Integrins and their ligands in rheumatoid arthritis.

Abstract

Integrins play an important role in cell adhesion to the extracellular matrix and other cells. Upon ligand binding, signaling is initiated and several intracellular pathways are activated. This leads to a wide variety of effects, depending on cell type. Integrin activation has been linked to proliferation, secretion of matrix-degrading enzymes, cytokine production, migration, and invasion. Dysregulated integrin expression is often found in malignant disease. Tumors use integrins to evade apoptosis or metastasize, indicating that integrin signaling has to be tightly controlled. During the course of rheumatoid arthritis, the synovial tissue is infiltrated by immune cells that secrete large amounts of cytokines. This pro-inflammatory milieu leads to an upregulation of integrin receptors and their ligands in the synovial tissue. As a consequence, integrin signaling is enhanced, leading to enhanced production of matrix-degrading enzymes and cytokines. Furthermore, in analogy to invading tumors, synovial fibroblasts start invading and degrading cartilage, thereby generating extracellular matrix debris that can further activate integrins.

Full text

Page 1

Integrins are a large group of transmembrane proteins that
anchor the cell to the extracellular matrix (ECM) or other
cells. Upon binding, integrins remodel the ECM by
inducing the expression of certain proteases. Integrins
control cellular proliferation, migration, and invasion.
Whereas in some cases integrins can mediate these eff ects
on their own, they usually signal in the context of growth
factor or cytokine receptors [1]. Ligand binding to integrin
controls adhesion-dependent
unligated integrins enhance apoptotic pathways [2]. Th is is
one reason why dysregulated integrin expression or
downstream signaling pathways can be observed in almost
proliferation, whereas
all forms of cancer, and integrin levels often determine the
aggressiveness or propensity to metastasize.
Migration, invasion, and proliferation of synovial cells
are major problems in rheumatoid arthritis (RA) [3]. Th is
debilitating disease is characterized by an infl amed
synovial tissue with a massive infl ux of immune cells and
an infl ated synovial lining consisting mainly of synovial
fi broblasts (SFs) and macrophages that adhere to the
adjacent ECM. Th ese SFs are highly proliferative and
contribute signifi cantly to cartilage and bone destruction.
In some aspects, they can be considered ‘tumor-like’ as
SFs are resistant to many apoptotic pathways, show
increased proliferation, and produce high amounts of
matrix metalloproteinases (MMPs) because of dysregu la-
tion of the transcription factors AP-1, p53, and nuclear
factor-kappa-B (NF-κB) [4]. Fibroblasts, macrophages,
but also endothelial cells in synovial tissue show high
levels of integrin expression [5]. In RA, not only integrins
but also their ligands (for example, fi bronectin and
collagen as well as their degradation products) are up-
regulated [6]. Th is ‘overstimulation’ of cells by integrin-
mediated signals increases basal secretion of pro-
infl ammatory cytokines like interleukin-6 (IL-6) and IL-8
but also levels of various MMPs [7-9].
Th is review introduces the predominant integrins
expressed on synovial cells along with their binding
partners and demonstrates their signifi cance in disease
progression. We focused mainly on the β1, β3, and β5
integrins because altered levels of their ligands and
respective integrins themselves are one major factor that
fuels the destruction of cartilage in RA. Although
integrins infl uence RA signifi cantly, only a few reviews
that address integrins in RA have been published. A
Medline search for ‘integrins’ and ‘arthritis’ yielded 108
(compared with ‘integrins’ and ‘cancer’ with over 1,200
reviews) reviews, most of which outlined single integrins
or cell types. Two reviews in 1993 and 1995 – by Postigo
and colleagues [10] and Liao and Haynes [11], respec-
tively – were the last comprehensive articles describing
the infl uence of integrins on the pathogenesis of RA.
Both articles, however, failed to describe the intricacies
in integrin biology, from the generation of integrin
ligands from the ECM to the subsequent triggering of
integrin signaling cascades that fi nally lead to the genera-
tion of pro-infl ammatory factors and cartilage
Abstract
Integrins play an important role in cell adhesion to
the extracellular matrix and other cells. Upon ligand
binding, signaling is initiated and several intracellular
pathways are activated. This leads to a wide variety
of eff ects, depending on cell type. Integrin activation
has been linked to proliferation, secretion of matrix-
degrading enzymes, cytokine production, migration,
and invasion. Dysregulated integrin expression is often
found in malignant disease. Tumors use integrins to
evade apoptosis or metastasize, indicating that integrin
signaling has to be tightly controlled. During the
course of rheumatoid arthritis, the synovial tissue is
infi ltrated by immune cells that secrete large amounts
of cytokines. This pro-infl ammatory milieu leads to an
upregulation of integrin receptors and their ligands
in the synovial tissue. As a consequence, integrin
signaling is enhanced, leading to enhanced production
of matrix-degrading enzymes and cytokines.
Furthermore, in analogy to invading tumors, synovial
fi broblasts start invading and degrading cartilage,
thereby generating extracellular matrix debris that can
further activate integrins.
© 2010 BioMed Central Ltd
Integrins and their ligands in rheumatoid arthritis
Torsten Lowin* and Rainer H Straub
REVIEW
*Correspondence: torsten.lowin@klinik.uni-regensburg.de
Laboratory of Experimental Rheumatology and Neuroendocrine Immunology,
Department of Internal Medicine, University Hospital Regensburg, Franz-Josef
Strauß Allee 11, 93053 Regensburg, Germany
Lowin and Straub Arthritis Research & Therapy 2011, 13:244
http://arthritis-research.com/content/13/5/244
© 2011 BioMed Central Ltd

Page 2

degrada tion. To our knowledge, this is the fi rst review
that demonstrates the whole integrin machinery in
arthritis, including ligands, receptors, and eff ector cells.
Integrin biology
A cell needs to sense the composition of the extracellular
environment to elicit appropriate responses. Integrins, a
family of heterodimeric proteins, serve as sensors of the
ECM. Integrins consist of an α subunit that determines
ligand specifi city and a β subunit that initiates intra-
cellular signaling events. Whereas most integrins bind
almost exclusively to ECM molecules, β2 hetero dimers
bind other adhesion molecules, mediate cell-cell contacts,
and are important components of the immune system. In
contrast to the β2 integrin, which is expressed only on
immune cells, other β subunits have a much more diverse
expression. In the synovium, fi broblasts, macrophages,
and endothelial cells are just a few examples of cells that
express the β1, β3, and β5 subunits. Th e ECM-binding
integrins mediate cellular processes such as adhesion,
migration, tissue invasion, and chemotaxis but also
cytokine and MMP production.
Within the protein sequences of fi bronectin, collagen,
and vitronectin, integrins bind to arginine-glycine-
aspartic acid (RGD) motives. Interaction with the RGD
sequence is an important switch for integrin activation
and subsequent cellular changes. Integrins can transduce
signals from outside the cell to the interior and vice versa,
allowing the cell to respond dynamically to a wide variety
of stimuli. Growth factors, hormones, and cytokines as
well as the composition of the ECM are able to modulate
integrin signaling and affi nity [12]. When integrins are
activated by ligands, several signaling events are initiated.
Besides the focal adhesion kinase (FAK), the mitogen-
activated protein kinase (MAPK) pathway, the phos-
phatidyl inositol 3-kinase (PI3K), and c-Jun N-terminal
kinases (JNKs) are activated [13]. Growth factors and
cytokines employ the same signaling pathways as
integrins, demonstrating an intricate interplay between
the two. Integrins are linked to the cytoskeleton by
coupling to adaptor proteins such as vinculin and talin.
Th erefore, changes in integrin activation lead to changes
in the actin cytoskeleton, a prerequisite for adhesion,
migration, and invasion on ECMs.
Integrin knockouts and related pathologies
Given that various integrin combinations recognize the
same ECM molecule (for example, fi bronectin is recog-
nized by α4β1, α5β1, ανβ1, ανβ3, and α3β1 and weakly by
others), one would expect some redundancy in integrin
function. In fact, this is the case when collagen-binding
integrin subunits α1 or α2 are knocked out. When α1 is
knocked out, corresponding mice show decreased vascu-
la ri zation, and isolated endothelial cells from these
animals show reduced proliferation whereas α2 knock-
outs demonstrate only a subtle phenotype. Th e picture is
diff erent when regarding the knockout of fi bronectin-
binding integrin subunits. Knockout of either integrin α3,
α4, α5 or αν is lethal during embryogenesis and respec-
tive mice die shortly after birth. Also, the fi bronectin-
binding integrins have a more tissue-specifi c distribution
than collagen-binding integrins, making target cells more
susceptible to specifi c knockouts.
Besides having a role in development, integrin muta-
tions lead to a variety of other defects. Leukocyte adhe-
sion defi ciency type 1 (LAD-1) is an autosomal recessive
disorder based on point mutations within the β2 integrin,
resulting in either its absence on the cell surface or the
inability to associate with the corresponding α subunit.
Th is leads to reoccurring bacterial infections, suggesting
an impaired function of macrophages and neutrophils.
Another autosomal recessive (although an autoimmune
form exists) disease is the Glanzmann thrombasthenia, in
which the platelet integrin αIIbβ3 fails to bind to
fi brinogen and fi brin. Th is step is important to form
blood clots after injury, and failure to do so leads to
prolonged bleeding.
Integrins are also involved in the generation and
perpetuation of cancer. In several tumors, expression of
specifi c integrin subunits is associated with malignancy.
In melanoma, increased expression of ανβ3 and α5β1
promotes vertical growth and lymph node metastasis. In
non-small-cell lung carcinoma, high α5β1 levels predict
decreased patient survival. In general, alterations in
integrin activation or expression promote tumor growth
and metastasis. However, in some cases (for example,
α2β1 in breast cancer), integrins can be protective by
decreasing cell dissemination [14]. Th is integrin increases
diff erentiation of epithelium and enhances ECM binding,
thus decreasing migration.
Integrins in synovial tissue
When the integrin distribution in the synovial tissue is
analyzed, several factors need to be considered. First,
integrin expression and function diff er depending on cell
type. Second, the location of the cell is important as
expression levels diff er at the synovial lining compared
with the sublining. Every cell in the synovial tissue
expresses a specifi c subset of integrins, depending on
lineage and origin. Several groups have investigated
synovial tissue samples and isolated cells to study integrin
expression. Th e integrin subunit β1 is most widely ex-
pressed since it can be found on macrophages, lympho-
cytes, endothelial cells, and fi broblasts [5,15,16]. Th e
fi bronectin receptor α5β1 and the laminin receptor α3β1
were also found on every synovial cell type, whereas
α4β1, which besides fi bronectin also binds VCAM-1, was
found only on lymphocytes. Endothelial cells and
Lowin and Straub Arthritis Research & Therapy 2011, 13:244
http://arthritis-research.com/content/13/5/244
Page 2 of 8

Page 3

fi bro blasts also express the collagen receptor α1β1 and
the vitronectin receptor ανβ3 [17]. In synovial tissue,
integrin expression also depends on the location of the
particular cell. Expression of most integrins is similar
throughout the synovial tissue but is diff erent at the
synovial lining layer [5]. Th is is the area where synovial
cells adhere to cartilage and activated fi broblasts and
macrophages degrade ECM and invade cartilage. Th e
laminin receptor α6β1 is expressed by fi broblasts at the
lining layer but not by macrophages, which usually
express this receptor [18]. At the synovial lining, we
found an increase in α5, αν, and β1 integrin levels
compared with the sublining area. Th is clearly indicates
that increased integrin expression is associated with a
more ‘destructive’ phenotype of the corresponding cell.
Figure  1 depicts the integrin-ligand interactions and
their consequences (for example, proliferation and secre-
tion of pro-infl ammatory mediators) described in the
next three sections. Th e most important cell types (fi bro-
blasts, endothelium, T cells, B cells, macrophages, and
Th 17 cells), including their individual reaction to integrin
engagement, are depicted (Figure 1). Th e individual inte-
grin levels on each cell type are presented in Table 1. Th is
table refl ects our own observations of tissue samples
stained for individual integrins and fl ow cytometric
analyses as well as literature data.
The collagen-binding integrins α1β1 and α2β1
Collagens are widely expressed in synovial tissue, and,
during the course of RA, a signifi cant proportion of
patients develop antibodies against at least one collagen
subtype. Th e collagens II, IX, X, and XI are restricted to
cartilage, whereas all other forms can also be found
within the ECM. Th e classical collagen-binding integrins
rely on the triple-helical structure for binding [19].
However, if collagen is proteolytically degraded, RGD
sequences are exposed and other RGD-binding integrins
can bind. In this case, ανβ3, αIIbβ3, and α5β1 can be
activated by collagen-derived RGD sequences [20].
Four diff erent integrin combinations recognize native
collagen: α1β1, α2β1, α10β1, and α11β1. Integrin α10β1
is important for chondrogenesis, whereas α11β1 has a
role in the recognition and organization of interstitial
collagen matrices during development. During the course
of arthritis, only α1β1 and a2β1 seem to play a role; this is
because these are the two major collagen-binding inte-
grins in the synovial tissue. Although all of the above-
mentioned subunits bind to collagen, their respective
specifi cities diff er for each collagen subtype; for example,
integrin α1 preferentially binds collagen type IV.
Th e integrin α1 was found to be expressed at the
synovial lining and in blood vessels of arthritic joints.
Interestingly, expression was increased compared with
osteoarthritis and trauma controls, suggesting that the
pro-infl ammatory milieu in RA promotes integrin α1
synthesis [21]. However, our group also found an increase
of this adhesion receptor on synovial fi broblasts after
treat ment with cortisol [5]. An increase of α1 – in analogy
to integrin α5, which is also upregulated by cortisol –
poten tially increases adhesion to collagen. Owing to
cortisol action, the increase in integrin expres sion is un-
coupled from integrin signaling because intra cellular signal-
ing pathways are silenced by glucocorticoid treatment.
Lymphocytes also express integrin α1, but only synovial
lymphocytes show increased expression of this receptor
[22]. One important modulator for α1 levels is tumor
necrosis factor (TNF) because anti-TNF therapy reduces
the number of α1-positive eff ector memory T cells that
contribute signifi cantly to infl ammation by producing
large amounts of interferon-gamma (IFN-γ). Besides
adhesion to collagen, α1 mediates several other eff ects,
depending on cell type. On endothelial cells, vascular
endothelial growth factor (VEGF) strongly upregulates
α1, which is a prerequisite for the formation of new blood
vessels. Inhibition of the integrin/collagen binding by
specifi c antibodies reduced the formation of new blood
vessels but did not alter pre-existing vasculature [23]. As
angiogenesis is important for the progression of arthritis,
its inhibition could be an attractive target in the therapy
of RA. Engagement of integrin α1 on macrophages and T
cells stimulates migra tion and cytokine production [24].
Th e role of α1 on synovial fi broblasts has not been
elucidated yet, but studies from dermal fi broblasts
suggest that α1 controls collagen synthesis, migration,
and proliferation on colla gen substrates. In a murine
anti-collagen II antibody-induced arthritis model,
inhibition of integrin α1 reduced cartilage degradation
and leukocyte infi ltration, demon strating an important
role for this integrin in infl amma tion [25].
Integrin α2β1 has functions similar to those of α1β1
(for example, VEGF-induced proliferation of endothelial
cells) but is much weaker in expression in the synovial
tissue. Ligation of α2β1 by collagen type I or II, but not
collagen type IV, augmented IFN-γ production in T cells.
In contrast to CD4+ and CD8+ T cells, which upregulate
α1β1 after activation, Th 17 cells upregulate α2β1, which
is thought to be a co-stimulatory molecule necessary for
IL-17 production [26]. In a model of experimental
autoimmune encephalomyelitis, an antibody against α2
suppressed clinical signs and central nervous system
infl ammation because of reduced extravasation of immune
cells. Since leukocyte infi ltrates also fuel synovial infl am-
mation, one would expect that α2 inhibition might also
be benefi cial in the joint.
Laminin-binding integrins α3β1 and α6β1
Laminins are a major component of the basal lamina and
usually are secreted by epithelial cells, endothelium, and
Lowin and Straub Arthritis Research & Therapy 2011, 13:244
http://arthritis-research.com/content/13/5/244
Page 3 of 8

Page 4

organogenic fi broblasts. Laminin is a trimeric molecule
consisting of an α-chain, a β-chain, and a γ-chain that can
combine to form 15 diff erent laminin isoforms [27].
Several integrin hetero dimers recognize laminin as a
ligand, but the affi nity for each laminin subtype diff ers.
Laminin binds to integrins α3β1, α6β1, α7β1, and α6β4,
but only the fi rst two are present in the synovial tissue. In
the synovial tissue, α6β1 is strongly expressed in the
synovial lining layer by fi broblasts, whereas α3β1 is also
expressed by lymphocytes, macrophages, and endothelial
cells [18]. Not only the number of laminin receptors but
also the production of laminin itself is upregulated in the
synovial lining. Besides endothelial cells, SFs produce and
secrete laminin, and its deposition at the synovial lining
Figure 1. Engagement with fi bronectin-, laminin-, or collagen-binding integrins leads to the production and secretion of
pro-infl ammatory factors. Binding is indicated by the arrows. Cell type, ligand (for example, fi bronectin or fi bronectin-derived peptides), ligand
occupation, and integrin expression level determine the cellular response. BAFF, B-lymphocyte activating factor; CCL2, chemokine (C-C motif)
ligand 2; IFN-γ, interferon-gamma; IL, interleukin; MMP, matrix metalloproteinase; TNF, tumor necrosis factor; VCAM-1, vascular cell adhesion protein
1; VEGF, vascular endothelial growth factor.
Lowin and Straub Arthritis Research & Therapy 2011, 13:244
http://arthritis-research.com/content/13/5/244
Page 4 of 8

Page 5

is increased in RA [28]. Along with collagen and fi bro-
nectin, laminin promotes adhesion and proliferation of
synovial cells and lymphocytes. Furthermore, integrin
binding to laminin increases the expression of MMP-3
and MMP-10, which is further augmented when trans-
forming growth factor-beta (TGF-β) is present [29].
Although no studies have focused on the contribution of
laminin and its respective integrin receptors on the
progres sion of RA, results from tumor research indicate
that upregulation of α6β1 or α3β1, at least in most cases,
increases cell invasion and metastasis. As a consequence,
increased laminin deposition and increased integrin
expression on synovial lining cells increase MMP
secretion by these cells, resulting in greater joint damage
and invasion into cartilage.
Fibronectin-binding integrins α4β1, α5β1, aνβ3,
and αvβ5
Fibronectin is a large glycoprotein consisting of two
mono mers linked by disulfi de bonds. Owing to alter-
native splicing, fi bronectin exists in 20 diff erent isoforms.
Th e molecule is folded into globular domains, each of
which has a diff erent function. In these domains, repeat-
ing amino acid sequences form the so-called type I, II,
and III repeats. Th ese sequences interact with other
proteins; for example, type I repeats bind to fi brin, and
regions in type III repeats contain the RGD motive that
binds integrins. Fibronectin can be divided, on the basis
of solubility, into two classes: soluble plasma fi bronectin
and insoluble cellular fi bronectin, the latter of which is
organized into fi brils as a scaff old for the ECM [30].
Fibronectin levels are elevated in cartilage and synovial
fl uid of patients with osteo arthritis, whereas in RA,
fi bronectin is highly expressed at the synovial lining and
invading pannus [31].
About 12 integrin heterodimers bind to fi bronectin but
only some combinations are found in the synovial tissue.
Th e classical fi bronectin receptor α5β1 recognizes only
fi bronectin, whereas ανβ3 and ανβ5 also bind to other
ECM molecules or cellular proteins. Th e α5 integrin is
expressed by all cells in the synovial tissue, and α5 levels
are increased in the synovial lining layer [5]. Lymphocytes
have been shown to attach to other synovial cells by
binding to the fi bronectin coating on their cell surface by
α4β1 and α5β1 and to some extent by ανβ3 integrins [32].
Adhesion to fi bronectin increases proliferation and
survival of chondrocytes and protects synovial cells from
Fas-induced apoptosis [33]. In addition, Zeisel and
colleagues [34] found an increase of MMP-3 production
by SFs after stimulation with a bacterial ligand of α5β1,
suggesting that enhanced integrin ligation increases joint
destruction in RA. Matrix-degrading enzymes in synovial
fi broblasts are induced by small integrin-binding pep-
tides derived from the degradation of fi bronectin and
other ECM molecules. Werb and colleagues [7] demon-
strated that only fi bronectin-derived fragments, but not
intact fi bronectin, increased the expression of MMP-1
and MMP-3. Besides having a role in cell survival and
prolifera tion, α5β1 also regulates cytokine and growth
factor production. In SFs, ligation of α5β1 increased
synthesis of B-lymphocyte activating factor (BAFF) [35].
Increased BAFF synthesis not only promotes the
proliferation of B cells and immunglobulin class-
switching but possibly acts as an autocrine mechanism of
SFs to stimulate their own NF-κB activity. Besides BAFF,
direct ligation of α5β1 induces the expression of NF-κB-
controlled genes in fi broblasts and endothelial cells. Most
of these genes – like IL-8 and heparin-bound epidermal
growth factor (HB-EGF) – or endothelin-1 control
angiogenesis or infl ammation and their enhanced
expression aggravate RA.
Naïve and memory T cells depend on binding of a4β1
and α5β1 to fi bronectin to promote proliferation [32]. In
this context, SFs assist in T-cell activation, as T cells bind
to the fi bronectin coating on their surface and α4β1
transmits co-stimulatory signals. In RA, infi ltrated T cells
in the synovial tissue display higher surface levels of α4β1
compared with peripheral T cells and localize to sites of
tissue injury by binding to proteolytically degraded
cartilage fragments. Th e most important function of
α4β1 on lymphocytes is adhesion to endothelium and
subsequent transmigration to sites of injury.
Table 1. Surface integrin levels of the predominant cell types in the rheumatoid arthritis synovium
α1 α2 α3 α4 α5 α6 αν β1 β2 β3 β5
Synovial fi broblast ++ − +(+) − +++ + +++ +++ − ++ ?
Macrophage + + + +++ +++ (+) +++ +++ ++ +++ +
Endothelium ++ + ++ + +++ + +++ +++ − +++ +
T lymphocyte (Th1) ++ + ++ +++ +++ + +++ +++ ++ +++ +
Th17 cell − ++ ++ ++ +++ + +++ +++ ++ ? ?
B lymphocyte ++ + ++ +++ +++ + ++ +++ ++ ? ?
For most integrins, expression level data in the literature are given as ‘high’ or ‘strong’. This makes it hard to estimate integrin levels on diff erent cell populations. We
consulted several publications and, albeit subjectively, categorized integrin expression levels on a range from strong to weak expression (indicated by one, two, or
three plus signs). ?, There is no available data in the literature about the expression of respective integrins.
Lowin and Straub Arthritis Research & Therapy 2011, 13:244
http://arthritis-research.com/content/13/5/244
Page 5 of 8