers (remaining antibodies), using normal rabbit serum instead of primary antibody, and by omitting the primary antibodies.
Immunostaining was evaluated using an Axioskop microscope from Zeiss (Jena, Germany).
Electron Microscopy
For electron microscopy specimens from a tonsil containing AL amyloid and multiple MGCs were fixed in a mixture of 2% formalin/2.5% glutaraldehyde (pH 7.2, overnight, 4°C) and then in 3.125% glutaraldehyde (7 hours, 4°C). Following standard procedures of tissue processing for electron microscopy, the specimens were finally embedded in Lowicryl using the K4M kit (Plano, Wetzlar, Germany). Specimens for postembedding immunoelectron microscopy were not postfixed with OsO4. Polymerization took place throughout 24 hours at -30°C and was initiated by UV light. Semithin sections (1 µm) were stained with toluidine blue. Ultrathin sections (80 to 120 nm) were mounted on copper grids and counterstained with 3% aqueous uranyl acetate (30 minutes, room temperature) and contrasted with 1% aqueous lead citrate (15 minutes, room temperature).
For postembedding immunoelectron microscopy, ultrathin sections (120 nm) were mounted on formvar-coated nickel grids (200 mesh, Plano). Immunoelectron microscopy was performed in triplicate as described previously.25 The specificity of immunostaining was controlled by omitting the primary antibody. The sections were air-dried and examined using a Zeiss EM900 electron microscope.
Preparation of Amyloid Fibril Proteins
Amyloid fibril proteins were prepared as described by Skinner and
colleagues.31
Briefly this was as follows: 
6 g of
amyloidotic tissue, which had been stored at -80°C, was homogenized
in 60 ml of aqueous 0.9% NaCl solution for 30 seconds using an
Ultra-Turrax. The homogenate was centrifuged at 2,500 x
g for 30 minutes at 4°C. The supernatant was discarded and
the pellet was resuspended in 60 ml of 0.9% NaCl for 30 seconds,
centrifuged, resuspended for 15 seconds in 60 ml of sodium citrate
buffer (0.05 mol/L sodium citrate, 0.01 mol/L Tris, pH 8.0), and
centrifuged again. Subsequently the supernatant was discarded and the
pellet was resuspended in 60 ml of 0.9% NaCl solution for 15 seconds
and centrifuged. The last washing step was repeated until the optical
density (OD 280) of the supernatant was <0.1. Thereafter the pellet
was resuspended in 50 ml of deionized H2O
(dH2O) and centrifuged for 200 minutes at
2,500 x g (4°C). This step was repeated three times.
The supernatants collected from the last three washing steps contained
amyloid fibril and precursor proteins (AFPPS) and
they were stored in aliquots at -20°C until further use (see below).
The final pellet had a whitish top layer and also contained both
amyloid fibril and precursor proteins (AFPPP) as
demonstrated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and Western blotting. This was also stored
at -20°C until further use.
Degradation Experiments
In vitro degradation experiments with recombinant cathepsin K were performed as follows. Samples from the pellet containing amyloid fibril and precursor proteins (AFPPP) were dissolved in water by heating for 30 minutes at 100°C. Samples from the supernatant containing amyloid fibril and precursor proteins (AFPPS) were concentrated in a speed-vac without heating. Both AFPPP or AFPPS were then dispersed at a concentration of 5 mg/ml in 100 mmol/L sodium acetate buffer (substituted with 5 mmol/L EDTA, 2.5 mmol/L dithiothreitol, pH 5.5) or 50 mmol/L Tris-HCl buffer (substituted with 5 mmol/L EDTA, 2.5 mmol/L dithiothreitol, pH 7.4). Degradation was performed at 37°C and was started by the addition of cathepsin K (0.8 nmol/L). The reaction was stopped by the addition the cysteine protease inhibitor E-64 (200 µmol/L). Omission of cathepsin K and incubation in the presence of 200 µmol/L E64 served as a control. All experiments were performed in triplicate.
In situ degradation experiments were performed using frozen sections (8 µm) of unfixed amyloidotic tissue. Specimens were shortly hydrated in 100 mmol/L of sodium acetate buffer (substituted with 5 mmol/L EDTA, 2.5 mmol/L dithiothreitol, pH 5.5). Degradation was performed at 37°C and was started by the addition of cathepsin K (0.8 nmol/L) in 100 mmol/L sodium acetate buffer. The reaction was stopped by jet-washing in 50 mmol/L Tris-HCl buffer and the specimens were fixed in 3% p-formaldehyde (substituted with 5% CaCl2) for 5 minutes. Immunostaining was performed as described above. Omission of cathepsin K and incubation in the presence of 200 µmol/L E64 served as a control.
SDS-PAGE and Western Blotting
Proteins were resolved in 16.5% polyacrylamide gels according to Schägger and von Jagow32 and visualized by staining with Coomassie blue or silver stain.33 Using Western blotting, proteins on unstained polyacrylamide gels were transferred onto a polyvinylidene difluoride membrane (Immobilon-PSQ, pore size 0.1 µm; Millipore, Bedford, MA) using the tank-blotting system from Bio-Rad Laboratories (München, Germany) according to the manufacturer’s instructions. Transferred proteins were visualized by Coomassie blue staining. Immunostaining of transferred proteins was performed using the Vectastain ABC alkaline phosphatase kit (Camon) and antibodies directed against AA fibril protein (monoclonal, clone mc1) and cathepsin K. Immunostaining was visualized with BCIP/NBT (BioTrend Chemikalien GmbH, Köln, Germany). N-terminal amino acid sequencing was performed by WITA GmbH (Teltow, Germany).
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Results |
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In six (55%) patients with AA amyloidosis and seven (58%)
patients with AL amyloidosis, a variable number of multinucleated
CD68-immunoreactive foreign body-type giant cells was found adjacent to
amyloid deposits (Figure 1)
. Some of
these had a vacuolated cytoplasm and asteroid bodies. Cases of AL
amyloidosis occasionally showed intracellular immunostaining for
-light chain as described previously.29
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Cathepsin K was detected in 41 (77%) of 53 specimens from 22
(96%) patients. Immunostaining of variable intensity was found in
thyroid epithelium, bile duct epithelium, renal tubular epithelium,
polymorphonuclear cells, smooth muscle cells of vessel walls, and
fibroblasts (not shown). Except for thyroid epithelium, none of these
cells showed a spatial relationship to amyloid deposits. However,
strong immunostaining for cathepsin K was found in MGCs adjacent to
amyloid deposits in six (55%) patients with AA amyloidosis and seven
(58%) with AL amyloidosis. The number of immunoreactive MGCs varied
and some were immunonegative (Figure 1)
. Immunostaining of amyloid
deposits was present in five (45%) patients with AA amyloidosis and
three (25%) patients with AL amyloidosis and it was mainly confined to
deposits adjacent to or surrounded by MGCs (Figure 1)
. AA amyloidosis
showed cathepsin K-immunoreactive MGCs in the spleen (four patients),
liver (three patients), and kidney (two patients). AL amyloidosis
showed cathepsin K-immunoreactive MGCs in the liver of two patients,
and in the kidney, spleen, tonsil, breast, lymph node, and lung each of
one patient. No differences were found between amyloid deposits of -
and -light chain origin.
No immunostaining was found using normal rabbit serum instead of primary antibody, or after omission of the primary antibody.
Electron Microscopy
Electron microscopy of ultrathin sections from a tonsil containing
AL amyloid showed rigid nonbranching amyloid fibrils of 10 to 12 nm
diameter and indefinite length, and MGCs with abundant vesicular
storage granules. Postembedding immunolabeling with the antibody
directed against cathepsin K yielded immunostaining that was closely
spatially related to amyloid fibrils (Figure 2)
. Additional intracellular
immunostaining was found in the storage granules of MGCs (Figure 2)
.
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Unfixed frozen sections of spleen with green birefringent AA
amyloid deposits were incubated for 4 hours with 0.8 nmol/L cathepsin K
at pH 5.5, both with and without E64. Incubation without cathepsin K
served as a further negative control. The results of in situ
proteolysis were controlled by immunohistochemistry using antibodies
directed against AA amyloid and amyloid P component. Without protease
pretreatment the amyloid deposits showed immunostaining of the margins
only (Figure 3)
. The staining pattern
obtained with the antibody directed against AA amyloid changed after
pretreatment with cathepsin K in that immunostaining was also observed
in the center of the deposits (Figure 3)
. Co-incubation with E64
reversed this effect of cathepsin K. Immunostaining for amyloid P
component was unaffected by cathepsin K pretreatment and was
predominantly confined to the margins of the deposits (Figure 3)
.
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In vitro degradation experiments used amyloid fibril
and precursor proteins (AFPP) prepared from the spleen of a patient who
had suffered from generalized AA amyloidosis.28
After
SDS-PAGE and Western blotting (see below) two fibril proteins of 5.8
and 6.9 kd were identified (Figure 4)
.
N-terminal sequencing showed that both fibril proteins had identical
N-terminal ends (SFFSFL) homologous to the N-terminal region of serum
amyloid A starting at position 2. In addition,
AFPPS and AFPPP contained
trace amounts of precursor protein (SAA) and several bands that were
larger than 12 kd as shown by SDS-PAGE and Western blotting (Figure 4)
.
These bands were interpreted as oligomeric fibril proteins because they
disappeared after prolonged heat-denaturation and the amount of
detectable fibril proteins increased accordingly (not shown).
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. After 10 minutes of incubation,
multiple additional bands ranging from 4 to >49 kd were detected by
SDS-PAGE and Western blotting (Figure 5)
. The number of detectable
bands decreased throughout time and after 4 hours both
AFPPP and AFPPS were
completely degraded. Proteolytic activity at pH 7.4 lasted for just 10
minutes (Figure 5)
; the 5.8- and 6.9-kd bands of the fibril proteins
disappeared and, as observed at pH 5.5, multiple bands were found
spread out over the entire lane. However, this pattern showed no
further changes after 0.5, 2, 4, and 8 hours of incubation (Figure 5)
.
The proteolytic activity at pH 7.4 was not prolonged by the addition of
0.15% (w/v) solutions of any of the following to the incubation
medium: chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate,
or heparan sulfate. The degradation profile as demonstrated by SDS-PAGE
was identical to degradation in the absence of glycosaminoglycans
(GAGs; not shown). Cathepsin K also completely degraded native SAA, as
present in SAA-rich high-density lipoproteins (not shown) at pH 5.5 and
7.4. Again at pH 7.4 degradation lasted for just a short period of time
and was not influenced by the addition of GAGs.
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. Unfixed tissue samples were not available
for in vitro degradation experiments with AL amyloid. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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12 kd (Figure 4)
. Proteolysis after amyloid has
formed may have no effect or it may facilitate lateral growth of the
fibril or, alternatively, it may lead to the degradation of amyloid. This investigation is the first to show that cathepsin K is expressed by MGCs adjacent to amyloid deposits and also that it is present extracellularly with a close spatial relationship to amyloid fibrils. Based on these findings we assume that cathepsin K, under certain conditions, may be involved in the pathology of AA and AL amyloidosis. MGCs represent a specific response to the presence of amyloid deposits rather than being the cause of amyloid formation because several patients studied here had amyloid deposits without MGCs. Cathepsin K is found only in association with MGCs and therefore its expression is not a prerequisite for amyloidogenesis to occur, but it may exert an effect on the degradation of amyloid deposits. To test this hypothesis a series of degradation experiments was performed.
In vitro degradation experiments using crude amyloid fibril
extracts demonstrated that cathepsin K is an adequate fibril
protein-degrading protease. This cysteine protease completely degraded
crude fibril extracts within 8 hours, irrespective of whether the
fibril protein was present as a monomer or as an oligomer (Figure 5)
and degradation occurred even at pH 7.4. These experiments show that
cathepsin K is able to degrade amyloid fibril proteins extracellularly
at a physiological pH albeit for a short period of time. It has been
shown by others that the activity of cathepsin K is influenced by GAGs.
In the presence of chondroitin 4-sulfate the hydrolysis of soluble and
insoluble collagen by cathepsin K is increased mainly by stabilizing
the protease. Chondroitin 6-sulfate, dermatan sulfate, and heparan
sulfate had no effect.20
We were unable to show that these
GAGs had any effect on proteolytic activity at neutral pH. This may be
due partly to the fact that GAGs co-purify with amyloid fibrils when
using the water-wash procedure of Skinner and colleagues31
and hence proteolytic activity of cathepsin K at neutral pH may be
because of the presence of GAGs. GAGs have been shown to accumulate in
all amyloids studied41,42
and they may be a structural
component of the fibril43
and exert an effect on
fibrillogenesis.41
The composition of GAGs associated with
amyloid deposits varies between different amyloid diseases, patients,
and organs studied. For example, the relative amount of chondroitin
sulfate among amyloid-associated GAGs may range from 25 to 70% with
the amount of other GAGs, such as heparan sulfate or dextran sulfate,
changing accordingly.44
Some of the GAGs are bound to a
protein backbone as proteoglycan. A variable amount and composition of
GAGs within amyloid deposits may influence the proteolytic activity of
cathepsin K. Using SAA-rich high-density lipoproteins (that are
initially devoid of GAG) instead of AFPP as a substrate, we were unable
to detect any effect of GAGs on the proteolytic activity of cathepsin K
at pH 7.4. This finding is in agreement with our previous report
showing that the GAG-enhancing effect on the proteolysis of protein
substrates is primarily restricted to acidic pH
conditions.20
In situ experiments using frozen sections containing intact
AA amyloid fibrils showed that cathepsin K is most likely to change the
antigenicity of the fibril protein by retrieving epitopes. These are
retained in native material but not in formalin-fixed specimens
because, in contrast to immunostaining of frozen sections, staining was
homogeneous on formalin-fixed and paraffin-embedded specimens (Figure 1)
. This antigen retrieval, as observed on frozen sections, may
indicate proteolytic attack of the amyloid fibril protein itself
because immunostaining for amyloid P component was unchanged. Amyloid P
component is probably a structural constituent of amyloid fibrils and
present in the deposits of every amyloid disease studied so
far.45
The deposition of amyloid is not irreversible. Progression of
generalized amyloidosis can be delayed or stopped by treatment of the
underlying disease, and deposits may also
regress.10,46-48
Identification of proteases that are
involved in this degradation process and specifying their modes of
action may help in the development of new treatments, particularly in
situations in which treatment of the underlying disease has shown only
little if any benefit, eg, in primary AL amyloidosis. We have
demonstrated here that cathepsin K is a possible candidate; among M
it is a unique protease in that it is expressed only by MGCs and
epithelioid cells (Röcken C, Bühling F, unpublished
observation). Also, it is the most potent mammalian elastase yet
described and has unique collagenolytic activity and the potential to
degrade AA amyloid fibril proteins, as shown in this study. Additional
evidence for the putative significance of MGCs in amyloidosis comes
from earlier animal studies performed by Wright and
colleagues,10
who have found an association between
amyloid resorption and the presence of MGCs. However, further studies
are required to investigate the mechanisms by which M occurring in
amyloid deposits can be stimulated to become MGCs that synthesize and
secrete cathepsin K.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Deutsche Forschungsgemeinschaft (grants RO 1173/3-1 and SFB 387/B-7), Bonn Bad-Godesberg, Germany; and the Wilhelm Vaillant-Stiftung, München, Germany.
Accepted for publication November 29, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin in cases of generalized AA and AL amyloidosis. Virchows Arch 2000, 437:521-527[Medline]
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