ARTHRITIS & RHEUMATISM
Vol. 52, No. 11, November 2005, pp 3448–3459
Involvement of Subchondral Bone Marrow in
Rheumatoid Arthritis
Lymphoid Neogenesis and In Situ Relationship to
Subchondral Bone Marrow Osteoclast Recruitment
Serena Bugatti,1 Roberto Caporali,1 Antonio Manzo,2 Barbara Vitolo,1 Costantino Pitzalis,2
and Carlomaurizio Montecucco1
Objective. To evaluate the presence and immuno-
histochemical characteristics of subchondral bone mar-
row inflammatory infiltrate in rheumatoid arthritis
(RA) and to determine the in situ relationship between
marrow inflammation and osteoclast recruitment.
Methods. Bone samples and paired synovia from
8 RA patients undergoing joint surgery were analyzed by
immunohistochemistry and in situ hybridization for
specific lymphoid neogenetic features, such as T and B
cell composition, follicular dendritic cell (FDC) net-
works, peripheral lymph node addressin (PNAd)–
positive high endothelial venules, and lymphoid chemo-
kine expression. Osteoclasts were identified as
multinucleated tartrate-resistant acid phosphatase
(TRAP)–positive and cathepsin K–positive cells adher-
ent to the bone surface.
Results. An inflammatory infiltrate with perivas-
cular aggregates of variable size was detected in 7
(87.5%) of 8 synovial samples and in paired bone
samples. Lymphoid neogenetic features typical of rheu-
matoid synovium were also recognized in the bone
marrow. PNAdⴙ blood vessels were found in 4 of 8
patients, CD21ⴙ FDC networks in 2 patients,
Supported by Guy’s, King’s and St. Thomas’ Charitable
Foundation and IRCCS Policlinico S. Matteo.
1
Serena Bugatti, MD, Roberto Caporali, MD, Barbara Vi-
tolo, BSc, Carlomaurizio Montecucco, MD: University of Pavia,
IRCCS Policlinico S. Matteo, Pavia, Italy; 2Antonio Manzo, MD,
Costantino Pitzalis, MD: Guy’s, King’s and St Thomas’ School of
Medicine, Guy’s Campus, London, UK.
Address correspondence and reprint requests to Carlomauri-
zio Montecucco, MD, Cattedra di Reumatologia, IRCCS Policlinico S.
Matteo, P.le Golgi 2, 27100 Pavia, Italy. E-mail: montecucco@
smatteo.pv.it.
Submitted for publication February 25, 2005; accepted in
revised form July 21, 2005.
3448
DOI 10.1002/art.21377
© 2005, American College of Rheumatology
CXCL13ⴙ cells in 5 patients, and CCL21ⴙ cells in 6
patients. TRAP-positive and cathepsin K–positive oste-
oclasts were identified on both the synovial and marrow
sides of the bone surface. Bone marrow samples show-
ing a higher degree of inflammation were characterized
by a significantly increased number of osteoclasts ad-
herent to the subchondral bone.
Conclusion. Our data demonstrate that lymphoid
aggregates with lymphoid neogenetic features are detect-
able on the subchondral side of the joint in established RA.
Moreover, the local inflammation/aggregation process
appears to be related to osteoclast differentiation on the
marrow side of subchondral bone, supporting a func-
tional role of the bone compartment in local damage.
Rheumatoid arthritis (RA) is a chronic inflam-
matory polyarthritis characterized by persistent inflam-
mation of the synovium and destruction of the joint (1).
The hallmark of the disease is the development of
juxtaarticular erosions, which are typically located in
bare areas, at the interface between articular cartilage
and bone. Of note, this region is adjacent to the under-
lying bone marrow. Osteoclasts have been shown to be
the main cell type responsible for focal bone loss in RA
(2,3), and their differentiation and activation occur in
the same molecular pathways (RANK/RANKL system)
that control normal bone remodeling (3–6).
Local bone erosion is driven by the inflamed
synovial tissue. In addition to the well-known pathoge-
netic role of synovial pannus, it also has been suggested
that the infiltrated synovium contributes to the process
of joint damage by creating the microenvironment in
which inflammatory immune cells can interact, exert
effector functions, and up-regulate inflammatory medi-
SUBCHONDRAL BONE MARROW INVOLVEMENT IN RA 3449

Table 1. Demographic and histologic features of the study population*

Bone marrow scores

Disease RF Anti-CCP Erosions in Inflammation score, Osteoclast count,

Patient/age/sex duration, years status status hands/feet Surgery site/procedure mean ⫾ SEM mean ⫾ SEM
RA1/63/F 15 Pos. Pos. Yes Wrist/arthrodesis 1.9 ⫾ 0.3 2.1 ⫾ 0.4
RA2/52/F 13 Pos. Pos. Yes Knee/joint replacement 1.0 ⫾ 0.0 1.9 ⫾ 0.2
RA3/79/F 10 Pos. Neg. Yes Hip/joint replacement 0.5 ⫾ 0.3 0.7 ⫾ 0.2
RA4/47/F 34 Pos. Pos. Yes MCP/joint replacement 1.5 ⫾ 0.3 1.8 ⫾ 0.2
RA5/39/F 12 Neg. Pos. Yes Knee/joint replacement 3.1 ⫾ 0.3 4.5 ⫾ 0.5
RA6/57/F 16 Pos. Neg. No Knee/joint replacement 0.2 ⫾ 0.2 0.1 ⫾ 0.1
RA7/31/M 8 Pos. Pos. Yes Hip/joint replacement 1.0 ⫾ 0.0 3.2 ⫾ 0.2
RA8/72/F 5 Neg. Neg. Yes MTP/joint resection 3.3 ⫾ 0.5 2.5 ⫾ 0.2
OA1/61/F – Neg. NA No Hip/joint replacement 0 0.4 ⫾ 0.2
OA2/67/F – Neg. NA No Hip/joint replacement 0 0.6 ⫾ 0.4
OA3/79/F – Neg. NA No Hip/joint replacement 0 0.1 ⫾ 0.1
OA4/69/M – Neg. NA No Hip/joint replacement 0 0.6 ⫾ 0.2

* RF ⫽ rheumatoid factor; anti-CCP ⫽ anti–cyclic citrullinated peptide; RA ⫽ rheumatoid arthritis; MCP ⫽ metacarpophalangeal; MTP ⫽
metatarsophalangeal; OA ⫽ osteoarthritis; NA ⫽ not available.

ators (7). In accordance with this concept, the develop-
ment of lymphoid aggregates in RA synovium has been
correlated with a worse clinical and radiologic outcome
(8). Furthermore, it has been shown that patients with
synovitis characterized by aggregates or well-defined
germinal centers (GCs) have a different pattern of
cytokine production compared with that in the diffuse
forms of synovitis, in that increased local expression of
interleukin-1 (IL-1), IL-6, and tumor necrosis factor
(TNF) (9,10), which are known to be involved in oste-
oclast differentiation (11), is evident. Moreover, T lym-
phocytes infiltrating the synovium are an important,
although not unique, source of RANKL, which ulti-
mately leads to differentiation and activation of oste-
oclasts (12,13).
However, the synovial model of bone erosion is
not sufficient to entirely explain the pathogenesis of
joint damage, and the relationship between synovitis and
erosions still remains controversial (14–16). Recent at-
tention has been focused on the subchondral side of the
joint. An involvement of subchondral bone marrow has
been described in different animal models of inflamma-
tory arthritis (17,18). In humans, data from magnetic
resonance imaging (MRI) analyses of RA joints show
that bone marrow is affected early in the disease course
and spatially and temporally related to bone erosions
(19,20). However, the nature and role of such changes to
the bone marrow still remain unclear in human disease,
despite evidence of subchondral bone osteoclasts (21)
and bone marrow inflammatory infiltrate (22–25) in
established RA.
In the present study, we performed the first in
situ characterization of the inflammatory/organizational
features of subchondral bone marrow in RA, and de-
fined their local relationship to osteoclast distribution.
We found that lymphocyte infiltrates are consistently
present in the bone marrow compartment, where they
can acquire, as in the synovium, lymphoid neogenetic
features. Consistent with this observation, we demon-
strated, inside marrow lymphoid aggregates, the expres-
sion of the chemokines CXCL13 and CCL21, which are
physiologically involved in the functional organization of
secondary lymphoid organs (26,27). We also observed
that these lymphoid aggregates are significantly related
to histologic evidence of increased subchondral oste-
oclast recruitment.
PATIENTS AND METHODS
Patients. Eight patients fulfilling the American College
of Rheumatology (formerly, the American Rheumatism Asso-
ciation) 1987 revised criteria for the classification of RA (28)
were included in the study. Four patients who underwent total
joint replacement for hip osteoarthritis (OA) were used as
controls. The demographic and clinical features of the patients
and controls are summarized in Table 1.
Collection, processing, and storage of the synovial and
bone specimens. Bone samples and paired synovial tissues
were obtained from RA patients at the time of arthrodesis,
joint resection, or joint replacement. Samples of articular
cartilage and subchondral bone obtained from OA patients
during total hip arthroplasty served as controls for the bone
study. For each patient, more than 1 sample of synovial and
bone tissue was collected. Human tonsils were used as positive
controls for immunohistochemistry and in situ hybridization.
All procedures were performed for diagnostic or therapeutic
purposes after the patients gave their informed consent, with
approval provided by the local ethics committee.
All samples were fixed in 10% paraformaldehyde (pH
7.0) for 24 hours at room temperature (RT). Bone samples
3450
underwent decalcification in 10% formic acid at RT for the
time necessary to reach the appropriate consistency. Consec-
utive 5-␮m–thick sections of each sample were mounted on
L-polylysine–coated slides and kept at RT for further analysis.
For histologic evaluation, 1 slide for each sample was stained
with hematoxylin and eosin (H&E).
Immunohistochemistry. Formalin-fixed, paraffin-
embedded tissue sections were deparaffinized and rehydrated
through graded ethanol solutions. Once hydrated, sections
were heated for 35 minutes at 96°C in Dako Target Retrieval
Solution (S1700; Dako, Carpinteria, CA) or, for CD21 antigen
detection, were incubated for 20 minutes at 37°C with pre-
warmed 0.05% Pronase (S2013; Dako) in Tris buffered saline
(TBS) (pH 7.6). The sections were then washed in TBS and
incubated for 10 minutes with Protein Block Serum Free
(X0909; Dako).
The following primary antibodies were used: rabbit
anti-human CD3 polyclonal Ig (N1580; Dako), mouse anti-
human CD20 (IgG2a, clone L26; Dako), mouse anti-human
CD21 (IgG1, clone 1F8; Dako), goat anti-human CXCL13
polyclonal IgG (AF801; R&D Systems, Minneapolis, MN),
goat anti-human CCL21 polyclonal IgG (AF366; R&D Sys-
tems), mouse anti-human CD31 (IgG1, clone 1A10; Novocas-
tra, Newcastle-upon-Tyne, UK), rat anti-human/mouse periph-
eral lymph node addressin (PNAd) (IgM, clone MECA 79;
PharMingen, San Diego, CA), mouse anti-human tartrate-
resistant acid phosphatase (TRAP) (IgG2b, clone 26E5; No-
vocastra), mouse anti-human cathepsin K (clone CK4; Novo-
castra), mouse anti-human parathyroid hormone receptor
(PTH-R) (clone 3D1.1; Santa Cruz Biotechnology, Santa Cruz,
CA), mouse anti-human CD235a, glycophorin A (IgG1, clone
JC159; Dako), and mouse anti-human CD61 platelet glyco-
protein IIIa (IgG1, clone Y2/51; Dako). Sections were then
incubated with the appropriate biotinylated secondary anti-
body (E0431 swine anti-rabbit Ig, E0413 rabbit anti-mouse Ig,
E0466 rabbit anti-goat Ig, or E0468 rabbit anti-rat Ig; all from
Dako) for 30 minutes followed by streptavidin–biotin
complex–alkaline phosphatase (K0391; Dako) for an addi-
tional 30 minutes. Staining of sections was developed using a
New Fuchsin substrate kit (K0625; Dako), counterstained with
Mayer’s hematoxylin (1.09249; Merck, Rahway, NJ), and
mounted with Aquamount mounting medium (BDH, Poole,
UK). Primary and secondary antibodies were diluted in Dako
Antibody Diluent (S3022; Dako). Negative-staining control
experiments were performed either by omitting the primary
antibody or by using a control isotype-matched antibody.
In situ hybridization. In situ hybridization was per-
formed on RA synovium to detect production of the 2 chemo-
kines CXCL13 and CCL21. Sense and antisense 35S-labeled
CXCL13 or CCL21 RNA probes, 368 bp or 374 bp in length,
respectively, were generated by in vitro transcription (Roche
Molecular Biochemicals, Indianapolis, IN). The CXCL13
probe corresponded to position ⫺24 to 344 of the CXCL13
sequence, and the CCL21 probe corresponded to position
6–368 of the CCL21 sequence. Formalin-fixed, paraffin-
embedded tissue sections were dewaxed, rehydrated in graded
ethanol solutions, and subjected to in situ hybridization as
previously described (29). Finally, the sections were dipped in
Kodak photo emulsion NTB-2 (Eastman Kodak, Rochester,
NY) and exposed in complete darkness for 5 weeks at 4°C;
development and fixation were performed according to the
BUGATTI ET AL
instructions provided by Kodak. Sections were counterstained
with hematoxylin.
Cellular analysis and scoring. Specific lymphoid fea-
tures of cellular aggregates in RA synovium and subchondral
bone marrow were assessed by staining formalin-fixed,
paraffin-embedded consecutive sections for CD3, CD20,
CD21, PNAd, CXCL13, and CCL21. On RA and OA bone
samples, immunostainings for TRAP, cathepsin K, and PTH-R
on serial sections were added to the above-mentioned series of
stainings.
Aggregates were classified as segregated if T cell–B
cell enrichment areas were predominant, classified as positive
for follicular dendritic cell (FDC) networks when a CD21-
positive cluster of reticular cells was detectable, and classified
as positive for CXCL13, CCL21, and PNAd when at least 1 cell
or 1 blood vessel was found to be positive within the aggregate.
Osteoclasts were identified as multinucleated (at least
2 nuclei) TRAP⫹ cells adherent to the bone surface. Cathep-
sin K was used as an additional discriminative marker for
osteoclasts, to evaluate their proteinase expression. Osteo-
blasts were identified as cuboid-shaped cells on bone surfaces,
both by H&E staining and after immunostaining for PTH-R.
The degree of infiltration into the bone marrow was
assessed using a semiquantitative scoring system based on
H&E analysis and immunostaining for CD3 and CD20 (mar-
row inflammation score 0 ⫽ normal tissues; score 1 ⫽ minimal
infiltration; score 2 ⫽ mild infiltration; score 3 ⫽ moderate
infiltration; score 4 ⫽ marked infiltration; score 5 [never
observed in our samples] ⫽ severe infiltration [30]). The mean
number of osteoclasts was determined at 40⫻ magnification in
5 fields, in areas of active bone resorption (if present) both on
the synovial side and on the subchondral marrow side of the
joint, in sections consecutive to the ones used for determina-
tion of the marrow inflammation score (30). For each patient,
at least 2 different cutting levels (150 ␮m apart) in at least 2
independent samples were studied. A parallel inflammation
score and osteoclast count were determined in each cutting
level analyzed, resulting in a total of 39 distinct joint areas
analyzed in the whole study population. The relationship
between the synovial osteoclast and the marrow osteoclast was
defined within each patient by calculating the mean osteoclast
number in all available sections.
Statistical analysis. Correlation between the bone
marrow osteoclast number and marrow inflammation score
was evaluated by Spearman’s rank correlation test, using
INSTAT software (GraphPad Software, San Diego, CA).
RESULTS
Features of RA synovial samples. Synovial tissues
from 8 RA patients were analyzed for organizational
features of the inflammatory infiltrate. Although the
degree of tissue cell infiltrate was different among
different patients, in 7 of 8 specimens perivascular
clusters of CD20⫹ B cells and CD3⫹ T cells could be
clearly distinguished. Such lymphoid clusters exhibited
either an intermixed distribution of T and B cells or a
fully developed compartmentalization, with a central
SUBCHONDRAL BONE MARROW INVOLVEMENT IN RA 3451

Figure 1. Paraffin-embedded sequential sections of rheumatoid arthritis synovial tissue showing positivity
for CXCL13 protein (A) and mRNA (B) and CCL21 protein (C) and mRNA (D) by immunostaining (A
and C, red) and in situ hybridization (B and D, black). CXCL13⫹ and CCL21⫹ cells were localized in
sublining areas, characterized by mononuclear inflammatory infiltrate with a strong association with large
cellular aggregates. Note the stringent matching between protein and mRNA expression for both
chemokines. (Original magnification ⫻ 20.)

area of B cell enrichment surrounded by a peripheral
area of T cells. T lymphocytes were also diffusely
distributed in the synovium sublining, while B lympho-
cytes were mostly present within clusters. In one sample
we observed the presence of a CD21⫹ FDC network,
indicative of GC reaction (31). PNAd⫹ vessels with the
morphologic characteristics of high endothelial venules
(HEVs) were observed inside and/or at the periphery of
larger aggregates in 6 (75%) of 8 patients.
In 7 (87.5%) of 8 patients, intraaggregate expres-
sion of CXCL13 could be seen, mainly in B cell areas of
larger aggregates (Figure 1A). The only sample without
cellular CXCL13 was characterized by a low degree of
infiltration. With regard to CCL21 expression, cells
positive for CCL21 could be distinguished in 7 (87.5%)
of 8 patients, with their location in perivascular areas
and in T cell areas of some aggregates (Figure 1C),
whereas CCL21 could not be detected in the only tissue
also lacking CXCL13. In situ hybridization and immu-
nohistochemical experiments performed on parallel sec-
tions demonstrated a consistent matching for the expres-
sion patterns of CXCL13 and CCL21 protein and
messenger RNA (mRNA) (Figures 1A–D), confirming
that the lymphoid chemokines CXCL13 and CCL21 are
produced inside lymphoid aggregates in RA synovium.
Signs of local inflammation in RA subchondral
bone marrow. Preliminary H&E analysis showed a vari-
able degree of cellularity in all RA bone samples.
However, this cellularity had different organizational
forms. The 3 samples obtained from the wrist, meta-
carpophalangeal (MCP), and metatarsophalangeal
(MTP) joints exhibited mononuclear aggregates within
noninfiltrated adipose marrow (Figure 2A), whereas the
5 samples from large joints (hip and knee) had a diffuse,
3452 BUGATTI ET AL

Figure 2. Histologic evaluation of subchondral bone marrow inflammation in rheumatoid arthritis (RA).
Staining of sections of subchondral bone marrow (with hematoxylin and eosin [H&E]) revealed 2 different
patterns of mononuclear infiltration. Tissues obtained from the wrist, metacarpophalangeal (MCP), and
metatarsophalangeal (MTP) joints showed patchy mononuclear infiltrates within uninflamed adipose marrow
(A), whereas tissues from the knees and hips showed heterogeneous cell infiltrates among adipocytes (D). This
heterogeneous population exhibited morphologic features of hematopoietic bone marrow, with myeloid cells,
erythroblasts, and megakaryocytes (inset in D; original magnification ⫻ 100). Immunostaining for CD20 and
CD3 on sequential paraffin-embedded sections of RA samples obtained from nonhematopoietic sites (wrist,
MCP, and MTP joints) (B and C, red) revealed that patchy infiltrates detectable by H&E were mainly composed
of B and T lymphocytes. In RA samples obtained from hematopoietic sites (knees and hips), sequential
immunostaining for CD20 and CD3 (E and F, red) showed that lymphocyte aggregates of B and T cells were
detectable within the hematopoietic bone marrow. (Original magnification ⫻ 10 in A–D; ⫻ 20 in E and F.)

heterogeneous cellular population occupying the
spaces among adipocytes in subchondral marrow (Figure
2D). This diffuse population of cells observed in large
joints showed morphologic features of hematopoietic
bone marrow, with myeloid cells, erythroblasts, and
megakaryocytes being identified (Figure 2D, inset). Im-
munohistochemical staining for glycophorin and CD61
confirmed the hematopoietic nature of these cells (re-
sults not shown).
Immunohistochemical experiments using mark-
ers of B cells and T cells (CD20 and CD3, respectively)
revealed that lymphoid aggregates were present in all
but 1 bone specimen from the RA joints. In samples
obtained from nonhematopoietic sites (wrist, MCP, and
MTP joints), lymphoid aggregates were located within
the adipose bone marrow (Figures 2B and C), whereas
the bone sites that normally exhibit hematopoietic activ-
ity (hip and knee) showed lymphoid aggregates within
the hematopoietic population (Figures 2E and F). In
control OA samples, hematopoietic bone marrow was
detectable in only a few samples from each patient
(average 50% of samples per patient), and no B cell–T
cell aggregates were detected (Figures 3A and B).
Marrow aggregates in RA bone were more fre-
quently located in the superficial bone marrow, in
proximity to sites where synovial tissue invaded the
subchondral bone and came in contact with the under-
lying bone marrow (Figure 3C). Notably, this superficial
distribution was not exclusive, since some lymphoid
aggregates were also present in deep marrow, appearing
in areas distant from the synovial–marrow junction
(Figure 3D).
Histologic study of the bone–pannus junction.
Since the RA bone samples had portions of synovial
tissue from the junction zone, we were prompted to
extend the histologic and immunohistochemical charac-
terization to this area. Despite the presence of marrow
lymphoid aggregates in proximity to areas of synovial
invasion, the adjacent synovial pannus in these areas was
mainly fibrous, and immunostaining for B and T lym-
SUBCHONDRAL BONE MARROW INVOLVEMENT IN RA 3453

Figure 3. Histologic staining of sections (with hematoxylin and eosin [H&E]) from patients with osteoarthritis (OA).
No histologic erosions were evident (A), and when immunostaining for CD20 was performed, no B cells were
detectable in OA subchondral bone marrow (B). A serial section stained for T cells (CD3) on the same OA sample
also demonstrated the absence of lymphoid aggregates (inset in B; original magnification ⫻ 20). H&E staining of
rheumatoid arthritis (RA) sections showed a marrow lymphoid aggregate in superficial bone marrow, in proximity to
the invasive synovial pannus (C). Note that the bone surface is interrupted by pannus penetration; arrows indicate the
histologic erosion. Immunostaining of RA sections for CD20 (D, red) revealed the presence of a marrow lymphoid
aggregate in bone marrow distant from areas of synovial invasion, with no histologic evidence of bone erosions.
Lymphoid aggregates, predominantly constituted of B cells (E, red) together with rare T cells (F, red), were observed
at the synovial pannus–bone marrow boundary in an area of bone marrow invasion. sp ⫽ synovial pannus; b ⫽ bone;
c ⫽ cartilage; m ⫽ bone marrow. (Original magnification ⫻ 10.)

phocytes did not reveal significant lymphoid infiltration.
However, in some cases large lymphoid aggregates could
be observed even more superficially, within the infiltrat-
ing synovial tissue (Figures 3E and F).
Signs of lymphoid neogenesis in bone marrow
infiltrates. Since the presence of lymphoid aggregates
was demonstrated in the marrow environment, we ascer-
tained whether these structures could acquire, as in the
synovial tissue, lymphoid neogenetic features, such as
the presence of some degree of T cell–B cell compart-
mentalization, PNAd⫹ vessels, CD21⫹ FDC networks,
and the local expression of the lymphoid chemokines
CXCL13 and CCL21.
PNAd⫹ blood vessels were found in 4 (50%) of 8
patients (Figure 4A). However, PNAd⫹ blood vessels
were much less frequent (average of 1 vessel per sample)
than those found in paired synovia, and were predomi-
nantly located inside the aggregates closer to the super-
ficial subchondral bone, near the bone–pannus junction.
Sequential staining with a panvascular marker (CD31)
confirmed the vascular nature of the PNAd⫹ structures
(results not shown).
CD21⫹ networks were observed in 2 patients
(25%) (Figure 4B). Notably, in 1 case a CD21⫹ FDC
network was also present in the paired synovial tissue.
The 2 bone samples with marrow aggregates in which
CD21⫹ FDCs could be observed were characterized by
T cell–B cell compartmentalization (Figure 4B, insets),
and 1 of the 2 was observed to have a PNAd⫹ blood
vessel.
CXCL13-producing cells were found inside some
marrow aggregates (Figure 4C) in 5 (62.5%) of 8
samples. Consistent with our previous observations in
RA synovium (32), CXCL13⫹ cells were detectable
both inside the aggregates lacking CD21⫹ FDC net-
works and inside the aggregates with a GC reaction.
Similarly, CCL21-producing cells (Figure 4D) were ob-
served in some aggregates in 6 (75%) of 8 samples.
CCL21 expression showed a scattered distribution
within cellular aggregates, which did not appear to be
3454 BUGATTI ET AL

Figure 4. Lymphoid neogenetic features of subchondral bone marrow aggregates. A, Peripheral lymph
node addressin–positive blood vessel inside a lymphoid aggregate. B, A network of CD21⫹ follicular
dendritic cells inside an aggregate of large size, indicating the presence of a germinal center reaction. In
the same aggregate, the analysis of serial sections stained for CD20 and CD3 reveals some degree of T
cell–B cell compartmentalization (insets in B; original magnification ⫻ 10). C, CXCL13-expressing cells
inside a marrow lymphoid aggregate; also shown at high magnification (inset in C; original magnifica-
tion ⫻ 100). D, CCL21-expressing cells inside the same aggregate as in C, in a sequential section; also
shown at high magnification (inset in D; original magnification ⫻ 100). (Original magnification ⫻ 40 in
A and B; ⫻ 20 in C and D.)

related to vascular structures. No CCL21⫹ vessels were
found inside the subchondral bone marrow. Analysis of
serial sections demonstrated the possible coexistence of
CXCL13 and CCL21 ectopic expression in the same
marrow aggregates (Figures 4C and D).
Correlation of the degree of subchondral bone
marrow inflammation with osteoclast recruitment on
subchondral bone. Following morphologic analysis and
detection of the osteoclast marker TRAP, we identified
multinucleated cells both on the synovial side and on the
marrow side of bone in RA samples (Figures 5A and B).
In control OA samples, just a few, scattered multinucle-
ated TRAP⫹ cells were present in subchondral loca-
tions, as expected in physiologic bone-remodeling con-
ditions (results not shown).
We thus assessed the frequency of bone marrow
lymphoid aggregates for a correlation with the number
of multinucleated TRAP⫹ cells in the subchondral
location. The analysis was performed in every patient by
comparing the histologic score for bone marrow infiltra-
tion with the osteoclast number in adjacent joint areas.
Within each patient we observed similar trends between
the marrow inflammation score and the osteoclast score,
when different samples and different cutting levels were
analyzed (Table 1).
In analyses comparing the extent of bone marrow
inflammation and subchondral osteoclast density in dif-
ferent joint areas within each patient, tissue areas show-
ing a higher degree of marrow aggregates were charac-
terized by an increased number of osteoclasts adherent
to the subchondral bone (Figure 5C). This association
was highly significant, as inferred by Spearman’s rank
correlation (r ⫽ 0.7991, 95% confidence interval 0.6409–
0.8922, P ⬍ 0.0001). Accordingly, osteoclasts were more
SUBCHONDRAL BONE MARROW INVOLVEMENT IN RA 3455

Figure 5. Detection of osteoclasts on both the synovial side (A) and the subchondral bone marrow side
(B) of the joint in rheumatoid arthritis (RA). A high-magnification view of the multinucleated
tartrate-resistant acid phosphatase (TRAP)–positive cells (osteoclasts, indicated by arrow) on the synovial
side is shown (inset in A). Serial staining for TRAP and cathepsin K was performed on subchondral
marrow osteoclasts (insets in B). Note that all TRAP⫹ multinucleated cells display cathepsin K
immunostaining with high intensity (asterisks in insets of B). b ⫽ cortical bone; m ⫽ bone marrow; s ⫽
synovium. (Original magnification ⫻ 40 in A; ⫻ 100 in B.) A positive correlation (Spearman’s r ⫽ 0.7991,
95% confidence interval 0.6409–0.8922, P ⬍ 0.0001) was found between the mean ⫾ SEM inflammation/
aggregation score and the local frequency of osteoclasts in subchondral bone marrow (C). The distribution
of subchondral bone marrow and synovial osteoclasts was compared in each RA patient (D). Bars show
the osteoclast count calculated in the different joint areas in each patient. In most cases the frequency of
synovial osteoclasts was only slightly higher than that of subchondral bone marrow osteoclasts.

numerous at bone sites next to the aggregates, even
though they could also be detected far from the local
inflammation. The only bone sample lacking lymphoid
aggregates was characterized by a very low osteoclast
score, similar to that found in OA joints. This sample
had been obtained from a patient who did not show
radiographic signs of erosions (see Table 1).
By comparing synovial and subchondral bone
marrow osteoclasts, we found that in all but 1 patient,
osteoclasts were more numerous on the synovial side,
although the difference was not marked (Figure 5D). In
some cases bone marrow osteoclasts exhibited morpho-
logic differences from those on the synovial side, in that
osteoclasts on the marrow side were flatter and con-
tained less nuclei. However, osteoclasts in both compart-
ments showed the same proteinase activity, since they
3456
displayed cathepsin K expression with similar intensity.
Serial staining for TRAP and cathepsin K on marrow
osteoclasts is represented in Figure 5B (insets).
On endosteal bone surfaces of RA samples,
accumulation of cuboid PTH-R⫹ osteoblasts was ob-
served in close association with areas of bone erosion.
Osteoblasts could be found both next to marrow lym-
phoid aggregates and on endosteal surfaces devoid of
underlying marrow inflammation. However, a significant
osteoblast population was observed in only those sam-
ples showing histologic erosions and marrow lymphoid
aggregates. In the control OA samples, the osteoblast
frequency had a wider variability. In 2 OA patients
(OA2 and OA4 in Table 1), endosteal bone was lined
with a significant number of osteoblasts, whereas osteo-
blasts were almost completely absent in the other 2
patients (OA1 and OA3 in Table 1).
DISCUSSION
In the present study, we show that inflammatory
changes similar to those found in RA synovium may
occur in the subchondral bone marrow of the involved
RA joints. Subchondral bone marrow lymphoid aggre-
gates were detectable in all patients with erosive arthri-
tis, whereas this feature was not observed in patients
with noninflammatory arthritis, such as OA. Moreover,
the process of local inflammation seemed to be related
to the recruitment of osteoclasts at the marrow side of
subchondral bone, thus suggesting a potential role of the
bone compartment in local damage.
One limit of our analysis is that we studied only
samples obtained during surgical procedures carried out
in patients with longstanding disease. Consequently, we
do not know whether the subchondral changes observed
are an early phenomenon in RA, which would imply
involvement in the pathogenesis of erosions, or whether
they are actually a late consequence of advanced disease.
Histopathologic studies of early bone lesions, however,
are difficult because of obvious problems in finding
adequate samples from patients with early arthritis.
Nevertheless, an involvement of subchondral bone mar-
row in RA at its onset is indirectly supported by MRI
studies, which have shown that bone marrow edema is
an early finding and functions as a predictor of radio-
graphic damage (19,20,33). Since regions of bone edema
show some MRI aspects similar to those of active
synovitis, it has been postulated that both may have a
similar inflammatory basis (20).
To our knowledge, only sporadic histologic de-
scriptions of the subchondral side of the bone in RA are
BUGATTI ET AL
available. More than 20 years ago, Wyllie (22) observed
that lesions in the subchondral marrow of metatarsal
and metacarpal heads in advanced RA were character-
ized by infiltrates of macrophages, lymphocytes, and
plasma cells. Subsequently, other authors suggested that
inflammatory changes occur in RA bone marrow (23–
25), and that proteinases and proinflammatory cytokines
are present in the subchondral region (25,34–36). How-
ever, a systematic analysis of the subchondral side has
not been performed.
We describe herein our findings that, at least in
longstanding RA, subchondral bone marrow exhibits
cellular aggregates, mainly comprising B and T lympho-
cytes. Bone marrow lymphoid aggregates seem to be a
specific feature of inflammatory arthritis, since they
were not detectable in the OA control samples (only a
diffuse hematopoietic cell population was observed).
Unfortunately, no data on this feature in normal joints
are available. It is of interest to point out that, in
epiphyseal regions of long bones, which is a normal
hematopoietic site in adults, the hematopoietic bone
marrow was more prevalent in RA samples compared
with OA samples. This could be attributable either to
the more advanced age of the OA patients or to local
production of hematopoietic growth factors by the in-
flamed synovial tissue in RA. One possible candidate for
the latter role is granulocyte–macrophage colony-
stimulating factor, which is an important regulator of
myelopoeisis and a mediator of inflammation and local
tissue damage in RA (37,38), thus representing a key
element of a possible hematopoietic growth factor net-
work active at sites of inflammation.
Despite the common finding of signs of marrow
inflammation, our bone samples differed in the degree
of lymphoid aggregation. In fact, in some cases only a
few aggregates were present, whereas in other cases
there was a large amount of lymphoid aggregates within
the subchondral bone marrow. For each patient, how-
ever, the degree of marrow inflammation was similar
between samples and at different cutting levels, thus
suggesting that in RA, the process of marrow inflamma-
tion might be heterogeneously regulated, as indicated by
the organizational features of synovial inflammation
(39).
Of note, intramarrow lymphoid aggregates were
predominantly found in the superficial bone marrow, at
the interface between synovial tissue and the subchon-
dral bone marrow. Moreover, we observed that synovial
pannus, usually devoid of lymphoid cells, could become
highly inflamed at sites of bone marrow invasion, in
conjunction with the presence of large lymphoid aggre-
SUBCHONDRAL BONE MARROW INVOLVEMENT IN RA
gates. These findings suggest that bone marrow inflam-
mation is a consequence of the invasion of juxtaarticular
bone marrow by synovial tissue. This would be consistent
with recent findings in animal models. Human TNF–
transgenic mice, for instance, were observed to have
inflammatory bone marrow infiltrates of B cells adjacent
to inflamed joints, located at the interface between
synovial inflammatory tissue and bone marrow and only
occurring when the cortical bone barrier was disrupted
by local bone erosions (18). However, in our samples,
the simultaneous presence of some lymphoid aggregates
in deep areas of the bone marrow, at sites apparently
distant from synovial invasion, does not exclude the
possibility of an independent inflammatory process in
RA subchondral bone. This is supported by indirect
evidence that periarticular osteoporosis imaged on plain
radiographs and bone marrow edema demonstrated on
MRI occur before the development of detectable ero-
sions (19,20).
At present we do not know whether these mar-
row aggregates that are apparently unrelated to synovial
invasion could arise de novo from within the bone
marrow or whether they gain access to the bone marrow
through other anatomic structures, allowing communi-
cation between the 2 compartments. In collagen-induced
arthritis (CIA), cortical bone canals connecting the bone
marrow to the synovium have been shown to increase in
size in early inflammatory lesions and to mediate the
transmigration of mesenchymal cells from marrow to
synovium (40). In this hypothetical dynamic, the same
canals could also promote the early exchange of inflam-
matory cells between the 2 sides.
It recently has been noted that the synovial
inflammatory infiltrate may undergo a process of orga-
nization in which it acquires the structural features of
secondary lymphoid organs (32,39). Such a phenome-
non, known as lymphoid neogenesis, has been described
in RA and in many other chronic inflammatory/auto-
immune conditions (41). Consistent with the currently
available data (32), our synovial samples were charac-
terized by a variable degree of lymphoid organization. A
follicular synovitis with CD21⫹ networks was observed
in one case. PNAd⫹ HEVs were found in the majority
of synovial tissues, and all but 1 sample exhibited the
lymphoid chemokines CXCL13 and CCL21, both at the
protein level and at the mRNA level.
More interestingly, our bone marrow study re-
vealed that a similar organizational process also occurs
in the subchondral side of the joint. Although a follicle
with GCs was previously described in RA bone marrow
(23), no other features apart from the morphologic
3457
aspect were used to characterize this structure. In 2 cases
we detected a network of CD21⫹ FDCs, indicating the
presence of a true GC reaction (31), and in half of our
samples, PNAd⫹ HEVs were distinguishable. Further-
more, CXCL13- and CCL21-expressing cells were found
within some marrow aggregates. These results demon-
strate, for the first time, that in the marrow environment,
a process of lymphoid neogenesis can take place, with
the achievement of a high degree of structural organi-
zation and the local ectopic expression of the lymphoid
chemokines CXCL13 and CCL21.
An important issue is whether the stromal envi-
ronment, which has been shown to promote both the
survival and the accumulation of lymphocytes in the
synovium (42), has a similar functional role in the
marrow environment. Although this has not been spe-
cifically addressed in the present study, previous studies
have shown that stromal cells from subchondral bone
marrow of RA and OA patients may produce inflamma-
tory chemokines, which are up-regulated by IL-1␤ and
TNF␣ (35).
Consistent with previous findings (21,43), we
detected bone-adherent osteoclasts not only on the
synovial side, but also on the marrow side. We observed
a positive correlation between the degree of subchondral
lymphoid aggregation and the recruitment of oste-
oclasts, with the more infiltrated samples containing a
larger amount of osteoclasts on the bone marrow side.
To our knowledge, this association between marrow
lymphoid aggregates and subchondral erosions has not
been previously described in RA, and is in accordance
with the data obtained from animal models of arthritis.
In CIA, GCs have been recognized in the subchondral
bone, and their presence is closely associated with areas
of bone erosion (17). More recently, bone erosions filled
with osteoclasts located next to bone marrow infiltrates
have been described in human TNF–transgenic mice
(18).
This evidence allows us to assume that both
synovial mechanisms and marrow inflammatory changes
might lead to the development of bone erosions in RA.
Subchondral bone osteoclasts in our samples seemed to
play an important role in bone damage, since their
number was only slightly lower in absolute terms than on
the synovial side. Moreover, cathepsin K immunostain-
ing was similar in both sites, suggesting that bone
marrow osteoclasts have an essential proteinase (44) and
may participate in local erosion. However, further func-
tional studies are needed to address this issue.
The presence of osteoblasts on the subchondral
side of the joint at sites of bone erosions and in samples
3458
characterized by marrow inflammation suggests that
bone damage in RA might be associated, at least in part,
with an attempt at healing and is consistent with the
occurrence of enhanced endosteal bone formation in
human TNF–transgenic mice that exhibit marrow lym-
phoid infiltrates (18). Evidence that radiographic bone
erosions in RA may occasionally show some degree of
repair has been presented (45). Osteoblast recruitment
could exert a direct effect through a coupling mechanism
between endosteal osteoclasts and osteoblasts (46).
However, we cannot rule out the possibility that local
production of chemokines, including CXCL13, within
marrow aggregates may provide additional signals for
recruitment and activation of osteoblasts (47,48).
We have thus shown that an inflammatory lym-
phoid infiltrate with a variable degree of organization,
similar to that occurring in synovial tissue, is a charac-
teristic feature of RA subchondral bone marrow, at least
in advanced disease. Subchondral aggregates are primar-
ily located in superficial bone marrow, near the invading
synovial inflammatory tissue, but they can also be de-
tected in deep sites that appear to be distant from the
cartilage–pannus junction. This latter finding raises the
hypothesis that subchondral bone marrow inflammation
might, at least in part, develop independent of the
propagation of synovial tissue. In any case, since our
findings indicated a positive correlation between the
degree of bone marrow inflammation and the recruit-
ment of subchondral bone osteoclasts, it appears that
this inflammatory process has an active role in local
bone damage.
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