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Interaction between Synovial Inflammatory Tissue and Bone
Marrow in Rheumatoid Arthritis
1
Esther Jimenez-Boj,* Kurt Redlich,* Birgit Tu¨rk,* Beatrice Hanslik-Schnabel,
†
Axel Wanivenhaus,
†
Andreas Chott,
‡
Josef S. Smolen,* and Georg Schett
2
*
Rheumatoid arthritis (RA) leads to destruction of cartilage and bone. Whether rheumatoid arthritis also affects the adjacent bone
marrow is less clear. In this study, we investigated subcortical bone marrow changes in joints from patients with RA. We describe
penetration of the cortical barrier by synovial inflammatory tissue, invasion into the bone marrow cavity and formation of
mononuclear cell aggregates with B cells as the predominant cell phenotype. B cells expressed common B cell markers, such as
CD20, CD45RA, and CD79a, and were mature B cells, as indicated by CD27 expression. Plasma cells were also present and were
enriched in the regions between aggregates and inflammatory tissue. Moreover, molecules for B cell chemoattraction, such as
BCA-1 and CCL-21, homing, mucosal addressin cell adhesion molecule-1 and survival, BAFF, were expressed. Endosteal bone
next to subcortical bone marrow aggregates showed an accumulation of osteoblasts and osteoid deposition. In summary, we show
that synovial inflammatory tissue can reach the adjacent bone marrow by fully breaking the cortical barrier, which results in
formation of B cell-rich aggregates as well as increased formation of new bone. This suggests that bone marrow is an additional
compartment in the disease process of RA. The Journal of Immunology, 2005, 175: 2579 –2588.
R
heumatoid arthritis (RA)
3
is due to a chronic inflamma-
tory process of the synovial membrane. This membrane,
the innermost part of the joint capsule, is normally a fine
strand of only a few cell layers. It bridges the two neighboring
bones and inserts at periosteal regions close to the articular carti-
lage. This insertion site, where articular cartilage, periosteum, and
synovial membrane come into close contact is also called the junc-
tion zone. In RA, the synovial membrane is transformed into a
hypertrophic inflammatory tissue. This is based on an influx of
inflammatory cells, such as monocytes, T cells, and B cells, from
the blood stream as well as hyperplasia of resident synovial cells.
An important feature of synovial inflammatory tissue is its ca-
pacity to invade neighboring structures, such as cartilage and bone
(1–3). This invasive synovial tissue is also termed “pannus.” This
property makes RA the most disabling joint disease, since it leads
to structural damage of the joint, which finally leads to loss of
function and disability. Mineralized tissue, such as mineralized
cartilage and bone, is considered as primary target of synovial
inflammatory tissue, since at the aforementioned junction zone, it
is directly located underneath the inflammatory tissue of the in-
serting synovial membrane and the periosteum (2). Resorption of
subchondral bone, which appears in radiography, is a criterion for
the classification of RA and assessment of radiographic bone dam-
age has become an important tool for monitoring RA patients in
clinical studies and daily practice (4, 5).
The ability of synovial tissue to invade bone is closely linked to
the generation of osteoclasts (6– 8). Differentiation of osteoclasts
appears to be enhanced in the RA synovial membrane, since there
is an increased influx of mononuclear cells serving as osteoclasts
precursors and a plethora of signals, such as TNF and receptor
activator of NF-
B ligand, which stimulate osteoclast differentia-
tion (9, 10). In animal models, osteoclasts are essential for arthritic
bone resorption and blockade of osteoclasts has emerged as a pow-
erful tool to interfere with structural bone damage in arthritis
(11–13). Thus, osteoclasts are the tools of the inflamed synovial
membrane to invade bone. Whether cortical bone can actually be
completely penetrated by the inflamed synovial tissue, and what
the consequences of such penetration into the marrow space might
be, is less clear.
The cortical bone layer, which separates bone marrow from sy-
novial tissue, is comparatively thin, suggesting that an attack by
osteoclasts from the outside could penetrate cortical bone and open
the marrow space for synovial tissue. Such interaction could entail
profound changes of cellularity of synovial inflammatory tissue,
since the marrow space harbors vast amounts of leukocytes and
their precursors, and vice versa. To test this hypothesis, we studied
specimens from RA patients subjected to joint replacement sur-
gery, which contained not only synovial inflammatory tissue but
also neighboring cartilage, bone, and bone marrow. We performed
a histochemical and immunohistochemical study to search for and
define the nature of penetration of synovial inflammatory tissue
into the marrow space in human RA patients.
Materials and Methods
Patients and tissue specimens
Twelve patients, fulfilling the American College of Radiology criteria for
diagnosis of RA were analyzed in this study (4). All 12 patients were
routinely scheduled for joint replacement surgery because of refractory RA
in the affected joint. All joints showed clinical signs for active synovitis
(pain, swelling, and stiffness). Age, sex, and the years of disease duration
were recorded from all subjects. In addition, the number of tender and
*Division of Rheumatology, Department of Internal Medicine III,
†
Department of
Orthopedic Surgery, and
‡
Department of Pathology, Medical University of Vienna,
Vienna, Austria
Received for publication March 7, 2005. Accepted for publication May 23, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported by the START prize of the Austrian Science Fund (to
G.S.).
2
Address correspondence and reprint requests to Dr. Georg Schett, Division of Rheu-
matology, Department of Internal Medicine III, University of Vienna, Wa¨hringer Gu¨rtel
18-20, A-1090 Vienna, Austria. E-mail address: georg.schett@meduniwien.ac.at
3
Abbreviations used in this paper: RA, rheumatoid arthritis; ESR, erythrocyte sedi-
mentation rate; DMARD, disease-modifying antirheumatic drug; MAdCAM-1, mu-
cosal addressin cell adhesion molecule-1; TRAP, tartrate-resistant acid phosphatase.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
swollen joints, the presence of radiographic bone erosions in the replaced
joint, and the levels of C-reactive protein (mg/dl), erythrocyte sedimenta-
tion rate (ESR) (mm/h), and rheumatoid factor (U/L) were assessed in these
patients. Moreover use of steroids and disease-modifying antirheumatic
drugs (DMARDs) including biological agents was recorded. Resected ma-
terial consisted of juxtaarticular bone, articular cartilage and synovial tissue
attached to bone and cartilage in all 12 patients. In addition, four joints
from healthy patients receiving amputation surgery of the lower limb after
traumatic injury were collected. All participants gave written informed
consent. After explanation, the material was immediately placed into 0.9%
NaCl and fixed within1hin4.0% formalin. Fixation lasted for at least
18 h.
Preparation of tissue sections
After fixation, joints were longitudinally cut into two equally sized pieces.
One part was used to prepare decalcified paraffin-embedded tissue sections.
Material was fixed in 4.0% formalin overnight and then decalcified in 14%
EDTA (Sigma-Aldrich) at 4°C (pH adjusted to 7.2 by addition of ammo-
nium hydroxide) until the bones were pliable. Ten paraffin sections (2
m)
were cut sequentially throughout the joint and used for the histochemical
analyses. Twenty additional serial sections were cut for immunohistochem-
ical analysis (see below). The other part was used for preparation of un-
decalcified plastic-embedded tissue sections. Material was fixed in 70%
ethanol, dehydrated in 100% methanol, and embedded in methylmetacry-
late (K-Plast; MDS GmbH), as previously described (14, 15). Ten sequen-
tial sections 3-
m thick throughout this part of the joint were made on a
Jung microtome (Jung).
Antibodies
Monoclonal Abs against the following Ags were used: CD3 (1:50, clone
PS1; Novocastra), CD5 (1.20, clone 4C7; Novocastra), CD20 (1:200, clone
L26; DakoCytomation), CD21 (1:20, clone 1F8; DakoCytomation), CD23
(1:40, clone 1B12; Novocastra), CD27 (1:80, clone 137B4; Novocastra),
CD45RA (1:100, clone 4KB5; DakoCytomation), CD68 (1:100, clone PG-
M1; DakoCytomation), CD79a (1:25, clone JCB117; DakoCytomation),
CD138 (⫽Syndecan-1, 1.40, clone B-B4; Serotec), CXCL-13 (⫽BCA-1,
1:30, clone 53610; R&D Systems), Ki-67 (1:50, clone MIB-1; DakoCyto-
mation), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1)
(1:20, clone 355G8; Zymed Laboratories). Rabbit polyclonal Abs against
the following Ags were used: IgD (1:100; DakoCytomation), IgM (1:100;
DakoCytomation),
L chain (1:1200; DakoCytomation),
L chain (1:
1200; DakoCytomation), and myeloperoxidase (1:1200; DakoCytomation).
Goat polyclonal Abs were used for labeling BAFF (⫽BlyS, 1:25; R&D
Systems) and CCL21 (⫽6cKine, 1:20; R&D Systems).
Immunohistochemistry
All joint specimen were assessed by immunohistochemistry. For Ag re-
trieval, sections subjected to microwave in citrate buffer at 600 W for 12
min (CD5, CD20, CD21, CD79a, CD138, CCL21, IgD, IgM,
L chain,
Ki-67,
L chain, myeloperoxidase) or autoclaved in citrate buffer at 1 bar
for 20 min (CD3, CD5, CD23) or treated with 500
g/ml of either pro-
teinase XIV (CD27; Sigma-Aldrich), proteinase XXIV (CD21, CD68;
Sigma-Aldrich), or proteinase K (Roche; BAFF, CXCL13, MAdCAM-1)
at 37°C for 5 min. When rabbit polyclonal Ab was applied as a first Ab,
sections were blocked with 1:10 diluted goat serum for 20 min, if goat
polyclonal Ab was used blocking was performed with 1:10 diluted rabbit
serum for 20 min. Normal Ig from mouse, rabbit, and goat, respectively,
was used as control. After incubating with the first Ab for 1 h, sections
were washed and labeled with the following biotin-conjugated detection
Abs for 1 h: goat anti-mouse Ig, goat anti-rabbit Ig, and rabbit anti-goat Ig
(all 1:200; Santa Cruz Biotechnology). Detection was performed by using
Vectastain ABC kit standard (Vector Laboratories) and diaminobenzidine
(Fluka).
Histochemistry
Paraffin-embedded tissue sections were stained by H&E staining, tartrate-
resistant acid phosphatase (TRAP) staining (leukocyte acid phosphatase
kit; Sigma-Aldrich) for identification of osteoclasts and toluidine blue for
detecting cartilage proteoglycan content. Plastic embedded tissue sections
were stained by Goldner trichrome, von Kossa and Movat pentachrome,
according to standard protocols (15, 16).
Histological assessments
Number and size of subcortical bone marrow cell aggregates associated
with penetrated synovial tissue were determined from 10 sequential decal-
cified paraffin sections and 10 sequential plastic-embedded undecalcified
sections. For assessing the size of these subcortical bone marrow cell ag-
gregates, area of each single aggregate was measured by histomorphometry
using OsteoMeasure system (OsteoMetrics) and results were summarized
to define the total area of bone marrow affected by aggregates. For control
purposes, number and size of bone marrow cell aggregates, which were not
in association with synovial tissue penetration and were not directly local-
ized subcortically, were assessed. Width of cortical defects at sites of sy-
novial tissue penetration into the marrow space was also measured. All
measurements were done by histomorphometry using OsteoMeasure sys-
tem. Immunohistochemistry was done on 20 serial parraffin-embedded sec-
tions from each specimen. For each cell surface marker and each aggregate,
the fraction of positively labeled cells was assessed and a mean ⫾ SEM
was calculated for all patients. These analyses were performed in subcor-
tical bone marrow aggregates and for control purposes in cell aggregates
within synovial tissue as well as in the inflamed synovial membrane. Os-
teoid deposits in the subcortical region were assessed on Movat-labeled
sections of undecalcified tissue at two different sites, at the bone surface
next to subcortical bone marrow aggregates and at a site devoid of such
aggregates. The fraction of surface covered by osteoid from total bone
surface was measured and calculated in both compartments by using his-
tomorphometry. Finally, standard osteomorphometry measures were ap-
plied for assessing juxtaarticular trabecular bone in healthy normal subjects
and RA patients. The following parameters were measured: the fraction of
bone volume of total volume (BV/TV), trabecular thickness (Tb.Th), num-
ber (Tb.N), and separation (Tb.S), number of osteoclasts and osteoblasts
per bone perimeter (N.Oc/B.Pm, N.Ob/B.Pm), and the fraction of bone
surface covered by osteoclasts and osteoblasts (OcS/BS, ObS/BS).
Statistical analysis
Data are shown as means ⫾ SEM. Number and size of subcortical bone
marrow aggregates, size of cortical penetration, cortical width, and bone
surface covered by osteoid were compared by Mann-Whitney U test. For
correlating the size of subcortical bone marrow aggregates to markers of
disease activity, Spearman’s correlation coefficients and Bonferroni cor-
rection were calculated. A value of p ⬍ 0.05 was regarded as statistically
significant.
Results
Patients characteristics
Of the 12 patients studied, eight were female and four were male
(Table I). Mean (⫾SEM) age was 60 (⫾3) years, and mean disease
duration was 18.9 (⫾2.8) years, indicating long-standing RA.
Most patients had active disease as revealed by the number of
tender (mean ⫾ SEM number of tender joints: 6.8 ⫾ 2.9) and
swollen joints (4.4 ⫾ 1.3) and/or elevated acute phase response
(C-reactive protein, 4.8 ⫾ 2.7 mg/dl; ESR, 51 ⫾ 10 mm/h). Eleven
patients were rheumatoid factor positive and its mean (⫾SEM)
level was 286 (⫾127) U/L. Ten patients received DMARD therapy
with methotrexate, five of them as a monotherapy, one in combi-
nation with leflunomide, two with anakinra, a rIL-1 receptor an-
tagonist, and two with TNF blockers (infliximab and etanercept).
The remaining two patients received monotherapy with anakinra
or leflunomide. Low dose oral glucocorticoids were taken by eight
of 12 patients. Material originated from joint replacement surgery
of the metacarpophalangeal and proximal interphalangeal finger
joints in five patients, the metatarsophalangeal joints in another
five patients, and the knee as well as the wrist joint in each patient.
Cortical penetration leads to mononuclear cell aggregates in the
adjacent bone marrow
To search for a possible cortical penetration of synovial inflam-
matory tissue into the bone marrow, we generated axial sections of
the joints to allow a simultaneous assessment of cartilage, sub-
chondral bone and bone marrow. Cortical penetration was found in
nine of 12 patients and originated from subchondral bone erosions
(Fig. 1). Areas with cortical penetration by the pannus were asso-
ciated with a widespread resorption of subchondral mineralized
cartilage and bone, separating unmineralized articular cartilage
from underlying bone and characterized by small regions of bone
marrow invasion (Fig. 1A). Articular cartilage covering these areas
2580 SYNOVIAL INFLAMMATORY TISSUE AND BONE MARROW IN RA
has lost most of its proteoglycan content as shown by toluidine
blue stain (Fig. 1B). Where synovial tissue had penetrated bone, it
faced a mononuclear cell aggregate. This innermost invading part
of synovial inflammatory tissue was clearly separated from the
mononuclear cell aggregates in the bone marrow, which in turn
formed a barrier between the pannus and normal bone marrow
(Fig. 1, A–C). This interface was localized close to the inner layer
of cortical bone and filled most of the marrow space, where cor-
tical penetration had occurred (Fig. 1C). Mononuclear cell marrow
aggregates were absent when bone marrow was covered by an
intact cortical bone layer, even if eroded from the outside (Fig. 1,
D and E). When cortical bone was still present, numerous oste-
oclasts associated with synovial inflammatory tissue were local-
ized at the outer side, whereas the inner endosteal region was not
affected (Fig. 1, E and F).
Subcortical bone marrow aggregates in RA are linked to
penetration of cortical bone
To characterize bone marrow changes in RA in more detail, we quan-
titatively assessed the number and size of subcortical bone marrow
aggregates. An average number of three (mean ⫾ SEM 3.0 ⫾ 0.9)
sites of cortical penetration with consecutive bone marrow aggregates
were observed in each joint (Fig. 2A). In healthy joints, no such le-
sions were found. The average size of subcortical bone marrow ag-
gregates was 0.06 mm
2
, whereas no region of the subcortical bone
marrow was covered by such aggregates in normal joints (Fig. 2B).
The mean diameter of the cortical destruction sites was 236 ⫾ 27
m,
which is big enough to allow a meaningful interaction between cells
from synovial tissue and bone marrow (Fig. 2C). Moreover, cortical
width was significantly diminished in the vicinity of cortical penetra-
tion sites (mean ⫾ SEM, 99 ⫾ 5
m) compared with normal cortical
width (162 ⫾ 13
m), suggesting that bone resorption had signifi-
cantly weakened the cortical barrier (Fig. 2D).
Interestingly, distant from the site of penetration, bone marrow
cell aggregates, when found, were of much smaller size than those
at the penetration site.
Subcortical bone marrow aggregates are associated with high
disease activity
To clarify whether clinical features of RA influence cortical bone
penetration and formation of bone marrow aggregates, we next
correlated clinical parameters of RA with the area of subcortical
FIGURE 1. Cortical penetration and bone marrow invasion in RA. Histological sections from metacarpal heads of patients with RA. A, H&E stained
section showing invasion of synovial inflammatory tissue into the subchondral space previously filled with mineralized cartilage (black arrowhead).
Unmineralized cartilage appears on the top as a pink band; cortical bone barrier appears at the bottom as dark pink band, which is penetrated by synovial
tissue leading to formation of a mononuclear aggregate in the bone marrow (black arrow). B, Toluidine blue staining showing extensive proteoglycan loss
in articular cartilage. C, Close-up view of the interface of synovial inflammatory tissue (top) and bone marrow aggregate (bottom) at site of cortical
penetration (black arrow). D, Weakened but intact cortical barrier with no changes of the underlying bone marrow (H&E stain). E, The same regions stained
by TRAP showing osteoclasts (purple staining, black arrow) resorbing cortical bone from the outside. F, Close-up view of osteoclasts stained for TRAP
(black arrow). A, B, D, and E, Original magnification, ⫻50; C and F, original magnification, ⫻400.
Table I. Characteristics of rheumatoid arthritis patients
a
No. Sex Age DD CRP ESR SJC TJC RF DMARD BIOL GC Joint region
1 F 68 31 2.5 74 4 0 116 MTX ANA Yes MTP
2 M 56 25 2.2 58 0 0 11 LEF No MCP
3 F 69 25 3.0 62 6 28 92 MTX IFX Yes MCP
4 F 45 13 8.1 40 9 12 1030 MTX ANA Yes MCP
5 F 63 23 ND 34 0 0 ND MTX No PIP
6 F 75 3 0.8 9 0 0 49 MTX ⫹ LEF No MTP
7 F 45 7 1.4 88 1 1 486 MTX Yes PIP
8 F 72 17 0.8 47 12 14 94 MTX Yes Knee
9 M 53 20 28.7 117 3 2 0 ANA Yes Wrist
10 M 52 12 0.6 34 9 11 19 MTX ETA Yes MTP
11 F 55 32 0.3 3 ND ND 972 MTX No MTP
12 F 69 ND ND ND ND ND ND MTX Yes MTP
a
DD, Disease duration (years); SJC, swollen joint count (n from 28 joints); TJC, tender joint count (n from 28 joints); RF, rheumatoid factor (U/L); GC, glucocorticoids;
MTX, methotrexate; LEF, leflunomide; BIOL, biological drug; ANA, anakinra; IFX, infliximab; ETA, etanercept; MTP, metatarsophalangeal joint; MCP, metacarpophalangeal
joint; ND, not determined; PIP, proximal interphalangeal joint.
2581The Journal of Immunology
bone marrow aggregates. For comparative purposes clinical pa-
rameters were also related to the area of small bone marrow ag-
gregates, which were distant from cortical bone and were thus not
attached to synovial inflammatory tissue. Subcortical infiltrates
were more prominent in patients with signs of higher diseases
activity, as indicated by a high number of tender or swollen joints
(swollen joints: Spearmans r ⫽ 0.64; p ⬍ 0.05) as well as a high
acute phase response (C-reactive protein: r ⫽ 0.71; p ⬍ 0.05),
measured by C-reactive protein level and ESR (Table II and Fig. 3,
A and C). In addition, high titers of rheumatoid factor were also
associated with the presence of subcortical aggregates (r ⫽ 0.64;
p ⬍ 0.05; Fig. 3E). In contrast, small bone marrow aggregates
were not associated with any of these variables (Fig. 3, B, D, and
F). Age, sex, disease duration, as well as the joint region the ma-
terial had been taken from, were not relevant for the presence of
aggregates (Table II). Also, the use of steroids and the type of
DMARD or biological drug therapy did not appear to affect the
size of subcortical bone marrow aggregates.
B lymphocytes dominate subcortical bone marrow aggregates
Next, we turned to characterize cell-specific surface marker ex-
pression in subcortical bone marrow aggregates by immunohisto-
chemical analysis. Interestingly, B cells, as detected by CD20 ex-
pression, were by far the most frequent cell type (mean ⫾ SEM:
55 ⫾ 8%) (Fig. 4, A and E, and Fig. 5A). This was confirmed by
concomitant expression of other pan-B cell markers, such as
CD45RA (Figs. 4I and 5A) and CD79a (Fig. 5A), both of which
were found in a similar frequency in subcortical bone marrow
aggregates. T cells, as detected by positive labeling for CD3 (35 ⫾
4%) and CD5 (37 ⫾ 3%) were found in considerable lower fre-
quency and only few macrophages were present in subcortical
bone marrow aggregates (8 ⫾ 1%) (Figs. 4, B, C, F, and G, and
5A). Follicular dendritic cells, as detected by CD21 expression
(3 ⫾ 1%) and neutrophils, as detected by labeling for myeloper-
oxidase (0.8 ⫾ 0.6%), were almost completely absent (Figs. 4K
and 5A). This pattern of cellular composition of subcortical aggre-
gates was very similar among all patients investigated and was also
not different among the various DMARD therapies.
A more detailed analysis of B cell markers revealed abundance
of CD27, indicating that the main proportion of B cells are mature
B cells (53 ⫾ 2%) (Figs. 4J and 5A). In contrast, expression of IgM
(18 ⫾ 4%), representing immature and mature naive B cells, as
well as IgD (5 ⫾ 1%), representing mature naive B cells was much
less frequent (Fig. 5A). CD23 expression was virtually absent (1 ⫾
0.5%) in subcortical bone marrow aggregates. Plasma cells, as
detected by CD138 labeling were especially found at the periphery
of subcortical bone marrow aggregates and were usually found in
between aggregate and synovial inflammatory tissue (Fig. 4, D and
H). Plasma cell attributed to 8 ⫾ 4% of total cells in aggregates
(Fig. 5A). Less than 5% of cells showed signs of proliferation, as
detected by expression Ki-67 Ag (Fig. 5A).
Compared with subcortical bone marrow aggregates, synovial
inflammatory tissue showed a different cellular composition (Fig.
5B). The proportion of B cells was significantly smaller and B cells
only attributed to up to 10% of synovial cells. The number of T
cells in synovial tissue was comparable to subcortical bone mar-
row aggregates (24 ⫾ 6%), whereas macrophages (27 ⫾ 4%) were
much more frequent. Plasma cell content was very scarce (1 ⫾
1%). In seven patients, mononuclear aggregates were present
within synovial tissue, whereas no such aggregates were found in
the remaining five patients. All of these aggregates were localized
in the synovial tissue of the joint space and were distant from
subchondral bone erosions and sites of cortical penetration. This
cellular composition showed some similarities with subcortical
bone erosions, although their proportion of B cells (20 ⫾ 4%) and
plasma cells (2 ⫾ 2%) was lower (Fig. 5C). Composition of B cells
was very similar with a majority of mature B cells. T cells were
found in a similar frequency, whereas the fraction of macrophages
was more prominent in synovial aggregates than subcortical bone
marrow aggregates.
Expression of molecules for B cell chemotaxis, homing, and
activation in the vicinity of subcortical bone marrow aggregates
Hypothesizing that invading synovial inflammatory tissue ex-
presses molecules, which facilitate accumulation of B cells in the
neighboring bone marrow, we next stained for molecules respon-
sible for B cell chemotaxis, homing, and activation (Fig. 6). Che-
moattractants for B cells, such as CXCL-13, also termed B cell
FIGURE 2. Subcortical bone marrow aggregates and alteration of cor-
tical bone in RA. Joint sections from healthy individuals and patients with
RA were assessed for number (A) and size (B) of subcortical aggregates in
the juxtaarticular bone marrow. Shown are the size of cortical penetration
(C) and the width of cortical bone (D) in healthy individuals and patients
with RA. All measures were performed by histomorphometry of H&E
stained sections. Data are mean ⫾ SEM from four healthy joints and 12
joints affected by RA. ⴱ, significant difference (p ⬍ 0.05).
Table II. Correlation of subcortical bone marrow aggregates with
clinical parameters of disease
Parameter
Subcortical Bone
Marrow
Aggregates
a
Bone Marrow
Aggregates
b
Spearmans
p Spearmans
p
Age 0.18 0.57 0.25 0.43
Sex ⫺0.03 0.92 ⫺0.03 0.92
Joint region 0.06 0.85 0.15 0.66
CRP 0.71 0.02 0.41 0.23
ESR 0.68 0.01 0.57 0.08
Swollen joint count 0.65 0.04 0.25 0.40
Tender joint count 0.62 0.04 0.46 0.20
Rheumatoid factor 0.64 0.02 0.16 0.64
Glucocorticoids 0.46 0.10 0.31 0.34
DMARDs ⫺0.05 0.87 0.13 0.72
a
Area of subcortical bone marrow aggregates linked to penetration of cortical
bone and attached to synovial inflammatory tissue.
b
Area of small bone marrow aggregates in the marrow space with no association
to cortical penetration or synovial tissue.
2582 SYNOVIAL INFLAMMATORY TISSUE AND BONE MARROW IN RA
chemoattractant protein-1, as well as CCL-21 were found ex-
pressed at the interface of synovial inflammatory and subcortical
bone marrow aggregates (Fig. 6, A and B). Moreover, numerous
MAdCAM-1-positive blood vessels, resembling high endothelial
venules were found within these aggregates (Fig. 6C). BAFF, also
termed BlyS, a molecule important for B cell survival, was also
expressed within inflammatory tissue next to bone marrow aggre-
gates, suggesting that accumulation of B cells is triggered by local
expression of molecules involved in B cell migration, homing, and
survival (Fig. 6D).
FIGURE 4. Immunophenotypical
characterization of subcortical bone
marrow aggregates. Serial histologi-
cal section from the specimen shown
in Fig. 1, A and B, stained by Abs
against CD20 for detection of B cells
(A and E), CD3 for T cells (B and F),
CD68 for macrophages (C and G)
and CD138 for plasma cells (D and
H). The images depicted in E–H
show close-up views of the respec-
tive images depicted in A–D. I, Stain-
ing by Abs against CD45RO as a
pan-B cell marker; J, CD27 for ma-
ture B cells; and K, CD21 for follic-
ular dendritic cells. L, Control stain-
ing with normal mouse Ig. Note
abundance of CD20 expression in
subcortical bone marrow aggregates
as well as the focused expression of
CD138 at the interface of synovial
inflammatory tissue and aggregates.
A–D and I–L, original magnification
⫻100; E–H, original magnification
⫻400.
FIGURE 3. Subcortical bone marrow aggregates are
associated with surrogate markers for disease activity.
The area covered by subcortical bone marrow aggre-
gates linked to cortical penetration of synovial inflam-
matory tissue (A, C, E) as well as the area covered by
small aggregates in the bone marrow, which are not
linked to cortical penetration (B, D, F) was correlated to
the number of swollen joints (A, B), the serum level of
C-reactive protein (C, D) and rheumatoid factor (E, F).
2583The Journal of Immunology
Bone marrow penetration and formation of subcortical bone
marrow aggregates leads to endosteal bone formation
To address whether cortical bone reacts upon penetration and ag-
gregate formation in the bone marrow, we investigated undecalci-
fied plastic sections of the same joints (Fig. 7, A–H). Interestingly,
the inner endosteal surface of cortical bone revealed wide areas
covered by osteoblasts (Fig. 7, E–G), whereas osteoclasts were
absent. Moreover, underneath these osteoblast-covered areas, new
bone formation, as visualized by the presence of osteoid seams
could be detected (Fig. 7, A, C, E–G). These areas were only found
in the vicinity of subcortical bone marrow aggregates, whereas the
endosteal surface distant from such sites was largely unaffected
(Fig. 7, B and D). Quantification of these areas revealed that ⬍5%
of bone surface at sites without subcortical bone marrow aggre-
gates was covered by osteoid seams, whereas up to 40% of en-
dosteal bone surface was covered at sites close to the aggregates
(Fig. 7H).
Severe loss of periarticular bone in RA is associated with high
bone turnover
Considering the radiographic signs of periarticular bone loss in
RA, we also investigated the structure of periarticular bone in this
group of RA patients and for comparative purpose also in healthy
controls. Histomorphometric analysis of undecalcified sections re-
vealed massive loss of juxtaarticular trabecular bone as evident
from a 4-fold decrease of bone volume in RA patients compared
with normal controls (Fig. 8A). Bone loss was based on a more
FIGURE 5. Differences in expression pattern of surface molecules upon
subcortical bone marrow aggregates, synovial inflammatory tissue, and sy-
novial tissue aggregates. Subcortical bone marrow aggregates (A), synovial
inflammatory tissue (B), and synovial tissue aggregates (C) were analyzed
for the following B cell markers: CD20, CD23, CD27, CD45RO, CD79a,
CD138, IgD, and IgM. In addition, markers for T cells (CD3, CD5), mac-
rophage (CD68), granulocytes (myeloperoxidase, MPO), and follicular
dendritic cells (CD21) were analyzed. Also cell proliferation was assessed
by staining for Ki-67. Note the preponderance of B cells in subcortical
bone marrow aggregates, of macrophages, granulocytes, and T cells in
synovial inflammatory tissue and T cells, B cells, and macrophages in
synovial tissue aggregates.
FIGURE 6. Expression of molecules for B cell migration, homing, and
activation. Histological sections of the interface between synovial inflam-
matory tissue and subcortical bone marrow aggregates stained for
CXCL-13 (also termed BCA-1) (A) and CCL21 (also termed 6cKine) (B).
C, Staining for MAdCAM-1 showing a transverse (black arrow) and lon-
gitudinal section (black arrowhead) through high endothelial venules
within subcortical bone marrow aggregates. D, Expression of BAFF (also
termed BlyS) at the interface between synovial inflammatory tissue and
subcortical bone marrow aggregate. Control staining with normal mouse
(E) and goat (F) Ig, as first Abs. A–F, original magnification ⫻400.
2584 SYNOVIAL INFLAMMATORY TISSUE AND BONE MARROW IN RA
than 2-fold decrease of the trabecular thickness leading to an al-
most 3-fold increased trabecular separation (Fig. 8, B–D). Whereas
dynamic histomorphometry revealed only very low bone turnover
in periarticular bone of normal individuals, as evident from the
scarcity of osteoclasts and osteoblasts, both cell types were dra-
matically increased in trabecular bone adjacent to inflamed joints
(Fig. 8, E–H).
Discussion
In this study, we describe the nature of bone marrow involvement
in RA. We show that inflammatory synovial tissue can completely
disrupt the cortical bone barrier resulting in a direct exposure of
the underlying bone marrow to the inflammatory infiltrate. The
consequence is a profound change of the adjacent bone marrow,
which is characterized by the replacement of fat-rich bone marrow
by a B cell-rich mononuclear cell aggregate. Such aggregates ap-
pear at the interface between invading synovial tissue and bone
marrow. Aggregates were only present if cortical bone had been
completely penetrated, but not underneath an intact cortical bar-
rier. Moreover, penetration of cortical bone and bone marrow B
cell aggregates were associated with an increased bone formation
at the endosteum, suggesting an attempt to repair the defect from
the inside (Fig. 9).
Although, destruction of juxtaarticular bone is a well known
feature of RA, which has been described since the 19th century,
the question, whether RA can affect the bone marrow space next to
inflamed joints has never been entirely clarified (17). This is pri-
marily based on the limited accessibility of material from human
joints, which besides the synovial membrane contains cartilage,
bone, and, most importantly, bone marrow. Most information from
the nature of synovitis comes from needle biopsies and synovec-
tomy, which however, does not include material from synovial
tissue invading bone or cartilage. Material from joint replacement
surgery is the only source containing sufficient amounts of material
to allow investigation of processes in deeper region of the joint. In
fact, studies on histological characterization of the junction zone
have elaborated that the synovial membrane has the potential to
invade subchondral regions of the joint (9, 18, 19). Moreover,
there is one case report of the occurrence of a T and B cell-rich
aggregate in this subchondral space (20). The subchondral region,
which represents the primary target of invading synovial tissue,
consists of wider areas of mineralized cartilage, which adjoins the
superficial unmineralized cartilage at its distal end (also called tide
mark) and the lamella of cortical bone at its proximal end. The
layer of cortical bone is comparatively thin and separates articular
cartilage from bone marrow. Thus, most of subchondral damage
seen histologically and radiologically is, in fact, due to loss of
mineralized cartilage, which in contrast to the unmineralized su-
perficial region of articular cartilage is susceptible to degradation
by osteoclasts. Whether the cortical barrier can be completely dis-
rupted and whether marrow space can be directly exposed to sy-
novial tissue had not been formally studied. Recent evidence from
magnetic resonance imaging studies, however, suggested the pos-
sibility of a bone marrow involvement in RA. Thus, bone marrow
alterations commonly termed as “bone marrow edema,” with high
signal intensity on STIR or T2-weighted fat suppressed images and
low signal intensity on T1-weighted images, are present in patients
with RA (21, 22). Extending these observations, our study pro-
vides direct histological evidence for cortical penetration and bone
marrow changes in RA.
We found localized mononuclear cell aggregates in the bone
marrow at sites of inflammatory tissue invasion. Surprisingly, B
cells were the leading cell type in these lesions. T cells were far
less frequent, and macrophages, granulocytes, and follicular den-
dritic cells were almost completely absent in these lesions. Thus,
these subcortical bone marrow aggregates constitute a subcompart-
ment with a unique cellular composition. Inflamed synovial tissue,
for example, has a completely distinct cellular composition with
abundant macrophages but only a small fraction of B cells. Even,
mononuclear cell aggregates within synovial inflammatory tissue
were different in cellular composition, showing a lower proportion
of B cells but more macrophages than bone marrow aggregates.
The majority of cells of the latter were mature B cells expressing
markers such as CD20, CD27, CD45RA, and CD79.
Although it has been known for many years that synovial tissue
of RA patients harbors B cells, the observation that the juxtaartic-
ular bone marrow serves as a pool of B cells in the direct neigh-
borhood of arthritis is interesting. It is known that B cells in the
FIGURE 7. Accumulation of osteoblasts and new bone formation at the endosteal bone surface next to subcortical bone marrow aggregates. A–D,
Undecalcified plastic embedded tissue sections stained by Movat pentachrome showing osteoid deposition (red, arrowhead) at the endosteal bone surface
next to subcortical bone marrow aggregates (A) and distant from aggregates (B). C and D show the respective close-up views. Endosteal surface from
undecalcified plastic embedded tissue sections stained according to Goldner trichrome (E), Movat pentachrome (F) and von Kossa (G) showing accumu-
lation of osteoblasts (black arrow) and osteoid deposition underneath (black arrowhead) close to subcortical bone marrow aggregates. A and C, Original
magnification ⫻100; B and D and E–G, original magnification ⫻400; C and F, original magnification ⫻400. H, Fraction of bone surface covered by osteoid
at endosteal sites with and without aggregates. Values are means ⫾ SEM; ⴱ, significant difference (p ⬍ 0.05).
2585The Journal of Immunology
synovial membrane of RA patients undergo affinity maturation,
somatic hypermutation, and terminal differentiation (23–28). B
cells are not only a source of rheumatoid factor production but also
support T cell activation and are producers of regulatory cytokines
such as IL-4 and IL-10 (29, 30). In ⬃60% of RA patients, aggre-
gates consisting of T and B cells are a major source of B cells in
the synovial tissue (23, 30). However, such aggregates are not
completely specific for RA; they are also found in ankylosing
spondylitis and even sometimes in osteoarthritis (31, 32). Synovial
aggregates develop early in RA and increase in frequency with
disease duration (33–35). Although, there are clear similarities be-
tween bone marrow aggregates and synovial aggregates, the
former do not represent classical germinal centers, as synovial ag-
gregates in less than one third of RA patients do (30, 36, 37).
Importantly, bone marrow aggregates did not contain a significant
number of follicular dendritic cells, which are a typical feature of
germinal centers.
At the interface between synovial inflammatory tissue and sub-
cortical bone marrow aggregates, plasma cell concentrations were
maximal. In contrast, only very limited numbers of plasma cells
were present in other compartments of the synovial membrane.
Plasma cells have been described to accumulate around germinal
center-like aggregates. This reflects the distribution as found in
subcortical aggregates, when synovial tissue comes in contact with
B cell rich bone marrow aggregates (24). As suggested by previous
experimental data, synovial tissue and neighboring mononuclear
aggregates appear to have an intensive cross talk. Thus, synovial
fibroblasts can support B cell survival by molecules such as
CXCL-12 (SDF-1) and VCAM-1, which is termed pseudoperi-
polesis (38– 42). Other chemokines expressed by synoviocytes
promote B cell recruitment, such as CXCL-13 (also termed
BCA-1) and CCL-21 (also termed 6Ckine) (30, 43–45). In fact,
expression of the two latter chemokines could be detected at the
front of synovial tissue invasion in the vicinity of subcortical ag-
gregates, and numerous MAdCAM-1-positive high endothelial
venules, which are important for B cell homing, were found within
aggregates (46). Moreover, synovial tissue is also a source of B
cell survival factors, such as BAFF (also termed BlyS), expression
of which was also found in synovial tissue close to aggregates (47).
These observations suggest that synovial tissue provide signals of
B cell chemotaxis, homing and activation, which allow the forma-
tion of bone marrow aggregates. Interestingly, as previously ob-
served in synovial B cells (48), B cells of subcortical bone marrow
aggregates were EBV-negative (data not shown).
Compelling evidence for new bone formation was found at sites
of bone marrow aggregates. Osteoblasts accumulated and covered
large areas of the endosteal bone with osteoid deposits. In contrast,
bone distant from these lesions showed only few scattered bone
formation sites. The exact same pattern of skeletal effects has been
observed in human TNF transgenic mice (15). There were no os-
teoclasts attached to the endosteum; however, numerous oste-
oclasts were embedded in synovial tissue attacking bone from the
subchondral side of compact bone. This observation indicates that
the synovial and subchondral side of the cortical bone is affected
by resorption, whereas formation, as an attempt to repair bone
comes from the inner endosteal area of bone. This observation also
supports the hypothesis that bone erosion starts from the outside
due to synovial inflammation rather than from the bone marrow.
The B cell-rich bone marrow aggregates, which occur exclu-
sively at sites of pannus penetration through cortical bone into the
bone marrow space, appear to “shield” bone marrow from the in-
vading synovial tissue by forming a physical barrier and attempt-
ing to elicit new bone formation.
Currently targeted therapy of B cells has become a promising
new tool for the treatment of RA. Rituximab, an Ab directed
against CD20 leads to depletion of B cells and has shown efficacy
in inhibiting signs and symptoms of RA (49, 50). Due to the strik-
ing accumulation of CD20-positive B cells, the subcortical bone
marrow aggregates will represent a target of rituximab therapy.
Although rituximab could thus eliminate a presumably protective
process, direct or systemic synovial effects of anti-CD20 may
counterbalance and exceed such potentially negative aspects of
FIGURE 8. Histomorphometric evidence for bone loss and high bone turnover in periarticular bone. Periarticular trabecular bone was assessed by
histomorphometry in a specimen from healthy normal individuals (normal, left bars) and patients with RA (right bars). The following parameters were
measured: percentage of bone volume of total volume (BV/TV) (A), trabecular thickness (Tb.Th) (B), trabecular number (Tb.N) (C), trabecular separation
(Tb.S) (D), percentage of osteoclast covered surface of bone surface (OcS/BS) (E), percentage of osteoblast covered surface of bone surface (ObS/BS) (F),
number of osteoclasts per bone perimeter (N.Oc/B.Pm) (G), and number of osteoblasts per bone perimeter (N.Ob/B.Pm) (H). Values are means ⫾ SEM;
ⴱ, significant difference (p ⬍ 0.05).
2586 SYNOVIAL INFLAMMATORY TISSUE AND BONE MARROW IN RA
this therapy. It will be interesting to learn about the effects of
rituximab on structural damage.
In summary, bone marrow can be regarded as a compartment,
which is actively involved in the disease process of RA. It harbors
cell aggregates if the cortical barrier is disrupted and is a source for
bone repair.
Disclosures
The authors have no financial conflict of interest.
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