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Rheumatoid arthritis _ targeting


									Progress in Cell Cycle Research, Vol. 5, 59-70, (2003)
(Meijer, L., Jézéquel, A., and Roberge, M., eds.)

chapter 7

Rheumatoid arthritis: targeting the proliferative fibroblasts
                        1                                2            3                           4
IJsbrand Kramer *, Auragun Wibulswas , Daniel Croft , and Elisabeth Genot
  Section of Cellular and Molecular Biology, European Institute of Chemistry and Biology,
  Bat B2, University of Bordeaux 1, 33405 Talence, France.
  Thammasat University (Rangsit campus), Patum Thani 12121 Thailand.
  Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104-6160, USA.
  Section of Cellular and Molecular Biology, European Institute of Chemistry and Biology,
  U441 INSERM , Avenue du Haut Lévêque, 33600 Pessac, France.
* To whom correspondence should be addressed

     Our flexible joints are synovial joints composed of bone, hyaline cartilage, synovial membrane, ligaments
and tendons. Rheumatoid arthritis is a disease that affects multiple synovial joints and involves inflammation
of the synovial membrane, often resulting in loss of function due to erosion of bone and cartilage. Inflammation
is accompanied by an influx of immune-competent cells and by aberrant proliferation of resident fibroblast-
like synoviocytes. Accretion of fibroblasts directly contributes to joint destruction, through enhanced
production of matrix-degrading enzymes, and indirectly, through excessive release of cytokines that boost the
immune system. Targeting the proliferative fibroblast could facilitate regeneration of synovial joints.

ARCHITECTURE OF THE SYNOVIAL JOINT                                 renders it rather permeable to solutes. For instance,
     Our flexible joints are synovial joints. They own             large molecules like hyaluronan leak out at a constant,
their name to the presence of a visco-elastic solution,            slow rate.
the synovial fluid, which resembles eggwhite, (syn                 RHEUMATOID ARTHRITIS
ovum). The synovial fluid is composed mainly of the
                                                                   Clinical symptoms and diagnosis
glycosaminoglycan hyaluronan, a polymer composed
                                                                         Rheumatoid arthritis is described as a pattern of
of repeating β-1,4-glucuronate-β-1,3-N-acetylglucosa-
                                                                   illness, of unknown aetiology, involving chronic
mine dis a c c ha r ide s . Hya l ur o na n is the ma j o r
                                                                   synovitis (inflammation of the synovial membrane) of
component of the synovial space and it provides
                                                                   multiple joints often resulting in loss of joint function
hydraulic resistance (shock absorbing) due to its fluid
                                                                   due to severe erosion of bone, cartilage and ligaments.
retaining capacity (1).
                                                                   A precise definition is based on a number of criteria
     Synovial joints comprise a number of separate tis-            laid out by the American College of Rheumatologists
sues: bone, hyaline cartilage, synovial membrane,                  in 1987 (4). Aspects that are considered are: at least
ligaments and tendons, together creating a structure               three joints with active disease causing morning
that moves with a remarkably low level of wear and                 stiffness, joint swelling, tenderness, radiographic
tear (Figure 1). The synovial membrane, the focus of               changes (Figure 2), prolonged symptoms, involvement
this chapter, plays an important role in the function of           of metatarsophalangeal joints and the presence of
the synovial joint. It is responsible for the production           auto-antibodies such as rheumatoid factor (RF,
of hyaluronan and it forms an interface between the                representing IgM directed against the Fc-region of IgG
blood circulation and the synovial fluid, and it is                antibodies) or IgG antifilaggrin auto-antibodies (a
implicated in the regulation of immune surveillance of             citrullinated antigen) (5, 6). Rheumatoid arthritis may
synovial joints. The synovial membrane is made up of               be distinguished from other forms of chronic arthritis,
a cellular inner layer, the synovial lining, mainly                such as osteoarthritis, by the characteristic pattern of
composed of macrophages (type-A synoviocytes) and                  juxta-articular cartilage and bone erosion and the
fibroblast-like cells (inner lining cells or type-B                occurrence of fever, weight loss, anorexia, severe
synoviocytes) (2). The macrophages are identified by               muscle atrophy and symmetry of joint involvement
their expression of CD68 (macrosialin) and IgG-Fc                  (reviewed in 7). Tenderness, or at times straight-
receptors, and by their capacity to phagocytose.                   forward pain, is a prominent feature of arthritic joints
Fibroblast-like synoviocytes are recognized through                and arises from sensory wind up in the spinal cord
their constitutive expression of the adhesion molecule             mediated b y i n f l a mma t o ry me d i a t o rs s u ch a s
VCAM-1 (3) and the fibroblast marker hydroxyproline.               prostanoids, bradykinin, protons and cytokines such
These cells are responsible for the production of the              as IL-1β, IL-15 and TNF-α that sensitize peripheral
extracellular matrix components, in particular                     nociceptors, causing greater activation of spinal
hyaluronan. The outer layer of the membrane, the                   sensory neurones for a given stimulus (8). Pain
sub-lining, is mainly composed of a collagenous                    perception occurs from innervated tissues such as
extracellular matrix containing few dendritic cells and            subchondral bone, synovial membrane, joint capsule,
a vascular bed. The cellular components of the                     ligaments, and muscle.
synovial membrane do not form extensive cellular
contacts, as in epithelial or endothelial tissues, and this


                                                                                                    Figure 1. Synovial joints in
                                                                                                    health and disease.
                                                                                                    Synovial joints comprise a
                                                                                                    number of tissues, bone, car-
                                                                                                    tilage, capsule and synovial
                                                                                                    membrane. The joint space
                                                                                                    is filled with a solution of
                                                                                                    hyaluronan. Characteristics
                                                                                                    of synovial joints obtained
                                                                                                    from patients with rheuma-
                                                                                                    toid arthritis are hyperplasia
                                                                                                    of the synovial membrane,
                                                                                                    as a consequence of prolife-
                                                                                                    ration of resident fibroblast-
                                                                                                    like synoviocytes and an
                                                                                                    influx of immunecompetent
                                                                                                    cells. Bone and cartilage are
                                                                                                    covered        by     pannus.
                                                                                                    Cartilage and bone show
                                                                                                    signs of erosion.

Figure 2. Progressive destruction of synovial joints.
A thinning of cartilage is manifested by a reduction of
the distance between the opposing bones. At a later stage
erosion of bone and cartilage is visible and the joint has
lost its characteristic architecture.

                                                               normal                 thinning                     erosion
                                                                joint                of cartilage                of bone and

                                                             Figure 3. A model depicting a dissociation between clinical symp-
                                                             toms, pain, swelling, and erosion of bone (as observed by radiogra-
                                                             phical analysis).

Prognosis; the relationship between clinical symp-                sive disease at 2 years are unlikely to show subsequent
toms and joint erosion                                            erosive damage but not all patients with erosion at 2 years
     The disease has erosive and non-erosive variants,            subsequently have progressive joint damage. Damage
each causing pain and dysfunction, but the latter not             and disability are unrelated in early disease (<5 years) and
causing visible erosions of bone and cartilage and                this relationship only becomes significant after 5 years
being self-limiting. After two years of clinical symp-            with a strong correlation after 12 years. From this it fol-
toms, there is a turning point in the development of              lows that early clinical symptoms are primarily influen-
erosions visible by radiographic analysis (9). However,           ced by disease activity (inflammation), pain and psycho-
subsequent progression of joint damage is variable and            social factors, and little by structural abnormalities (10).
non-predictable. It is true that patients with a non-ero-         The discrepancy between disease activity and erosion is

                                                                      CHAPTER 7 / CELL CYCLE AND RHEUMATOID ARTHRITIS

also highlighted by the observation that a large number of             19). The presence of elevated levels of inflammatory
persons over the age of 50 have some radiographic detec-               cytokines enables a number of aspects of inflamma-
table erosion without clinical symptoms. It thus                       tion: the influx of immune competent cells, the phago-
appears that at least two pathological processes are                   cytic function of neutrophils and macrophages, anti-
simultaneously at work in the joint, one causing clini-                gen presentation by dendritic cells, activation of T-
cal symptoms and one being responsible for erosions                    cells, differentiation of B-cells into plasma cells, pro-
(see Figure 3, adapted from 11). Novel criteria to defi-               duction of immunoglobulins and, last but not least,
ne prognosis in early arthritis are clearly needed to                  induction of expression of matrix metalloproteinases.
enable differentiation between patients who will deve-                 Abundant evidence has been provided that destruc-
lop a progressive erosive disease (and require an                      tion of cartilage is a consequence of the action of metal-
"aggressive" therapy) and those with a self-limiting or                loproteinases. These zinc-dependent endopeptidases
a non-erosive variant.                                                 cleave a large number of extracellular components
Changes in the synovial membrane                                       such as collagens and proteoglycans (such as aggregan
      In rheumatoid arthritis, the synovial membrane                   in cartilage) (20, 21). Elevated levels of matrix metallo-
undergoes a number of changes. Macroscopically, there is               proteinase-1 (MMP-1 or interstitial collagenase),
a thickening of the membrane and the development of                    MMP-3 (stromelysin-1) and MMP-9 are detected in the
numerous projections at the inner side (villi) (Figure 1). At          synovial membrane, the synovial fluid and at the sites
the microscopical level a massive increase in the number               of cartilage erosion (22). MMPs are produced by fibro-
of immune-competent cells and fibroblast-like cells is obs-            blast-like synoviocytes, scattered cells in the sublining
erved, a phenomenon commonly referred to as hyperpla-                  region and by synovial endothelial cells (23, 24). Other
sia (12). A redistribution of fibroblast-like synoviocytes is          proteinases, such as cathepsin and elastase, are also
also evident. These cells gradually invade deeper layers of            detected. These cysteine or serine proteinases are pro-
the synovial membrane and also the bone and cartilage,                 duced by fibroblast-like synoviocytes, macrophages
where they form a cell mass, together with macrophages,                and neutrophilic granulocytes and are involved in the
that covers these tissues and is therefore named pannus                degradation of a wide range of proteins, not exclusive-
(from Latin meaning cover or coat). The omnipresence of                ly components of the extracellular matrix (25, 26).
fibroblast-like synoviocytes in the synovial joint is striking              Various knock-out or knock-in mice confirm the
and gives the impression of a neoplastic disorder, as if the           participation of the above described mediators in
cells were indeed transformed. These cells have an increa-             murine models of arthritis and three examples are pre-
sed proliferative capacity and are highly invasive (revie-             sented here. Firstly, transgenic mice expressing human
wed in 13). We shall return to this subject in detail in later         TNF-α spontaneously develop erosive arthritis (27).
sections of this chapter.                                              Secondly, mice lacking MMP-3 (stromelysin-1) fail to
      The presence of immune-competent cells, like B- and              cleave collagen and have limited destruction of pro-
T-lymphocytes as well as monocytes, clearly illustrates the            teoglycans after the induction of joint inflammation
inflammatory component of this disease. B-cells are main-              with an emulsion of albumin/Freund's adjuvant and
ly present as highly differentiated plasma cells secreting             heat-inactivated Bordetella pertussis (antigen-induced
auto-antibodies like rheumatoid factor (anti-Fc-IgG IgM)               arthritis) (28). Lastly, mice lacking IL-6 resist the deve-
or anti-citrullinated flaggrine antibodies. They are found             lopment of a chronic response after antigen-induced
in the sub-lining of the synovial membrane in the vicinity             arthritis (29, 30). The above-mentioned observations
of dendritic cells (antigen-presenting cells). Natural killer          indicate that rheumatoid arthritis is certainly a conse-
cells and mast cells have been observed. Chondrocyte-                  quence of an elaborated network of cytokines and pro-
derived cells are also known to appear in the proliferating            teinases. Regardless of the origin of the pathology, the
pannus. The appearance of pathological synovial tissue is              disease could thus be described as "an inflammatory
conspicuous in early rheumatoid arthritis, for instance ele-           disorder that fails to resolve itself" (Figure 4) but as we
vated levels of VCAM-expressing cells, fibroblast-like                 will see later this is certainly an oversimplification. It is
synoviocytes, and large numbers of macrophages are pre-                true that removing or inactivating any one of the com-
sent in synovial tissue within one year after diagnosis                ponents of this network ameliorates the general condi-
(14). Lastly, infiltrating neutrophilic granulocytes are               tion, both in animal models and in humans, but the
also observed but these reside mainly in the synovial                  question remains whether or not this cures the disease.
fluid.                                                                 Auto-antigens and auto-immunity
Inflammatory cytokines and metalloproteases                                 Immune tolerance to self is not absolute. Given
     The clinical symptoms are accompanied by eleva-                   the breadth of protein epitopes in higher eukaryotes,
ted levels of pro-inflammatory cytokines such as IL1β,                 an immune system from which all potential self-reacti-
IL-6, IL-15 and TNF-α, and an insufficient presence of                 vity had been deleted would probably not respond to
anti-inflammatory cytokines such as IL-4, IL-10, IL-13,                anything. The self-reacting T cells essentially ignore
IL-1ra and TGFβ1 (15, 16, 17). All cells of the synovial               the self-antigens because they are seen with low affini-
membrane contribute to their production, with macro-                   ty, they occur at low abundance or because they are
phages and lymphocytes being dominant in the pro-                      hidden behind tissue or processing barriers. The self-
duction of IL-1β, IL-15 and TNF-β and fibroblast-like                  tolerance may be lost because of aberrant processing of
synoviocytes being the predominant source of IL-6 (18,                 proteins or because of the homing of infectious orga-


                                                                     Figure 4. Inflammatory mediators present in the diseased joint.
                                                                     In patients with rheumatoid arthritis the balance between pro-
                                                                     and anti-inflammatory mediators is disturbed. An excess of pro-
                                                                     inflammatory mediators creates a chronic inflammatory environ-
                                                                     ment causing clinical symptoms like joint swelling and pain but
                                                                     also a thinning of cartilage. Functional blockade of the pro-
                                                                     inflammatory mediators improves the general condition of the
                                                                     synovial joint.

Figure 5. The three phases of an auto-immune response causing rheumatoid arthritis.
Stage 1. Auto-antigens, spilled into cellular environment, are presented by antigen-presenting cells in the context of MHC classII.
This complex is recognized by the T-cell receptor (TCR) causing activation of naïve T-cells and resulting in clonal expansion of T-
helper cells. These play a role in the subsequent activation of macrophages but more importantly of B-cells, which become anti-self
antibody- producing plasma cells.
Stage 3. Antibody/antigen complexes accumulate and activate complement with the generation of the proteolytic fragments, C3bi
and C5a. The immune complexes bind to macrophages, together with C3bi, and initiate phagocytosis causing the production of toxic
oxygen metabolites and the secretion of metalloproteinases and inflammatory cytokines. The complement factors C3a and C5a cause
activation of mast cells, with release of histamine, and recruitment of phagocytes (through their chemokinetic action). Why synovial
joints are favourable sites for accumulation of immune complexes and why macrophages react so vigorously remains to be resolved.

nisms, viral, bacterial or mycoplasmale, into certain tis-           T-cells. Activated T-cells, in turn, communicate with
sues. The local production of inflammatory cytokines                 macrophages (TH1) to boost production of inflammato-
and the ensuing cellular damage, cause release of cyto-              ry cytokines and, importantly, with B-lymphocytes
solic components and will wake up self-reactive T-                   (TH2) to boost the production of antigen-specific
lymphocytes and confront them with their antigen but                 immunoglobulins (vital for the rapid elimination of
this time in an effective way (through presentation in               "foreign"). The antibody/antigen complexes that are
an MHC classII context by antigen-presenting cells                   formed, often coupled to components of the comple-
together with lots of co-stimuli) (31) (Figure 5). In the            ment system (C3bi), are deleterious because they inten-
case of arthritis, not only infection by a pathogen, but             sify the inflammatory response by stimulating macro-
also repeated mechanical insults may create a favoura-               phages. In the case of the synovial joint, they do this by
ble environment for the activation of normally tolerant

                                                                   CHAPTER 7 / CELL CYCLE AND RHEUMATOID ARTHRITIS

binding to the IgG-Fc type IIIa receptor (FcRIIIA or                but not from those with osteoarthritis, spondylarthro-
CD16a) and the C3bi receptor (CD11b or mac-1).                      pathy or other diagnoses (38, 39). Anti-citrullinated
Macrophages only carry FcRIIIA in the tissues normal-               filaggrin antibody-producing plasma cells are also
ly affected by rheumatoid arthritis, including joint                detected in rheumatoid synovial tissue and these fin-
lining, the lungs and the pericardial lining of the heart,          dings have raised the hypothesis that citrullinated pro-
so inflammation only occurs in these tissues.                       teins could trigger an autoimmune response in rheu-
Activation leads to phagocytosis of the complexes with              matoid arthritis. Post-translational modifications of
the subsequent release of proteinases (members of the               proteins have been reported to be the cause of other
elastase and cathepsin family), chemokines (32) and                 autoimmune diseases, mainly in systemic lupus ery-
inflammatory cytokines, such as IL-1α and TNF-α                     thrematosus and scleroderma.
(33). It also causes activation of the respiratory burst                 The autoimmune model has gained renewed
with release of toxic oxygen metabolites (34) (for a                attention with the fortuitous generation in mice of an
review on this subject, 35). The activation of comple-              illness resembling rheumatoid arthritis, obtained by
ment not only causes the production of C3bi, it also                crossing a T cell-receptor transgenic line (KRN) with
results in the cleavage of C5 into C5a and C5b. C5a, an             the auto-immune disease sensitive-NOD strain. After
anaphylatoxin and chemotaxin, localizes elements of                 two weeks, all offspring (K/BxN) developed joint dis-
the inflammatory response (through chemotaxis),                     ease (40). At that time point T- and B-cells infiltrated
degranulates neutrophils (thereby causing release of                into the synovial joints and the mice started to present
free oxygen radicals and prostaglandins), and also sti-             signs of inflammation. The reason why these mice
mulates Il-1 and Il-6 production. In contrast, C5b teams            developed arthritis was recognition of self. The auto-
up with C6, C7, C8 and C9 to form a pore-forming                    antigen is glucose-6-phosphate isomerase (GPI), and it
structure that causes leakage through the plasma                    is recognized, in the context of MHC classII, by the
membrane followed by cell death (the lytic pathway,                 (monoclonal) T-cell receptor. GPI is a glycolytic enzy-
an important step in protection against micro-orga-                 me and not at all a joint-specific antigen (41). Despite
nisms).                                                             GPI being a ubiquitously expressed antigen, anti-GPI
     The generation of C5a and the release of inflam-               immune complexes, combined with complement pro-
matory cytokines will cause further recruitment of                  ducts, appeared to accumulate at the cartilage surface
immune-competent cells thereby generating an inflam-                of synovial joints of the mice (and caused the delete-
matory response that lasts until the antibody-antigen               rious inflammatory response in the manner illustrated
complexes are eliminated. In the case of auto-immuni-               in the previous paragraphs) (42).
ty, this point will never be reached and a chronic                       Arthritis could also be provoked by transfer of
inflammatory condition will ensue.                                  anti-GPI immunoglobulins, obtained from diseased
      A number of auto-antibodies have been found in                mice, into healthy lymphocyte-deficient recipients.
patients with rheumatoid arhritis, none of which are                This finding provided ample evidence for the role of
directed against joint-specific epitopes. As already                auto-antibodies and innate immune players in the
mentioned, one of them is rheumatoid factor. This                   development of the disease. Finally, and importantly,
represents mainly IgM antibodies directed against the               in the immunoglobulin transfer experiments, not all
Fc portion of IgG. Rheumatoid factor is not highly spe-             recipient mice developed arthritis, and a search for
cific for rheumatoid arthritis nor is it a very useful pro-         genetic influences on susceptiblity highlighted one
gnostic factor for the development of the erosive                   genomic region that contained the complement factor
variant. Moreover, it can be found in other autoimmu-               C5 gene (43). Interestingly, this gene has also been
ne diseases, malignancies, chronic infections and even              identified as a susceptibility locus for murine experi-
healthy persons (36). Other auto-antigens that have                 mental allergic asthma (44a) and myasthenia gravis
been detected are directed against perinuclear factor               (destruction of motor endplate AchR) (44b). From this
(APF), keratin (AKA), collagen, Epstein-Barr nuclear                information it follows that rheumatoid arthritis could
antigen and proteins such as RA33, Sa and p68.                      be interpreted as an auto-immune disease, causing the
Antibodies against APF and AKA are of great interest.               formation of immune complexes, which in turn activa-
These are in fact directed against a polypeptide                    te complement with the production of complement
sequence containing a modified arginine residue, cal-               factors C3bi, C5a etc, resulting in a runaway activation
led citrulline. This epitope is present in filaggrin, abun-         of the innate immune response. This then results in
dant in keratinocytes, but is also present in other pro-            joint destruction. According to this model, therapy
teins of unknown identity. The conversion of arginine               should focus on prevention of the C5a product being
to citrulline in proteins is an enzymatic process that is           formed. Quite independently from these findings,
carried out by peptidylarginine deiminase (PAD), an                 beneficial effects of anti-C5a monoclonal antibodies on
enzyme that appears to be hormonally controlled (37).               collagen-induced arthritis in mice has already been
Through altered post-translational processing, a modi-              demonstrated (45).
fied putative auto-antigen emerges which bypasses
                                                                    CURRENT TREATMENT OF ARTHRITIS
immune tolerance. The presence of citrullinated pro-
teins has recently been detected in a small number of                   Based on the knowledge presented in the pre-
synovial cells from patients with rheumatoid arthritis              vious sections, therapy has focussed on the attenuation


         T-cell/B-cell expansion      - nucleotide metabolism inhibitors
                                      - disease-modifying anti-rheumatic drugs (DMARDS)
                                        (gold salts, D-penicillamine, sulfasalazine)
                                      - immunosuppressors
         Inflammation                 - disease-modifying anti-rheumatic drugs
                                      - non-steroidal anti-inflammatory drugs (NSAIDS)
                                        (aspirin, indomethacin or aceclofenac)
                                      - cytokine inhibitors
                                        (TNF-α antibody (Infliximab), TNF-α soluble receptor (Etanercept) and
                                        IL-1β receptor antagonist (Anakinra)
                                      - glucocorticoids

Table 1. Therapeutic strategies

of the immune response and more recently on the inac-              the above-mentioned treatments, surgery can have a
tivation of particular inflammatory cytokines.                     profoundly positive impact on their ability to carry out
Conventional therapy includes the so-called disease-               their daily activities . The usual indications for surgery
modifying anti-rheumatic drugs (DMARDS) like gold                  include pain and/or disability that are unacceptable
salts, D-penicillamine or sulfasalazine, the non-steroi-           and functionally limiting. Surgery of hand and wrist,
dal anti-inflammatory drugs (NSAIDS) like aspirin,                 shoulder, elbow, knee or total hip replacement are all
indomethacin or aceclofenac, and the nucleotide meta-              common treatments. In some case surgical removal of
bolite inhibitors such as methotrexate (Table 1). The              the synovial membrane, synovectomy, suffices.
treatment rapidly suppresses inflammation and causes
                                                                   THE TRANSFORMED FIBROBLAST-LIKE
a reduction in cytokine expression and mononuclear
cell infiltration. This effect could be explained by the
observed reduction in the production of TNF-α and                        The previous sections were intended not only to
lower levels of expression of the endothelial adhesion             give the reader information about rheumatoid arthritis
molecules, E-selectin and ICAM-1, instrumental in the              but also to provide the necessary context in which to
recruitment of immune-competent cells (46).                        place the final section dealing with cell proliferation
Combination therapies are also applied, comprising                 and disease. In this section we add another level of
sulfasalazine, methotrexate and the immune-suppres-                complexity, the contribution of the fibroblast-like
sing glucocorticoids such as prednisolone (over a per-             synoviocytes to the destruction of synovial joints, inde-
iod of 28 to 40 weeks). Patients treated with such com-            pendent of T- or B-cells, auto-antigens or complement.
bination therapy show less radiological detectable                 As mentioned in the first section of this chapter, the
damage at 4 years compared with those treated with                 onset of rheumatoid arthritis is characterized by an
"monotherapy". Novel therapies that have been tested               increasing number of fibroblast-like synoviocytes and
extensively over the last three years, are administra-             a redistribution of these cells throughout the synovial
tion of antibodies, soluble receptors or receptor anta-            joint. The origin of these cells is, however, not clear. At
gonists that block the action of TNF-α (Infliximab or              best they can be described as a heterogeneous popula-
Etanercept) or IL-1β (Anakinra) combined with metho-               tion comprising proliferating specialized joint-lining
trexate (47). Using these particular therapies, arrest of          cells, common interstitial fibroblasts and recruited pri-
joint damage was observed, even in patients who did                mitive mesenchymal cells (49).
not respond clinically (and retained symptoms of swel-             FIBROBLAST-LIKE SYNOVIOCYTES AS
ling, tenderness etc). In general, infiltration of T-cells         AGGRESSORS
(CD3+), B-cells (CD22+) and macrophages (CD68+)
was greatly diminished, as was the expression of adhe-                   These fibroblast-like synoviocytes, when derived
sion molecules on fibroblast-like synoviocytes and vas-            from the joints of patients with rheumatoid arthritis,
cular endothelial cells. (48). At present this treatment is        are often characterized as aggressive or transformed
reserved for patients who do not respond well to                   cells. This term should not be interpreted as these cells
conventional therapy. Thus far, limited adverse effects            being malignant because they are not. For instance,
have been recorded with antibody treatments,                       when kept in culture they reach senescence and exp-
amongst which are opportunistic infections, tuberculo-             ress cell cycle inhibitors that are related to fibroblast
sis (awakening of a latent infection), multiple sclerosis          senescence (in particular p16INK4a) (50). Moreover,
or intestinal perforation. Immediate re-appearance of              there is little evidence for rheumatoid arthritis-specific
clinical symptoms follows the arrest of treatment.                 gene mutations (with the exception of p53, see below)
                                                                   or gene rearrangements, nor is there evidence for
    Finally, in those patients who develop joint dama-             microsatellite instability, commonly detected in true
ge with deformity and functional limitation, despite               malignant cells. It is also not clear whether this "trans-

                                                                   CHAPTER 7 / CELL CYCLE AND RHEUMATOID ARTHRITIS

                                                                                   Figure 6. "Transformation" of fibroblast-like
                                                                                   synoviocytes. The inflammatory environment
                                                                                   brings about proliferation of resident fibro-
                                                                                   blast-like synoviocytes (formation of granula-
                                                                                   tion tissue) through (over)expression of genes
                                                                                   implicated in cellular proliferation and de-dif-
                                                                                   ferentiation. Prolonged exposure may cause
                                                                                   permanent changes, like mutations in the p53
                                                                                   gene, expression of splice variants of CD44.
                                                                                   The cells also acquire the unique capacity to
                                                                                   express p16INKa but this remains suppressed
                                                                                   in the synovial environment. The result is the
                                                                                   formation of a proliferative and invasive
                                                                                   fibroblast-like synoviocytes that contributes to
                                                                                   joint destruction, either directly, through
                                                                                   release of their matrix metalloproteases, or
                                                                                   indirectly, through release of inflammatory
                                                                                   cytokines that boost the immune system.

formed" phenotype is an exclusive synovial joint-phe-               med" fibroblast-like synoviocytes are an elevated level
nomenon or a characteristic of any granulation tissue               of expression of the oncogenes c-myc , c-fos and c-jun
that forms during injury or chronic inflammation (and               (59, 60) a capacity to form colonies in soft agar (ancho-
needed for the reconstruction of extracellular matrix).             rage-independent growth) (61), exorbitant release of
However, numerous studies indicate that the fibro-                  IL-6 in the absence of detectable levels of IL-1β or TNF-
blast-like synoviocytes derived from rheumatoid                     α (62) and an increased production of matrix metallo-
arthritic joints have undergone a series of permanent               proteases like MMP-1 and MMP-3 (63). Furthermore
changes and are clearly distinguishable from fibro-                 these cells express elevated levels of Frizzled (Fz5) and
blasts derived from non-diseased joints, from osteo-                its ligand Wnt-5A (64) as well as Notch-1 (65), HOXD9
arthritic joints or from other tissues (skin or lung fibro-         (66) and the EGF receptor ErbB-2 (67). The presence of
blasts) (Figure 6). Different experimental approaches               Notch-1, Wnt, Fz and HOXD9 is interpreted as a sign
have demonstrated an increased percentage of fibro-                 of cellular de-differentiation because these are genes
blast-like cells in S-phase in the rheumatoid joint (51,            expressed throughout embryological development
52 , 53, 54). The proliferative advantage persists when             and that play an important role in tissue development.
whole tissue specimens or dissociated synovial cells                      The "transformed" cells are also characterized by
are brought into culture (55, 56). Our studies have                 expression of CD44-splice variants, in different spli-
shown that VCAM-1-expressing-cells derived from                     cing combinations (68). Fibroblast-like synoviocytes
rheumatoid arthritic joints can be either fast or slow              (identified as the VCAM-1-positive fibroblast popula-
dividing. Fast dividers have a doubling time of 55                  tion) expressing variants containing exons CD44v7/8
hours whereas slow dividers double every 69 hours                   were shown to have a proliferative advantage over
(57). These estimations have been made throughout                   those that do not express them (57). These findings
the second week in culture in the presence of 10% fetal             confirm the previous mentioned observation that cell
bovine serum. Other studies have shown that, under                  mass in pannus can arise from a monoclonal origin and
similar conditions, cells from highly inflamed joints               that clonal expansion is an important cause of increa-
double every 42 hours whereas those from osteo-                     sed cellularity of the synovial membrane. Aberrant
arthritic joints take 72 hours (55). Proliferation in the           expression of CD44-splice variants is often associated
synovial joint may be a consequence of the presence of              with true cell transformation (69). Lastly, in fibroblast-
high levels of inflammatory cytokines but this does not             like synoviocytes of some patients, overexpression or
explain the difference in proliferation seen under cul-             mutation of the tumour-suppressor p53 have been obs-
ture conditions. After all, under culture conditions the            erved (70, 71). Good evidence has been provided that
cells all experience a highly inflammatory environ-                 dominant negative mutations of p53 increased their
ment because of the presence of serum (the soluble                  invasive capacity, their resistance to apoptosis and
component of clotted blood). Evidence has also been                 their production and release of cytokines and metallo-
provided that synoviocytes obtained from pannus                     proteinase (72, 73, 74). What perhaps is lacking is a clear
lesions exhibit a monoclonal pattern of restriction frag-           demonstration that fibroblast-like synoviocytes contai-
ment length polymorphism (RFLP) of the phosphogly-                  ning the p53 mutations colonize the synovial joint and
cerate kinase 1 (PGK-1) gene, suggesting that this cell             concentrate at the interface between pannus and cartilage
mass originates from locally proliferating cells. The               (because of their survival advantage plus invasive quali-
pannus cells had high levels of expression of TGF-β                 ties). In addition, no reports have shown that these cells
and PDGF (58). Other characteristics of the "transfor-              dominate cell culture if kept for some weeks. It should be


noted that numerous studies have not observed muta-                 viocytes in different forms of joint athropathies (85-87).
tions in patients with rheumatoid arthritis (75, 76).               Inhibition of proliferation reduces joint destruction
Cellular changes persist ex vivo                                          If transformation of the fibroblast-like synoviocy-
     When derived from rheumatoid arthritic joints                  tes contributes to joint destruction, inhibition of their
and brought into culture, the fibroblast-like synoviocy-            proliferative or invasive capacities should provide pro-
tes are highly invasive, whereas those derived from                 tection against joint destruction. In studying expres-
non-diseased joints are devoid of such activity. Cells              sion of cyclin-dependent kinase inhibitors (CDI's) in
from osteo-arthritic tissue have rather variable levels             fibroblast-like synoviocytes, it was found that during
of invasion but always well below those observed in                 serum starvation, cells derived from rheumatoid
patients with rheumatoid arthritis (77). Invasion did               arthritic joints had the unique quality of expressing
not require high concentrations of growth factors, indi-            p16INK4a, in addition to the expression of p21CIP1 and
cating an intrinsic quality of the cells rather than sti-           p27KIP1 (88). Expression of p16INK4a could not be detec-
mulation by environmental factors, and was compara-                 ted in fibroblast-like synoviocytes in the synovial
ble with v-fos-transfected rat fibroblast cells (a cell line        membrane. Because this inhibitor is implicated in the
used to study the mechanisms of cellular invasion of                induction of a differentiation programme resulting in
extracellular matrix) (78). In these studies it was shown           senescence of human fibroblasts, one could conclude
that entry of cells into Matrigel could be inhibited up             that the synovial joint environment represses fibro-
to 60% by prior treatment of the fibroblast-like syno-              blast differentiation (89). Under similar experimental
viocytes with antibodies recognizing the variant exons              conditions, cells obtained from other tissues or from
CD44v3 or CD44v6. The aberrant expression of CD44-                  other types of athropathies only expressed p21Cip1 and
splice variants thus has important functional conse-                p27KIP1. After serum starvation, fibroblast-like synovio-
quences and could play a role in the altered phenotype              cytes derived from rheumatoid arthritic joints, again
of the fibroblast-like synoviocytes. Importantly, inva-             unlike other fibroblasts, were unable to reactivate the
sion and destruction of cartilage can also occur in a               cell cycle machinery when given medium with a high
more physiological model comprising synovial tissue                 serum content. Ectopic expression of p16INK4a in fibro-
or isolated fibroblast-like synoviocytes co-implanted               blast-like synoviocytes, using an adenovirus infection
with human cartilage under the skin of severe combi-                technique, abrogated the proliferative response nor-
ned immunodeficient (SCID) mice (lacking T- and B-                  mally induced by the inflammatory cytokines TNF-α
lymphocytes). Cells obtained from non-diseased tissue               and IL-1β. To test whether this irreversible inhibition
do not cause erosion (79, 80). Various elements play a              could be of therapeutic advantage, similar experiments
role in the damage of cartilage: elevated levels of                 were performed in rats, after intra-articular application
cathepsins L and B as well as MMP-1 and MMP-13 (81)                 of the virus containing p16INK4a. The animals showed
and activation of plasminogen (82). The interplay bet-              reduced symptoms of arthritis upon treatment with
ween fibroblast-like synoviocytes and chondrocytes of               mycobacterium butyricum-containing adjuvant and his-
the cartilage is also of importance because inhibition of           tological analysis showed reduced mononuclear cell
chondrocyte protein synthesis reduced the invasive                  infiltration, less pannus formation and less thinning of
capacity of the synoviocytes (83). These studies show               cartilage. Interestingly, significant differences between
unmistakably that the transformed phenotype persists                p16INK4a-expressing and non-expressing animals emer-
even under conditions where inflammatory cytokines                  ged only after two weeks of the adjuvant treatment.
or auto-antigens are absent, and that fibroblast-like               This finding supports the notion that a synergy may
synoviocytes cause cartilage erosion independent of an              exist between the adjuvant-induced deleterious immu-
intact immune system. It therefore has been proposed                ne response (early symptoms) and the ensuing trans-
that, in parallel to a cytokine network, an oncogene                formation of the fibroblast-like synoviocytes (amplifi-
network acts as a pathogenic mechanism in synovial                  cation of damage in the long run). Similar experiments
joint-destruction (84). More studies are required to pre-           were performed, this time with the ectopic expression
cisely define these molecular alterations and to deter-             of p21CIP1 or p16INK4a, in a mouse model where arthritis
mine how they relate to disease development. For                    is induced by immunisation against collagen. Both
instance, among patients showing true symptoms of                   cyclin-dependent inhibitors proved to be beneficial.
rheumatoid arthritis for two years, some never deve-                They not only prevented synovial overgrowth but also
lop full-blown joint destruction. This indicates that if            improved the inflammatory environment by keeping
irreversible changes in the fibroblast-like synoviocytes            low the levels of IL-1ss, IL-6 and TNF-β (90).
do occur, they must occur, or not, at that particular                     Induction of fibroblast-like synoviocyte differen-
stage. If markers for such permanent changes were                   tiation has also been obtained by ectopic expression of
found, they could prove useful in the prognosis of the              Jun-D, a transcription factor known to counteract the
development of an erosive or non-erosive variant of                 effects of Ras-mediated cell transformation or c-Jun-
the disease. A number of laboratories have taken up                 and jun B-mediated fibroblast proliferation (91). As
the genomic, transcriptomic or proteomic approach to                with ectopic expression of the cyclin-dependent kinase
characterize further alterations in fibroblast-like syno-           inhibitors, induction of differentiation by Jun-D reduced

                                                                 CHAPTER 7 / CELL CYCLE AND RHEUMATOID ARTHRITIS

inflammatory cytokine expression but also diminished               hand-in-hand is the targeting of cyclo-oxygenase COX-
the content of MMPs, through a down-modulation of                  2. This enzyme is the inducible variant of cyclo-oxyge-
AP-1 activity. Beneficial effects of Jun-D expression in           nase, is highly expressed in inflamed tissues, and
murine models of arthritis have not yet been reported.             converts arachidonic acid into prostaglandins of which
     The aberrant expression of CD44-splice variants               PGD2, PGE1 and PGE2 are relatively stable end pro-
provides proliferative and invasive advantages to the              ducts. Of these, PGE2 has been linked to inflammation.
fibroblast-like synoviocytes. Aberrant expression of               Mice lacking COX-2, but not the mice lacking COX-1,
adhesion molecules may be one way in which trans-                  have a reduced incidence and severity of collagen-
formed cells obtain additional signals supporting cell             induced arthritis, with a reduction in both prolifera-
proliferation (92). In our studies, we have shown that             tion of synovial lining and infiltration of immune-com-
addition of anti-CD44v7/8 antibodies to fibroblast-like            petent cells (99). Transfer of antibodies obtained from
synoviocytes in culture, causes expression of the                  the diseased mice ("arthritogenic" auto-antibodies)
cyclin-dependent inhibitor p21CIP1 and of the growth               could not induce arthritis in the naive COX-2 -/- mice.
arrest and DNA-damage-induced proteins GADD45                      These findings could be interpreted as COX-2 being an
and GADD153 concomitant with an inhibition of cell                 important regulatory enzyme for the execution of both
proliferation. No signs of apoptosis were detected in              an acquired and an innate immune response.
the first 5 days of treatment (57). From these studies we          However, it may be more than that. A recent study has
have learned that adhesion molecules also can be tar-              shown that PGE2 is also a potent transactivator of the
gets to restrain excessive cell proliferation. The results         EGF receptor (100) and this mechanism plays a vital role
presented above, provide ideas or suggestions, of how              in the promotion of gastro-intestinal hypertrophy (cau-
we could possibly target proliferation of fibroblast-like          sing polyps). An additional interpretation could thus be
synoviocytes. The hard work, translating them into a               that the absence of COX-2 prevents proliferation of fibro-
therapy for rheumatoid arthritis, still lies ahead.                blast-like synoviocytes, thereby providing extra protec-
                                                                   tion against these potentially aggressive cells.
     Induction of apoptosis would be another way to
reduce or eliminate the excess of fibroblast-like syno-            A PHARMACOLOGICAL INTERVENTION
viocytes. This could be particularly advantageous                  THAT TARGETS CELL PROLIFERATION
when the synovial joints are already populated by                  AND REDUCES INFLAMMATION
large numbers of cells. It is known that transformation                 Rheumatoid arthritis can be described as a disea-
renders cells more susceptible to apoptosis, and trans-            se with at least three pathogenic processes; 1) forma-
formed cells have often put into place additional res-             tion of auto-antibodies/complement complexes, 2)
cue pathways (93). These rescue pathways provide                   excessive production of inflammatory cytokines and 3)
potential pharmacological targets as their blockade                "transformation" of fibroblast-like synoviocytes.
could impose apoptosis. There is evidence that the pro-            Attempts to interfere with any of these processes have
tein kinase PKB (Akt) provides such a rescue signal,               all proven to be beneficial in murine models of rheu-
and that its activation is elevated in cells derived from          matoid arthritis. In the case of the inhibitors of TNF-α
rheumatoid arthritic joints (compared with those                   or IL-1β, benefits have also been demonstrated in
obtained from patients with osteoarthritis) (94).                  humans suffering from the real disease. Drawbacks of
Fibroblast-like synoviocytes express Fas, a cell death             the current therapies are either limited protection
receptor, and apoptotic cells are apparent in the syno-            against joint destruction (DMARDs and glucocorti-
vial membrane of patients with rheumatoid arthritis                coids) or excessive costs (antibody treatment). The stu-
(95). When Fas binds to its ligand FasL, receptor clus-            dies presented in this chapter clearly provide a ratio-
ters are formed and these induce a death signal through            nale for the development of a pharmacological inter-
activation of intracellular proteinases called caspases. In        vention that targets the cell cycle of fibroblast-like
an attempt to test if Fas expression could be exploited to         synoviocytes (Table 2). Possible means to restrain the
kill fibroblast-like synoviocytes, irradiated Fas-ligand-          fibroblast-like synoviocytes are induction of expres-
expressing cells (introduced through transfection) were            sion of p16INK4a or p21CIP1, abrogation of cell-matrix
combined with fibroblast-like synoviocytes and co-                 interactions or interference with the transactivation-
implanted with cartilage under the skin of SCID mice.              effect of PGE2 (COX-2-dependent). These approaches
This treatment effectively eliminated fibroblast-like syno-        might not only reduce the direct damaging effect of
viocytes through cell-cell interaction (96).                       these cells on bone and cartilage, but also dampen the
    One example where inhibition of cell proliferation             inflammatory response. We are of the opinion that one
and attenuation of the immune response might go                    cannot ignore the accretion of fibroblast-like synovio-

         Inhibiting of the cell cycle         - Induction of expression of senescence genes (p16INK4a)
                                              - induction of expression of cell cycle inhibitors (p21CIP1, GADDs)
                                              - blockade of proliferation promoting signals through specific
                                                adhesion molecules (CD44-splice variants, integrins)
         Sensitising the cells to apoptosis   - inhibition of apoptosis rescue pathhways (PKB)

Table 2. Targeting the fibroblast-like synoviocytes


cytes in the diseased joint for the simple reason that                   20. Shapiro, S.D. (1998) Curr Opin Cell Biol 10, 602-608.
their exorbitant production of IL-6 makes the rheuma-                    21. van Meurs, J., van Lent, P., Holthuysen, A.,
toid arthritic joint an impossible environment in which                      Lambrou, D., Bayne, E., Singer, I., and van den
to control the immune system. Under these conditions,                        Berg, W. (1999) J Immunol 163, 5633-5639.
any attempt to reverse inflammation or abrogate the
                                                                         22. Cunnane, G., FirtzGerald, O., Beeton, C., Cawston, T.E.,
formation of auto-immune complexes resembles
                                                                             and Breshinan, B. (2002) Arthritis Rheum 44, 2263-2274.
"mopping the floor with the water tap wide open"
(Dutch proverb) (18, 97, 98).                                            23. McCachren, S.S. (1991) Arthritis Rheum 34, 1085-1093.
                                                                         24. Gravallese, E.M., Darling, J.M., Ladd, A.L., Katz, J.N.,
ACKNOWLEDGEMENT                                                              and Glimcher, L.H. (1991) Arthritis Rheum 34, 1076-1084.
     The authors would like to thank Paul-Peter Tak,                     25. Adkison, A.M., Raptis, S.Z., Kelley, D.G., and
Academic Medical Center Amsterdam, The Netherlands,                          Pham, C.T. (2002) J Clin Invest 109, 363-371.
for his comments on this manuscript.
                                                                         26. Hou, W.S., Li, W., Keyszer, G., Weber, E., Levy, R.,
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