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Electron microscopic demonstration of immunoglobulin deposition in rheumatoid cartilage.

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563
ELECTRON MICROSCOPIC
DEMONSTRATION OF
IMMUNOGLOBULIN DEPOSITION IN
RHEUMATOID CARTILAGE
HITOSHI ISHIKAWA, J. DONALD SMILEY, and MORRIS ZIFF
Horseradish peroxidase (HRPO) conjugated
with goat antihuman IgG, goat antihuman IgM, and
aggregated human IgG has been used as a enzymatic
marker to stain IgG, IgM, and rheumatoid factor in
rheumatoid cartilage. When HRPO-anti IgG and
HRPO-anti IgM were used, immunoglobulin deposits
were not observed in nonrheumatoid cartilage. However 7 of 8 rheumatoid cartilage specimens stained
with HRPO-anti IgG showed electron-dense deposits.
Three rheumatoid specimens stained with HRPOanti IgM showed similar findings. Both of 2 rheumatoid specimens also stained positively with H R P O
conjugated with aggregated IgG, a finding indicating
that rheumatoid factor was present. T h e deposits were
From the Department of Internal Medicine (Rheumatic
Diseases Unit), T h e University of Texas Southwestern Medical
School, Dallas, Texas.
Supported by U.S. Public Health Service Research Grant
AM 09989 and Training Grant AM 05154, and by an Arthritis
Foundation Clinical Study Center Grant.
Morris Ziff, Ph.D., M.D.: Professor of Internal Medicine,
T h e University of Texas Southwestern Medical School, Dallas,
and recipient, Research Career Award, U S . Public Health Service; J . Donald Smiley, M.D.: Professor of Internal Medicine, T h c
University of Texas Southwestern Medical School, Dallas; Hitoshi
Ishikawa: Fellow in Internal Medicine, T h e University of Texas
Southwestern Medical School, Dallas (present address: University
of Kobe Medical School, Kobe, Japan).
Address reprint requests to Dr. Morris Ziff, Department
of Internal Medicine, University of Texas Southwestern Medical
75235.
School, 5323 Harry Hines Boulevard, Dallas, Texas
Submitted for publication November 10, 1974; accepted
May 19, 1975.
Arthritis and Rheumatism, Vol. 18, No. 6 (November-December 1975)
seen between the collagen fibers of the superficial
layer of the cartilage to a maximal depth of 22 from
the surface (average: 7 p). T h e amorphous fibrinous
material o n the surface of the cartilage was also
stained. T h e demonstration of I&, IgM, and rheumatoid factor in the superficial zone of rheumatoid cartilage suggests that immune complexes are deposited
in the cartilage in this disease.
Using tlie immunofluorescent method, Cook
and coworkers (1,Z) have shown that immunoglobulins
(Ig) and tlie third component of complement are deposited in articular cartilage, menisci, and ligaments
of an antigen-induced arthritis in the rahbit and in
rheumatoid articular cartilage (3). They concluded
that antigen-antibody complexes were sequestered in
cartilage. I n this paper the deposition of Ig in rheumatoid cartilage has been further investigated by electron
microscopy. Horseradish peroxidase conjugated with
goat antihuman IgG (HRPO-anti IgG) and antihuman
Ighf (HRPO-anti IgM) has been used as an immunologic marker to stain for IgG and IgM in the cartilage.
I n addition, heat-aggregated human IgG conjugated
with H R P O (HRPO-IgG agg) was used to detect
rheumatoid factor in the cartilage.
MATERIALS A N D METHODS
Cartilage Specimens. Articular cartilage samples
from the medial condyle of the f e m u r were o b t a i n e d d u r i n g
ISHIKAWA E T AL
564
c
I
I
1
I
I
I
35
45
55
65
75
TUBE NUMBER
Fig 1. Sephadex G 100 separation of HRPO-anti IgC. Reaction
product of H R P O with anti-lgC was fractionated on a Sephadex
G 100 column ( 3 X 92 e m ) using phosphate-buffered saline, p H
7.3. I'olume per fraction: 3.0 ml. Absorbency at 280 m m . T h e
first peak was collected.
joint surgery from 8 patients with classic seropositive
rheumatoid arthritis (4), from 2 with osteoarthritis, and
from 1 with chondromalacia, and one sample was obtained
from a normal joint of a patient undergoing amputation
for osteogenic sarcoma. T h e rheumatoid cartilage specimens
were removed from areas that appeared normal on gross
inspection.
Antibody Preparation. Purified antihuman IgG was
obtained from a goat immunized with 50 mg of human IgG
in complete Freund's adjuvant and bled on the fourteenth
day after immunization. T h e serum was precipitated in 45%
saturated ammonium sulfate at O°C and then chromatographed on a Sepharose 4B column to which IgG had
been attached (5). Anti-IgG was recovered by elution with
glycine-HC1 buffer at p H 3.0. Purified goat antihuman IgM,
previously absorbed by passage through a Sepharose IgG
column, was obtained by elution from a similar solid immunoadsorbent coupled with human IgM. Rabbit antiHRPO serum was obtained from Cappel Lab, Downington,
Pennsylvania.
Heat-aggregated Human IgG. Heat-aggregated human IgG (IgG agg) was prepared from Cohn fraction I1
(Grand Island Biological Co, Grand Island, New York) by
the method of Christian (6) using 10 mg of lyophilized
Cohn fraction I1 (FII). IgG agg was centrifuged at 15,000
rpm for 30 minutes and the supernatants were conjugated
with HRPO.
Conjugation of Horseradish Peroxidase with Immunoglobulins. Horseradish peroxidase (HRPO, type VI
RZ 2.68) was obtained from Sigma Chemical Co, St. Louis,
Missouri. HRPO was conjugated with goat antihuman IgG
and antihuman IgM using carbodiimide (7). Fifty milligrams of HRPO and 50 mg of goat antihuman IgG o r anti-
human IgM were dissolved in 5 ml of distilled water and
400 mg of carbodiimide ( 1 -cyclohexyl-3-carbodiimide
methop-toluene sulfonate, Aldrich Chemical Lo, Inc, Milwaukee,
Wisconsin) were added. Reaction was permitted to proceed
with gentle agitation at room temperature for 30 minutes.
T h e mixture was then dialyzed in 0.1 M phosphate-buffered
saline (I'BS), p H 7.4, at 4°C overnight with two changes of
PBS to eliminate excess carbodiimide. Aggregated protein
was removed by centrifugation at 15,000 rpm for 20 minutes.
Conjugated protein was separated from unbound enzyme
by filtration (Figure 1) through a column (3 X 92 cm) of
Sephadex G 100 (8). Antigenic specificity of the conjugated
antibodies for the y-chain or the p-chain respectively was
confirmed by immunodiffusion and immunoelectrophoresis
against purified IgG and IgM (Figure 2).
T o prepare conjugates of HRPO with aggregated
human IgG, 10 mg of heat-aggregated FII were conjugated
with HRPO as described above. T h e specificity of HRPOIgG agg for IgM rheumatoid factor is shown in Figure 2.
Tissue Preparation for Immunoelectron Microscopy.
Unfrozen cartilage specimens approximately 1 mm3 were
fixed in 3":) glutaraldehyde buffered with 0.1 M PBS at
p H 7.4 for 1 hour at 4OC. T h e fixative was removed with
three changes of PBS for 30 minutes each. For the detection
of immunoglobulin in the cartilage, tissues approximately
40 to 60 p thick were shaved by hand perpendicular to the
true surface using a razor blade and then exposed to HRPOanti IgG, HRPO-anti IgM or HRPO-IgG agg for 2 hours
at 4°C with constant agitation on a mechanical shaker
(Eberbach Co, Ann Arbor, Michigan). After washing the
samples in three changes of PBS for 15 minutes, the tissues
were again fixed in 3% glutaraldehyde for 15 minutes. T h e
fixative was removed by washing with three changes of
buffer for 10-minute periods. For the detection of bound
HRPO, the method of Graham and Karnofsky (9) was used.
T h e tissues were incubated with 5 mg of 3,3'-diaminobenzidine (Sigma Chemical Co, St. Louis) in 10 ml of 0.05 M
Tris-HC1 buffer, p H 7.4, for 25 minutes. Following this
incubation 0.1 ml of 1% H,O, was added for 10 minutes.
T h e specimens were then washed in buffer, postfixed in 2%
osmium tetraoxide in PBS for 1 hour, dehydrated in graded
alcohol and propylene oxide, and embedded in Epon 812
o r hlaraglas. Sections were cut on an LKB microtome. They
were examined in an EMU-3G RCA electron microscope
with or without staining with uranyl acetate and lead citrate.
RESULTS
H R P O - A n t i Ig Staining of Nonrheumatoid
Cartilage. W i t h H R P O - a n t i JgG, H R P O - a n t i IgiM, a n d
HRPO-FII agg, immunoglobulin deposition was rarely
observed i n nonrheumatoid cartilage samples ( T a b l e
1). T h e cartilage surface, superficial zone, a n d a chondrocyte in t h e cartilage of a normal individual (Figure 3) showed no HRPO staining u p o n incubation
w i t h H R P O - a n t i IgG, a fact indicating the absence of
IMMUNOGLOBULIN DEPOSITION IN RHEUMATOID CARTILAGE
IgG in these areas. In a section from a patient with
chondromalacia (Figure 4) that was also treated with
HRPO-anti IgG, no staining for Ig was observed. Two
samples from patients with osteoarthritis (Figure 5 )
that had been treated with HRPO-anti IgG and
HRPO-anti IgM also showed no Ig deposits. Only irregular fissures in the cartilage surface and discontinuous collagen fibers in the matrix were prominent
features.
HRPO-Anti Ig Staining of Rheumatoid Cartilage. When sections from the patients with rheumatoid
arthritis, all classic and seropositive, were treated with
HRPO-anti IgG and HRPO-anti IgM, it was found
that IgG was stained in 7 of 8 cases and IgM in 3 of 3
(Table 1). The staining was observed (Figure 6) in the
superficial zone of the cartilage. Immunoglobulins
were stained in the top 5 to 20 p below the joint surface. Here the peroxidase activity was localized within
the matrix in clusters of variable size in areas of degraded matrix where the collagen meshwork appeared
loosely arranged (Figures 7 and 8). Fibrinous material
on the surface (Figure 6) also showed a strongly positive staining reaction. T h e relationship between Ig
deposition and the individual collagen fibers is shown
in Figure 9. There appeared to be no specific relation
to the collagen fibers. Some electron-dense deposits
were localized along collagen fibers but others were
found in the space between the collagen fibers.
In order to make certain that the peroxidasepositive areas were immunologically specific, cartilage
specimens were pretreated with unconjugated anti-IgG
or anti-IgM and peroxidase-conjugated reagents were
then applied (Figures 10 and 11). Sections stained directly with HRPO-anti IgG (Figure 10) were then
compared with the blocked control sections at the
same magnification. Peroxidase staining was minimal
in the control sections, a fact indicating that pretreatment with unconjugated anti-IgG had blocked the
specific staining reaction. The same results were obtained when sections stained with HRPO-anti IgM
were compared with and without pretreatment with
anti-IgM.
Staining of Rheumatoid Cartilage for Rheumatoid Factor. When cartilage specimens from rheumatoid patients were treated with HRPO-FII agg,
staining was seen in the matrix of the superficial zones
of the cartilage. T h e electron-dense particles were observed mainly between the collagen fibers but particles
overlying the collagen fibers were also seen (Figure 12).
This observation indicates the presence of rheumatoid
565
Fig 2. A. Immunoelectrophoresis in agarose of HRPO-anti IgG.
Upper well contains human IgG, lower well contains human A B
serum, and trough contains HRPO-goat antihuman IgC. Note
that a reaction is present only against IgC. B. Immunoelectrophoresis of HRPO-anti IgC. Well contains HRPO-goat antihuman IgC. Upper trough contains anti-HRPO; lower trough
contains rabbit antigoat serum. Note that arcs for both H R P O
and IgG are present. C . Immunodiffusion in agarose of H R P O goat antihuman IgM. I n the central well is HRPO-anti IgM and
in the outer wells are I ) human .4B serum; 2 ) goat antihuman
IgM; 3 ) human IgM; and 4 ) human IgG. S o t e that reactions are
present only against IgM. D. Immunodiffusion in agarose of
HRPO-lgC agg. In the central well is HRPO-IgG agg (first peak
of Sephadex G 200 column fractionation of heat-aggregated Cohn
fiaction I l ) . I n the outer wells are 1 ) rabbit anti-HRPO; 2 ) normal human A B serum: 3 ) rheumatoid factor obtained from a
patient with rheumatoid hyperviscosity syndrome (IgM fraction
was obtained by column chromatography using agarose A 5 M and
further purified by using Sephadex G 200 column); and 4 ) heat
agg F I I . Note that reactions are present only against rheumatoid
factor and anti-HRPO. There are lines of identity between 3 and
4 indicating identity of HRPO-lgG agg and IgG agg in their
reactions with IgM rheumatoid factor.
ISHIKAWA E T AL
566
Fig 3. Superficial zone (A) and a chondrocyte ( B ) in nornial cartilage that has been treated with HRPO-anti IgG show no peroxidase
staining activity indicating absence of IgG. T h e section was stained with both uranyl acetate and lead citrate. J.S. = joint space. Col
represents collagen (A and B both X 14,000). I n all figures the numbers over the standardization line represent the number of microns.
factor-containing deposits in rheumatoid cartilage.
Cartilage from a patient with osteoarthritis that had
been treated with HRPO-FII agg showed no staining
reaction (Figure 12).
DISCUSSION
Deposition of immunoglobulin (Ig) and com-
plement has been demonstrated in the cartilage of
animals with experimental antigen-induced arthritis
(1,2) by the immunofluorescent method and by radioautography. Ig has also been demonstrated in the
cartilage of patients with R A (3) by the immunofluorescent technique. I n this paper the localization of
these deposits in rheumatoid cartilage by the electron
microscope is described.
Table 1. HRPO-Anti Ig Staining of Cartilage
HRPO-Anti IgM
HRPO-Anti IgG
Diagnosis
Normal
Osteoarthritis
Chondromalacia
Rheumatoid arthritis
ND = not done.
No. of
Specimens
1
NO.
Positive
HRPO-FII Agg
No. of
Specimens
No.
Positive
No. of
Specimens
No.
Positive
-
1
0
ND
2
1
1
0
1
1
0
0
0
ND
-
8
7
3
3
2
2
2
0
IMMUNOGLOBULIN DEPOSITION I N RHEUMATOID CARTILAGE
Fig 4. A cartilage section from a patient with chondromalacia treated with HRPO-anti ZgG shows
no staining for ZgG. Fibrous tissue is invading the cartilage matrix. Uranyl acetate and lead
citrate double staining used. Top left is 8p fiom joint surface ( X 2,400).
567
ISHIKAWA E T AL
Fig 5. Cartilage sections from a patient with osteoarthritis. A. Section treated with HRPO-anti IgG. B. Section treated with HRPO-anti
ZgM. IL'o imrnunoglobulin staining can be detected i n either case. Irregular fissures in the cartilage surface and discontinuity of the collagen fibers in the matrix are prominent features. Electron-dense material is diffusely deposited in the matrix. However no H R P O staining i s seen. Uranyl acetate and lead citrate double staining used. J.S. = joint space. (A: X 18,000; B: X 23,000.)
Antibodies labeled with peroxidase have been
widely used to detect tissue antigens (10-12). This
method is specific (11-13) and sensitive (10-14). I n the
present paper the presence of Ig in cartilage has been
investigat.ed using anti-Ig conjugated with horseradish
peroxidase (HRPO-anti Ig) by the carbodiimide method (7,15). Like ferritin (16,17) and heavy metal (18),
the peroxidase staining method generates electrondense reaction products (9). Moreover, because the
peroxidase-anti Ig complex has a lower molecular
weight (19) than ferritin (20,21), it can penetrate tissue
more readily.
T h e demonstration of Ig in the superficial zone
of rheumatoid cartilage is dependent upon the penetration of peroxidase-conjugated antibody protein. Normally, cartilage matrix is relatively impermeable to
macromolecules (22-24). However it has been observed
(23,24) that loss of integrity of the matrix permits
diffusion of IgG into the cartilage.
T h e fact that immunoglobulins observed in the
superficial zone of rheumatoid cartilage specimens
were found at a depth of 5 to 20 (average: 7 p) from
the cartilage surface and were not found in deeper
zones suggests that they originated in the joint cavity.
It was in fact frequently observed that the cartilage
matrix in the areas where immunoglobulins were de-
IMMUNOGLOBULIN DEPOSITION IN RHEUMATOID CARTILAGE
569
Fig 6. T w o sections from a patient with classic seropositive rheumatoid arthritis. A. Cartilage was treated with HRPO-anti I g C . B. Cattilage was treated with HRPO-anti I g M . In each case I g was stained in the t o p 5 to 20 P below the joint surface. T h e peroxidase activity appears localized within the matrix in clusters of variable size in areas w h e w the inatrix appears degraded and the collagen meshwork loosely arranged (arrows). Note that the fibrinous material on the joint surface (A, arrows) shows a strongly positive reaction.
Without heavy metal staining (A: X 10,800; B: X 17,000).
posited was degraded and that the collagen fibrils
appeared depleted.
Immune complexes in the synovial fluid are
phagocytosed by polymorphonuclear cells and by
phagocytic lining cells of the synovium (25-26) with
resulting release of lysosomal enzymes from these cells
(27-37). These enzymes, which have the capacity to
degrade cartilage collagen (3 1,32) and proteoglycan
(33-35,38-40), presumably facilitate the sequestration
of these immune complexes within the cartilage. Extracellular release of cathepsin D from chondrocytes
has been recently demonstrated immunohistochemically by Poole et a1 (40) using specific anti-cathepsin
D antibodies. These authors have suggested that
cathepsin D and other tissue proteinases act synergistically in the extracellular catabolism of cartilage
components.
Deposits of Ig in the cartilage were most frequently seen in the matrix between the collagen fibrils
as well as along the collagen fibrils. However they
were sometimes seen overlying collagen fibrils in a
patchy distribution. I t appears likely from the ob-
ISHIKAWA E T AL
570
Fig 7 . Section
of
metal staining
(X
rheumatoid cartilage treated with HRPO-anti IgG. An enlargement
14,000).
served distribution of these immunoglobulins that,
once trapped in the matrix of the cartilage, they remain in this tissue for long periods of time enmeshed
in the three-dimensional network of densely packed
collagen fibrils.
Treatment of cartilage sections with HRPOIgG agg stained deposits in the cartilage matrix. This
indicates the presence of rheumatoid factor in these
deposits. T h e distribution of rheumatoid factor-containing deposits in the cartilage was similar to those
of the IgG and IgM staining deposits. T h e present
data do not shed light on the question of whether the
immune complexes presumably present in rheumatoid
cartilage are made u p entirely of rheumatoid factor
complexes Or whether Other immune complexes are
also present. However the density and frequency of the
of
this area i s given in Figure 8. Without heavy
deposits stained with HRPO-IgG agg were smaller
than those observed after staining for IgG and IgXI.
T h e present study provides evidence of Ig deposition in rheumatoid cartilage. As previously suggested (3), the Ig deposits may represent sequestered
antigen-antibody complexes. These may play a role in
the chronicity of the disease by slowly releasing sequestered antigen from the cartilage into the synovial
cavity.
ACKNOWLEDGMENTS
The authors would like to acknowledge the kindness
of Dr. K. Hirohata (Kobe, Japan) in sending various cartilage samples and Dr. M. Andreis in carrying out immunediffusion studies.
IMMUNOGLOBULIN DEPOSITION I N RHEUMATOID CARTILAGE
Fig 8. Higher magnification of the area surrounded by the rectangle in Figure 7. Peroxidase
activity is localized within the matrix in clusters of variable size in areas where the matrix
appears depleted and the collagen fibers are loosely arranged (X 48,000).
57 1
572
ISHIKAWA E T AL
Fig 9. Relationship between Ig deposits and individual collagen fibers in rheumatoid cartilage.
T h e seclion was slnined w i t h HRPO-anti ZgG. Although sortie electron-dense deposits are localized along collagen fibers, others are found between collage71 fibers. T h e irregularly round large
gray structures are collagen fibrils seen 018 end. Without heavy metal staining ( X 64,000).
IMMUNOGLOBULIN DEPOSITION I N RHEUMATOID CARTILAGE
573
Fig 10. Blocking of cartilage HRPO-anti IgG staining by pretreatment with unconjugated anti-IgC. Section on left shows rheumatoid
cartilage treated directly with HRPO-anti IgG. T h e section on the right shows a n adjacent section of cartilage pretreated with unconjugated anti-IgG and then treated with HRPO-anti IgC. It can be seen that pretreatment with unconjugated anti-IgG blocked the specific staining reaction because of a marked decrease in peroxidase staining. Without uranyl acetate and lead citrate staining (X 50,000).
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ISHIKAWA E T AL
574
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IMMUNOGLOBULIN DEPOSITION IN RHEUMATOID CARTILAGE
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