Syndecan-3 is selectively pro-inflammatory in the joint and contributes to antigen-induced arthritis in mice
© Kehoe et al.; licensee BioMed Central Ltd. 2014
Received: 5 February 2014
Accepted: 24 June 2014
Published: 11 July 2014
Syndecans are heparan sulphate proteoglycans expressed by endothelial cells. Syndecan-3 is expressed by synovial endothelial cells of rheumatoid arthritis (RA) patients where it binds chemokines, suggesting a role in leukocyte trafficking. The objective of the current study was to examine the function of syndecan-3 in joint inflammation by genetic deletion in mice and compare with other tissues.
Chemokine C-X-C ligand 1 (CXCL1) was injected in the joints of syndecan-3−/−and wild-type mice and antigen-induced arthritis performed. For comparison chemokine was administered in the skin and cremaster muscle. Intravital microscopy was performed in the cremaster muscle.
Administration of CXCL1 in knee joints of syndecan-3−/−mice resulted in reduced neutrophil accumulation compared to wild type. This was associated with diminished presence of CXCL1 at the luminal surface of synovial endothelial cells where this chemokine clustered and bound to heparan sulphate. Furthermore, in the arthritis model syndecan-3 deletion led to reduced joint swelling, leukocyte accumulation, cartilage degradation and overall disease severity. Conversely, CXCL1 administration in the skin of syndecan-3 null mice provoked increased neutrophil recruitment and was associated with elevated luminal expression of E-selectin by dermal endothelial cells. Similarly in the cremaster, intravital microscopy showed increased numbers of leukocytes adhering and rolling in venules in syndecan-3−/−mice in response to CXCL1 or tumour necrosis factor alpha.
This study shows a novel role for syndecan-3 in inflammation. In the joint it is selectively pro-inflammatory, functioning in endothelial chemokine presentation and leukocyte recruitment and cartilage damage in an RA model. Conversely, in skin and cremaster it is anti-inflammatory.
Syndecans (sdcs) are heparan sulphate proteoglycans (HSPG) composed of a core protein to which heparan sulphate (HS) glycosaminoglycan chains are covalently attached. These molecules form part of the glycocalyx, which comprises a network of membrane-bound proteoglycans and glycoproteins at the cell surface of endothelial cells [1–3]. There are four mammalian syndecans, designated syndecan-1 (sdc-1), -2, -3, and -4, which have protein cores with characteristic structural domains [4, 5]. The variable ectodomain, which is exposed to the extracellular environment, contains three to five HS and in some cases chondroitin sulphate chains, and is attached to the cell membrane via a hydrophobic transmembrane segment [6, 7]. In addition, there is an intracellular domain containing peptide sequences, which serve as substrates for cellular kinases, enabling syndecans to act as signaling molecules .
HSPGs have been shown to play a pro-inflammatory role [9–11]. For example, on endothelial cells they bind and present chemokines to blood leukocytes that leads to leukocyte integrin activation, crawling on the endothelial cell surface and extravasation [12–15]. This interaction involves chemokine immobilisation and concentration at the endothelial surface and stimulation of leukocyte migration into the tissue . Evidence also suggests that HS functions in chemokine transcytosis, which relays chemokines from basal to luminal surfaces of endothelial cells for their presentation to blood leukocytes [12, 17–19]. Furthermore, endothelial HS may act as an adhesion molecule, for example binding L-selectin during neutrophil rolling . In contrast, data also indicate that HSPGs may be anti-inflammatory, for example in disease models of nephritis and lung inflammation using sdc-1 and sdc-4 knockout mice [20–25]. Furthermore, removal of HS by heparanase leads to increased leukocyte adhesion to the cremaster endothelium by intravital microscopy, suggesting an anti-inflammatory function . Further work is needed to address the apparent contradictory roles of HSPGs in inflammation. Whether sdcs are pro- or anti-inflammatory may relate to the particular tissue where they are expressed or the inflammatory state.
Inflammation is a central feature of rheumatoid arthritis (RA) that affects around 1% of the population and can result in disability and morbidity. In RA, inflammation of the joint synovium is characterised by the infiltration and activation of leukocytes, which can lead to progressive destruction of cartilage and bone. Chemokines are involved in stimulating the infiltration of leukocytes into inflamed tissue and there is substantial evidence showing an involvement of these mediators and their receptors in RA . For example, chemokine C-X-C ligand 1 (CXCL1) and CXCL8 are abundant in the sera, synovial fluid and synovium in human RA [27–32]. They are produced by synovial macrophages and other cells and attract neutrophils primarily. Furthermore, sdcs have been shown to be expressed in arthritic joints and sdc-4 functions in joint destruction [33–36].
A CXCL8 binding site on endothelial HSPG has been demonstrated in the synovium of RA patients . In order to clarify which HSPG bound the chemokine, immunolocalisation of syndecans and glypicans revealed particularly strong expression of sdc-3 on RA synovial endothelial cells with quantitative PCR confirming endothelial expression. Furthermore anti-sdc-3 antibody and heparanase reduced CXCL8 binding to the endothelium. These data suggest a role for sdc-3 in synovial inflammation. Sdc-3 is the predominant syndecan in the nervous system, where it was first identified, and has been associated with the control of feeding behaviour and the generation of cerebellar fibrillar plaques in Alzheimer’s disease [37, 38]. Sdc-3 is also an HSPG of the musculoskeletal system. It has been found in the synovium in adult human joints  and is expressed by chondrocytes [39, 40]. In addition, sdc-3 is involved in limb morphogenesis and skeletal development and regeneration [41, 42]. Several studies have shown that it is expressed by endothelial cells in the synovium, lymph nodes and liver [33, 43, 44].
Nothing is currently known about the role of sdc-3 in inflammation, unlike sdc-1 and -4 [20–25]. The expression of a CXCL8 binding site on endothelial sdc-3 in human RA suggests a role for this HSPG in inflammatory disease  although in vivo studies are needed to substantiate this hypothesis. The current study addresses this question, whether genetic deletion of sdc-3 in mice alters leukocyte trafficking in response to murine CXCL1. This chemokine is the functional homologue to CXCL8, which is absent in rodents. The study also addresses if deletion of sdc-3 alters the severity and progression of disease in an RA model. The involvement of sdc-3 in leukocyte recruitment in the synovium was compared to that in the skin and cremaster muscle. This was to find out if sdc can play a different role in different tissues, which may help explain its apparent contradictory function in inflammation. We show that sdc-3 plays a dual role in inflammation depending on the tissue and vascular bed. In the joint it is pro-inflammatory, since its deletion leads to reduced leukocyte recruitment and the severity of arthritis. However, in the skin and cremaster it is anti-inflammatory, since its deletion leads to enhanced leukocyte interaction with the endothelium and recruitment. This is the first study to show a role for sdc-3 in inflammation and reveals its function is tissue-selective.
Experiments were undertaken in 7- to 10-week-old inbred C57Bl/6 wild-type (sdc-3+/+) and sdc-3 null (sdc-3−/−) mice. Sdc-3−/−mice were generated by Dr Ofer Reizes, Cleveland, USA ; they are viable, fertile and develop normally. Procedures were performed with ethical approval from the Home Office, UK, project licence PPL 40/3047.
Chemokine-driven leukocyte migration into the skin and joints
Mice were injected intradermally or intra-articularly in the knee joint space with recombinant murine CXCL1 (KC) (PeproTech, London, UK) 3 μg/site in phosphate-buffered saline (PBS) . PBS administration was used as a control. After four hours, the animals were sacrificed and skin biopsies or joints were processed for light microscopy. Leukocyte recruitment into the dermis and synovium was observed by light microscopy, neutrophils being identified by their lobed nuclear morphology. To quantitate leukocyte recruitment the number of neutrophils in the synovium was randomly counted in 10 fields of view at x780 magnification per section from sdc-3−/− (n = 8) and sdc-3+/+ (n = 8) mice.
Myeloperoxidase (MPO) assay
The MPO assay was used as a surrogate marker for the presence of neutrophils in skin tissue and was carried out as described . Briefly, excised pieces of skin from mice were snap frozen in liquid nitrogen and homogenized on ice in 500 μl of PBS with 0.01 M EDTA and a proteinase inhibitor mix (Sigma-Aldrich, Poole, UK) and 1 ml of 1.5% Triton X-100 in PBS. Samples were placed on a rotary shaker at 300 rpm on ice for 30 min, centrifuged at 12,000 × g for 10 min, and supernatants were collected. Total protein concentration for each sample was quantified by BCA Lowry assay (Thermo Scientific Pierce, Cramlington, UK). The protein concentration in all tissue extracts was adjusted to 0.9 mg/ml. MPO activity was determined by using the EnzChek MPO Activity Assay Kit (Invitrogen, Paisley, UK) according to the manufacturer’s instructions.
For CXCL1 (KC) and E-selectin detection in skin and joint samples we used a tyramide signal amplification kit  (Molecular Probes, Invitrogen). Briefly, formalin-fixed, wax-embedded sections of skin and joints were de-waxed, rehydrated, washed in PBS, and skin subjected to antigen retrieval in Tris-HCl buffer, pH 9.0, at 100°C in a water-bath for 20 min; for joints antigen retrieval was in 10 mM Tris-HCl buffer, 1 mM EDTA and 0.05% Tween 20, pH 9.0, overnight at 65°C. The endogenous peroxidase was blocked by incubation for 10 min with 3% H2O2 followed by incubation with 1% blocking reagent for 60 min at room temperature. Sections were incubated for 60 min with rabbit anti-murine CXCL1 polyclonal antibody (PeproTech) at 2 μg/ml or rat anti-murine E-selectin monoclonal antibody at 5 μg/ml (kindly supplied by Dr Alexander Zarbock, University of Munster, Germany). Sections were then treated with HRP-conjugated goat anti-rabbit or goat anti-rat secondary antibodies for 60 min, then Alexa Fluor™ 488 tyramide for 10 min. For sdc immunofluorescence, sections were treated with affinity purified rabbit anti-mouse sdc-3 (1:500)  goat anti-rabbit Alexa 594 antibody (Invitrogen) containing 10% mouse serum. Tissue sections were stained with DAPI for cell nuclei and analysed using a Leica IX51 microscope (Leica, Wetzlar, Germany). Control sections were negative when treated with rabbit or rat immunoglobulin (Ig)G instead of primary antibodies (added at the same concentrations) or when the primary antibodies were omitted.
For dual labelling, sections were treated with anti-E-selectin (as above) together with rabbit anti-von Willebrand factor (1:100; Dako, Ely, UK) followed by goat anti-rabbit Alexa 594 second antibody (Invitrogen) For quantitation of E-selectin expression, five vessels were randomly sampled per section of synovium (n = 6 sdc-3 null and wild-type mice with antigen-induced arthritis (AIA) at day 3) and skin (n = 8 sdc-3 null and wild-type mice). The numbers of vessels showing a luminal E-selectin distribution were counted. For quantitation of CXCL1 on endothelial cells five vessels were randomly sampled per skin (n = 8 sdc-3+/+and n = 9 sdc-3−/−mice) and joint (n = 6 sdc-3+/+and sdc-3−/−mice) section.
Heparanase treatment of sections was performed using a previously described method . Briefly, formalin-fixed, wax-embedded sections of CXCL1-injected joints were de-waxed, rehydrated, washed in PBS, and subjected to antigen retrieval as above. The sections were treated with 20 units/ml of heparanase I and 4 units/ml heparanase III (both Sigma-Aldrich, UK) in HBSS, or HBSS alone, for 1.5 hours at 37°C. After enzymatic treatment, the samples were rinsed twice with HBSS before CXCL1 immunolocalisation as described above.
The effects of sdc-3−/−on leukocyte rolling and stationary adhesion was also measured in vivo in the cremaster muscle microcirculation using intravital microscopy (PPL 40/2747) . Briefly, in anaesthetized (ketamine/xylazine; intraperitoneally (ip)) mice, the testis was exposed through a small scrotal incision and the cremaster muscle exteriorised, cleared of connective tissue and pinned across a glass coverslip on a specialised microscope stage. The muscle was continuously superfused with bicarbonate-buffered saline (131.7 mM NaCl, 4.69 mM KCl, 2.7 mM CaCl2, 2.1 mM MgCl2 and 14.44 mM NaHCO3, pH 7.4), equilibrated with 5% CO2 in N2 and maintained at 37°C. Prior to intravital observations, mice were either pre-treated with an intrascrotal injection of TNFα (500 ng in 200 μl; R&D Systems, Abingdon, UK) for three hours or the cremaster was superfused with CXCL1 (5nM in 500 ml; Peprotech) for 1 hour. Control mice received a PBS vehicle. Leukocyte-endothelial cell interactions were observed in single unbranched post-capillary venules (PCV; 20 to 50 μm diameter). Leukocyte rolling was determined by counting numbers of cells rolling along a 100 μm PCV segment within 60 seconds. A leukocyte was considered firmly adherent if it remained stationary for ≥30 seconds.
Induction of murine antigen-induced arthritis (AIA)
Experiments were performed in 7- to 8-week-old male mice. Murine AIA was induced as described . Briefly, mice were immunised subcutaneously with 1 mg/ml of methylated bovine serum albumin (mBSA) emulsified with an equal volume of Freund’s complete adjuvant and injected intraperitoneally with 100 μl heat-inactivated Bordetella pertussis toxin (all reagents from Sigma-Aldrich). The immune response was boosted one week later. Twenty-one days after the initial immunisation, murine AIA was induced by intra-articular injection of 10 mg/ml mBSA in PBS in the right knee (stifle) joint. For a control, the same volume of PBS was injected into the left knee joint.
Animals were inspected daily for arthritis development by measuring knee joint diameters using a digital micrometer. The difference in joint diameter between the arthritic (right) and non-arthritic control (left) in each animal gave a quantitative measure of swelling (in mm).
Animals were killed at the indicated times after induction of arthritis. Joints were fixed in neutral buffered formal saline, and decalcified with formic acid at 4°C before embedding in paraffin. Mid-sagittal serial sections (7 μm thickness) were cut and stained with haematoxylin and eosin (H&E). Two independent observers blinded to the experimental groups scored sections. Synovial hyperplasia, cellular exudate and cartilage depletion were scored from 0 (normal) to 3 (severe); synovial infiltrate was scored from 0 to 5 [48, 49]. Cartilage damage was scored on serial haematoxylin/safranin O-stained sections. All parameters were subsequently summed to give an arthritis index (mean ± SEM).
Differences between groups were compared by Mann-Whitney U or unpaired t tests, with P <0.05 being deemed as significant.
Sdc-3 deletion reduces neutrophil recruitment in CXCL1-injected joints
Reduced chemokine presentation by synovial endothelial cells in sdc-3−/−mice
Serial sections of CXCL1-injected joints were treated with heparanase I and III to degrade heparan sulphate prior to CXCL1 immunolocalisation. Use of these enzymes resulted in lack of CXCL1 immunofluorescence in endothelial cells of wild-type and sdc-3−/−synovial blood vessels (Figure 2D). Quantitation revealed that for wild type the mean number of endothelial CXCL1 clusters per blood vessel after heparanase digestion was 2.8 ± 0.7 (mean ± SE, n = 6), which was significantly lower than without heparanase (28.7 ± 3.2, see Figure 2F) (P <0.0001; t test). For sdc-3 null mice the mean number of endothelial CXCL1 clusters per blood vessel after heparanase was 2.7 ± 1.8 and 8.7 ± 1.8 without heparanase (both mean ± SE, n = 5) (Figure 2F) and these values did not significantly differ. These heparanase data suggest that the heparan sulphate chains of endothelial sdc-3 bind CXCL1 clusters. Controls in the absence of anti-CXCL1 were negative (Figure 2E). Sections were also immunostained for E-selectin. Although E-selectin was detected in synovial endothelial cells, it was less abundant than in skin endothelial cells, and there was no significant difference in E-selectin distribution between sdc-3−/−and sdc-3+/+mice in the presence or absence of CXCL1 (data not shown).
Less severe AIA in sdc-3−/−mice
Joint inflammation and cartilage damage on day 3 of antigen-induced arthritis
2.07 ± 0.23
2.67 ± 0.67**
0.63 ± 0.24
0.63 ± 0.24*
6.00 ± 0.75**
2.57 ± 0.15
3.97 ± 0.24
1.40 ± 0.31
1.40 ± 0.27
9.33 ± 0.74
Sdc-3 deletion provokes enhanced neutrophil recruitment in CXCL1-injected skin
Immunofluorescence using anti-murine sdc-3 showed that this HSPG was expressed in the endothelium of the dermis in wild-type mice (Figure 4C and D). To further investigate the potential mechanism of increased neutrophil recruitment after CXCL1 challenge adhesion molecule expression was examined. E-selectin immunolocalisation was performed in skin tissue sections (Figure 4E and F). This adhesion molecule is expressed by dermal endothelial cells and is involved in the rolling stage of leukocyte adhesion to the endothelium [52–55]. Using dual labelling E-selectin co-localised with von Willebrand factor as a marker of endothelial cells (Additional file 1C to E), and the proportion of von Willebrand factor positive dermal blood vessels that expressed E-selectin was >95% (n >15 vessels per wild-type and sdc-3−/−mouse). E-selectin exhibited a predominantly luminal or intracellular distribution in the endothelial cells of the dermis in sdc-3−/−and sdc-3+/+mice (Figure 4E and F; Additional file 1A and B). Quantification revealed that there was a two-fold increase in the numbers of vessels with a luminal E-selectin distribution in sdc-3−/−mice compared to wild type (P <0.008 Mann-Whitney test) following CXCL1 administration (Figure 4G). When PBS was administered instead of CXCL1, as vehicle-injected control, there was also significantly more vessels with a luminal E-selectin distribution in sdc-3 null mice compared to wild type (Figure 4G). In sdc-3−/−mice there was no significant difference in luminal E-selectin between CXCL1- and PBS-injected skin (Figure 4G), suggesting that this chemokine was not affecting E-selectin distribution. After injection of CXCL1, this chemokine could be detected as a uniform distribution in endothelial cells of dermal venules by immunofluorescence, however, there was no significant difference in the number or percentage of these cells positive for CXCL1 in sdc-3−/− (n = 8) and wild-type (n = 9) mice (data not shown); this suggests that CXCL1 presentation in skin may be occurring by a different proteoglycan than sdc-3. Control sections treated in the absence of E-selectin, von Willebrand or CXCL1 antibodies were negative.
Increased rolling and adhesion of leukocytes in cremaster venules in sdc-3 null mice
Interestingly, the basal number of adherent leukocytes was significantly (P <0.05) increased in unstimulated sdc-3−/−mice when compared to wild-type mice, with more than double the numbers of adherent cells observed (Figure 5B). As expected, TNFα stimulation significantly (P <0.05) increased leukocyte adhesion in wild-type mice when compared to unstimulated wild-type mice. However, this effect was more dramatic in the sdc-3−/−mice, with significantly increased leukocyte adhesion observed when compared to either unstimulated sdc-3−/− (P <0.05) or TNFα-stimulated wild-type (P <0.05) mice (Figure 5B). Indeed, when compared to unstimulated sdc-3−/−, a 2.2-fold increase in adhesion was observed. Although CXCL1 stimulation did not increase leukocyte adhesion in wild-type mice, it was associated with a significant increase in adhesion in sdc-3−/−mice when compared to CXCL1-stimulated wild type (P <0.05). This did not reach significance when compared to unstimulated sdc-3−/−, presumably reflecting increased basal adhesion in the sdc-3−/− (Figure 5B). All statistical comparisons for intravital microscopy were made by ANOVA followed by Tukey’s pairwise tests.
The current study demonstrated that sdc-3 played a role in inflammation, but interestingly, highlighted both pro- and anti-inflammatory properties for this proteoglycan depending upon the tissue and nature of the inflammatory insult. In the joint, chemokine administration resulted in reduced neutrophil influx in the synovium of sdc-3 null mice indicating that this HSPG is playing a pro-inflammatory role. This effect may be attributed to chemokine presentation by sdc-3 on synovial endothelial cells since deletion of this HSPG reduced the presence of chemokine CXCL1 on these cells. Furthermore, heparanase reduced the amount of endothelial CXCL1 suggesting the involvement of HS chains in binding CXCL1. The chemokine in synovial endothelial cells was not uniformly distributed but appeared to be bound to sdc-3 in clusters. Thus CXCL1 may be concentrated and immobilised into clusters at the endothelial surface for presentation to blood leukocytes. This is in agreement with Hardy et al.  who found a focal distribution of CCL2 bound to HS at the apical endothelial surface during leukocyte transendothelial migration in vitro. CXCL1 clusters were particularly reduced at the endothelial surface in sdc-3−/−mice whereas in the remainder of the cell in intracellular/abluminal locations this was not the case. This suggests that sdc-3 may be particularly involved in chemokine presentation whereas other molecules may play a more dominant role in transcytosis, such as the Duffy antigen/receptor for chemokines [45, 56]. The finding of sdc-3 binding and presenting CXCL1 in the current study is in agreement with our previous study . In human RA there is induction of a CXCL8 binding site on sdc-3 HS chains of synovial endothelial cells. Mice lack CXCL8 and CXCL1 is the functional equivalent in the murine system. Therefore taken together, these two studies suggest that sdc-3 may be involved in binding CXC chemokines and stimulating leukocyte trafficking into the RA synovium.
A pro-inflammatory function of sdc-3 was also apparent in a murine model of RA. Induction of AIA in the knee joint resulted in reduced joint swelling in sdc-3 knockout mice suggesting that sdc-3 contributes to the clinical manifestation of the disease. This HSPG is also involved in underlying inflammatory changes such as leukocyte accumulation into the synovium, which was reduced in sdc-3 null mice as was the overall histological severity of disease. The pro-inflammatory function of sdc-3 in AIA may be due to chemokine presentation by synovial endothelial cells. Furthermore a role for sdc-3 in joint damage, which is a major feature of RA, is implicated as shown by the inhibitory effects of sdc-3 deletion on cartilage damage. This involvement of sdc-3 in cartilage damage may be related to its pro-inflammatory function in the synovium, via leukocyte recruitment leading to cytokine or degradative enzyme release. However, the effects of loss of sdc-3 in the arthritis model may be mediated, at least in part, by cells other than endothelial cells, since sdc-3 is also expressed by chondrocytes [39, 40]. Further studies involving conditional deletion of sdc-3 in selected cell types and examining the effect on arthritis severity would be of interest in this respect.
Recent data suggest a role for sdc-4 in inflammatory arthritis [34, 35]. Using the human TNF transgenic mouse model (hTNFtg) of RA, sdc-4 was involved in the attachment and invasion of synovial fibroblasts into cartilage, contributing to cartilage destruction. Sdc-4 also regulates ADAMTS-5 activation and cartilage breakdown . This suggests that sdcs may be involved in various aspects of joint inflammation and damage in arthritis, with endothelial sdc-3 functioning in leukocyte recruitment and fibroblast sdc-4 in cartilage destruction.
Deletion of sdc-3 in the skin had the opposite effect compared to that in the joint. When CXCL1 was injected into the skin neutrophil recruitment was enhanced in sdc-3−/−mice compared to wild type suggesting that this HSPG plays an anti-inflammatory function in this tissue. The effect may be mediated, at least in part, by the adhesion molecule E-selectin since the luminal distribution of E-selectin increased in knockout animals suggesting increased expression of this adhesion molecule at the endothelial surface. This may lead to elevated neutrophil recruitment in the presence of CXCL1. E-selectin is expressed in normal skin venules where it is upregulated in skin inflammation [52–55]. Sdc-3 is part of the glycocalyx, which can form an anti-adhesive layer to blood leukocytes at the endothelial surface and it has been proposed that this may mask endothelial adhesion molecules inhibiting leukocyte-endothelial interactions [1, 10]. Steric hindrance may play a role in this process since the glycocalyx can reach microns in thickness whereas selectins only extend <50 nm from the endothelial surface [1, 57]. Therefore loss of sdc-3 in knockout mice may lead to the unmasking or altered expression of E-selectin at the luminal endothelial surface leading to increased leukocyte recruitment. This is in agreement with other studies that show that stimuli that degrade the glycocalyx or induce a more open mesh such as enzymes, cytokines, or ischaemia and reperfusion appear to uncover adhesion molecules, thereby allowing leukocytes to interact with the endothelium [1–3, 26]. For example, heparanase, which is a glycosidase that removes HS, causes increased leukocyte adherence at the endothelial surface in the cremaster venules of mice by intravital microscopy . In the present study, endothelial sdc-3 does not appear to be presenting the chemokine CXCL1 in the skin since there was no difference in the presence of this chemokine on dermal venules in wild-type and knockout mice and other HSPGs may be more involved in this mechanism. Thus in the skin sdc-3 may be involved in regulating leukocyte adhesion via altering the distribution or expression of the adhesion molecule E-selectin.
Since the data obtained from the skin demonstrated an anti-inflammatory role for sdc-3, we further investigated its role using the more direct approach of intravital microscopy, which allowed real-time dynamic images of leukocyte adhesion to be monitored in anaesthetised mice in vivo. Furthermore, the effects on sdc-3 deletion on leukocyte rolling could also be assessed, which was not possible on static sections. Increased numbers of rolling and adherent leukocytes in the venules of sdc-3−/−mice in response to either CXCL1 or TNFα was observed compared to wild type. These results suggest that sdc-3 has an inhibitory effect on leukocyte-endothelial interactions in response to inflammatory stimuli and are in accord with those in skin. Intravital microscopy has been performed in sdc-1 null mice following TNFα treatment where there is increased adhesion of leukocytes to endothelial cells of the mesentery venules [11, 58]. These intravital data, together with ours, indicate that sdc-3 and sdc-1 play similar roles in cremaster and mesenteric venules using similar inflammation models. In these tissues sdc-3 and sdc-1 appear to be negative regulators of leukocyte-endothelial interactions.
The anti-inflammatory role of sdc-3 in our models in skin and cremaster is similar to that of sdc-1 and -4 in inflammatory disease models. Sdc-1 gene deletion in mice reduces inflammation in models of allergic contact dermatitis, allergic lung disease, colitis and nephritis, with increased leukocyte recruitment and more severe disease [20, 21, 23, 24]. Similarly sdc-4 null mice exhibit increased inflammation and neutrophil recruitment in a model of pulmonary inflammation and lung injury . Thus our finding of sdc-3 having a pro-inflammatory role in synovium in a mouse model of RA is more unusual amongst the different sdc knockout models. Whether these HSPGs have pro- or anti-inflammatory functions may depend on the sdc, the tissue or cell-type where they are expressed and/or the type of inflammation. Furthermore, specific targeting of sdcs tailored to particular inflammatory diseases is called for if they are to be exploited therapeutically in human diseases. For example, blocking sdc-3 or sdc-4 in human RA would be of potential interest in reducing inflammation and joint destruction, whereas this strategy may have opposite effects in certain inflammatory conditions of the skin, lung, gut and kidney.
In the current study, sdc-3 was found to be expressed by endothelial cells in murine synovium and skin. This is in agreement with human tissues where endothelial sdc-3 was found particularly expressed in the endothelial cells of RA synovium . Interestingly, sdc-3 is also found in lymphoid tissue, where this HSPG perfectly delineates some of the high endothelial venules . These venules are the preferred sites of lymphocyte extravasation which, taken with the findings of the current study, suggests a role for this HSPGs in lymphocyte trafficking in the lymph nodes. Sdc-3 is also expressed by the endothelial cells in human liver .
Sdc-3 appears to have a tissue-selective role in inflammation being pro-inflammatory in the joint, which may be mediated by endothelial chemokine presentation. It is also involved in leukocyte accumulation and cartilage damage in joints with AIA. In the skin and cremaster it may be anti-inflammatory, contributing to the anti-adhesive properties of the endothelial glycocalyx. This study helps clarify the contradictory roles of HSPGs being reported as pro-and anti-inflammatory and suggests the importance of tissue-dependent functions of endothelial cells in the case of sdc-3. Furthermore, it suggests that targeting sdc-3 in the joint in inflammatory arthritis would be a therapeutic strategy.
chemokine C-X-C ligand 1
heparan sulphate proteoglycan
methylated bovine serum albumin
tumour necrosis factor.
We wish to acknowledge helpful discussions with Prof Simon Jones, University of Cardiff, and rheumatologists Drs Robin Bulter, Josh Dixey, Ayman Askari and Mark Garton, RJAH Orthopaedic Hospital. The help and support of staff at the Liverpool John Moores University Life Science Support Unit is gratefully acknowledged. We thank P. Evans, N. Harness, and M. Pritchard, RJAH Orthopaedic Hospital, for their expertise in histology.
Funding was from the Medical Research Council (UK), and the Institute of Orthopaedics and Rheumatology Trust Funds, RJAH Orthopaedic Hospital (UK).
- Reitsma S, Slaaf DW, Vink H, van Zandvoort MA, oude Egbrink MG: The endothelial glycocalyx: composition, functions, and visualization. Pflugers Arch. 2007, 454: 345-359.PubMed CentralView ArticlePubMedGoogle Scholar
- Henry CB, Duling BR: TNF-alpha increases entry of macromolecules into luminal endothelial cell glycocalyx. Am J Physiol Heart Circ Physiol. 2000, 279: H2815-H2823.PubMedGoogle Scholar
- Chappell D, Dörfler N, Jacob M, Rehm M, Welsch U, Conzen P, Becker BF: Glycocalyx protection reduces leukocyte adhesion after ischemia/reperfusion. Shock. 2010, 34: 133-139.View ArticlePubMedGoogle Scholar
- Choi Y, Chung H, Jung H, Couchman JR, Oh ES: Syndecans as cell surface receptors: Unique structure equates with functional diversity. Matrix Biol. 2011, 30: 93-99.View ArticlePubMedGoogle Scholar
- Bernfield M, Kokenyesi R, Kato M, Hinkes MT, Spring J, Gallo RL, Lose EJ: Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans. Annu Rev Cell Biol. 1992, 8: 365-393.View ArticlePubMedGoogle Scholar
- Carey DJ: Syndecans: multifunctional cell-surface co-receptors. Biochem J. 1997, 327: 1-16.PubMed CentralView ArticlePubMedGoogle Scholar
- Couchman JR: Syndecans: proteoglycan regulators of cell-surface microdomains?. Nat Rev Mol Cell Biol. 2003, 4: 926-937.View ArticlePubMedGoogle Scholar
- Couchman JR: Transmembrane signaling proteoglycans. Annu Rev Cell Dev Biol. 2010, 26: 89-114.View ArticlePubMedGoogle Scholar
- Parish CR: The role of heparan sulphate in inflammation. Nat Rev Immunol. 2006, 6: 633-643.View ArticlePubMedGoogle Scholar
- Celie JW, Beelen RH, van den Born J: Heparan sulfate proteoglycans in extravasation: assisting leukocyte guidance. Front Biosci. 2009, 14: 4932-4949.View ArticleGoogle Scholar
- Götte M: Syndecans in inflammation. FASEB J. 2003, 17: 575-591.View ArticlePubMedGoogle Scholar
- Middleton J, Neil S, Wintle J, Clark-Lewis I, Moore H, Lam C, Auer M, Hub E, Rot A: Transcytosis and surface presentation of IL-8 by venular endothelial cells. Cell. 1997, 91: 385-395.View ArticlePubMedGoogle Scholar
- Proudfoot AE, Handel TM, Johnson Z, Lau EK, LiWang P, Clark-Lewis I, Borlat F, Wells TN, Kosco-Vilbois MH: Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proc Natl Acad Sci U S A. 2003, 100: 1885-1890.PubMed CentralView ArticlePubMedGoogle Scholar
- Massena S, Christoffersson G, Hjertström E, Zcharia E, Vlodavsky I, Ausmees N, Rolny C, Li JP, Phillipson M: A chemotactic gradient sequestered on endothelial heparan sulfate induces directional intraluminal crawling of neutrophils. Blood. 2010, 116: 1924-1931.PubMed CentralView ArticlePubMedGoogle Scholar
- Hardy LA, Booth TA, Lau EK, Handel TM, Ali S, Kirby JA: Examination of MCP-1 (CCL2) partitioning and presentation during transendothelial leukocyte migration. Lab Invest. 2004, 84: 81-90.View ArticlePubMedGoogle Scholar
- Johnson Z, Proudfoot AE, Handel TM: Interaction of chemokines and glycosaminoglycans: a new twist in the regulation of chemokine function with opportunities for therapeutic intervention. Cytokine Growth Factor Rev. 2005, 16: 625-636.View ArticlePubMedGoogle Scholar
- Middleton J, Patterson AM, Gardner L, Schmutz C, Ashton BA: Leukocyte extravasation: chemokine transport and presentation by the endothelium. Blood. 2002, 100: 3853-3860.View ArticlePubMedGoogle Scholar
- Rot A: Chemokine patterning by glycosaminoglycans and interceptors. Front Biosci. 2010, 1: 645-660.View ArticleGoogle Scholar
- Wang L, Fuster M, Sriramarao P, Esko JD: Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses. Nat Immunol. 2005, 6: 902-910.View ArticlePubMedGoogle Scholar
- Xu J, Park PW, Kheradmand F, Corry DB: Endogenous attenuation of allergic lung inflammation by syndecan-1. J Immunol. 2005, 174: 5758-5765.View ArticlePubMedGoogle Scholar
- Rops AL, Götte M, Baselmans MH, van den Hoven MJ, Steenbergen EJ, Lensen JF, Wijnhoven TJ, Cevikbas F, van den Heuvel LP, van Kuppevelt TH, Berden JH, van der Vlag J: Syndecan-1 deficiency aggravates anti-glomerular basement membrane nephritis. Kidney Int. 2007, 72: 1204-1215.View ArticlePubMedGoogle Scholar
- Teng YH, Aquino RS, Park PW: Molecular functions of syndecan-1 in disease. Matrix Biol. 2012, 31: 3-16.PubMed CentralView ArticlePubMedGoogle Scholar
- Kharabi Masouleh B, Ten Dam GB, Wild MK, Seelige R, van der Vlag J, Rops AL, Echtermeyer FG, Vestweber D, van Kuppevelt TH, Kiesel L, Götte M: Role of the heparan sulfate proteoglycan syndecan-1 (CD138) in delayed-type hypersensitivity. J Immunol. 2009, 182: 4985-4993.View ArticlePubMedGoogle Scholar
- Floer M, Götte M, Wild MK, Heidemann J, Gassar ES, Domschke W, Kiesel L, Luegering A, Kucharzik T: Enoxaparin improves the course of dextran sodium sulfate-induced colitis in syndecan-1-deficient mice. Am J Pathol. 2010, 176: 146-157.PubMed CentralView ArticlePubMedGoogle Scholar
- Tanino Y, Chang MY, Wang X, Gill SE, Skerrett S, McGuire JK, Sato S, Nikaido T, Kojima T, Munakata M, Mongovin S, Parks WC, Martin TR, Wight TN, Frevert CW: Syndecan-4 regulates early neutrophil migration and pulmonary inflammation in response to lipopolysaccharide. Am J Respir Cell Mol Biol. 2012, 47: 196-202.PubMed CentralView ArticlePubMedGoogle Scholar
- Constantinescu AA, Vink H, Spaan JA: Endothelial cell glycocalyx modulates immobilization of leukocytes at the endothelial surface. Arterioscler Thromb Vasc Biol. 2003, 23: 1541-1547.View ArticlePubMedGoogle Scholar
- Szekanecz Z, Vegvari A, Szabo Z, Koch AE: Chemokines and chemokine receptors in arthritis. Front Biosci (Schol Ed). 2010, 2: 153-167.View ArticleGoogle Scholar
- Hosaka S, Akahoshi T, Wada C, Kondo H: Expression of the chemokine superfamily in rheumatoid arthritis. Clin Exp Immunol. 1994, 97: 451-457.PubMed CentralView ArticlePubMedGoogle Scholar
- Koch AE, Kunkel SL, Burrows JC, Evanoff HL, Haines GK, Pope RM, Strieter RM: Synovial tissue macrophage as a source of the chemotactic cytokine IL-8. J Immunol. 1991, 147: 2187-2195.PubMedGoogle Scholar
- Koch AE, Kunkel SL, Harlow LA, Mazarakis DD, Haines GK, Burdick MD, Pope RM, Walz A, Strieter RM: Epithelial neutrophil activating peptide-78: a novel chemotactic cytokine for neutrophils in arthritis. J Clin Invest. 1994, 94: 1012-1018.PubMed CentralView ArticlePubMedGoogle Scholar
- Koch AE, Kunkel SL, Shah MR, Hosaka S, Halloran MM, Haines GK, Burdick MD, Pope RM, Strieter RM: Growth-related gene product alpha. A chemotactic cytokine for neutrophils in rheumatoid arthritis. J Immunol. 1995, 155: 3660-3666.PubMedGoogle Scholar
- Koch AE, Volin MV, Woods JM, Kunkel SL, Connors MA, Harlow LA, Woodruff DC, Burdick MD, Strieter RM: Regulation of angiogenesis by the C-X-C chemokines interleukin-8 and epithelial neutrophil activating peptide 78 in the rheumatoid joint. Arthritis Rheum. 2001, 44: 31-40.View ArticlePubMedGoogle Scholar
- Patterson AM, Gardner L, Shaw J, David G, Loreau E, Aguilar L, Ashton BA, Middleton J: Induction of a CXCL8 binding site on endothelial syndecan-3 in rheumatoid synovium. Arthritis Rheum. 2005, 52: 2331-2342.View ArticlePubMedGoogle Scholar
- Korb-Pap A, Stratis A, Mühlenberg K, Niederreiter B, Hayer S, Echtermeyer F, Stange R, Zwerina J, Pap T, Pavenstädt H, Schett G, Smolen JS, Redlich K: Early structural changes in cartilage and bone are required for the attachment and invasion of inflamed synovial tissue during destructive inflammatory arthritis. Ann Rheum Dis. 2012, 71: 1004-1011.View ArticlePubMedGoogle Scholar
- Pap T, Bertrand J: Syndecans in cartilage breakdown and synovial inflammation. Nat Rev Rheumatol. 2012, 9: 43-55.View ArticlePubMedGoogle Scholar
- Echtermeyer T, Bertrand J, Dreier R, Meinecke I, Neugebauer K, Fuerst M, Lee YJ, Song YW, Herzog C, Theilmeier G, Pap T: Syndecan-4 regulates ADAMTS-5 activation and cartilage breakdown in osteoarthritis. Nat Med. 2009, 15: 1072-1076.View ArticlePubMedGoogle Scholar
- Reizes O, Lincecum J, Wang Z, Goldberger O, Huang L, Kaksonen M, Ahima R, Hinkes MT, Barsh GS, Rauvala H, Bernfield M: Transgenic expression of syndecan-1 uncovers a physiological control of feeding behavior by syndecan-3. Cell. 2001, 106: 105-116.View ArticlePubMedGoogle Scholar
- van Horssen J, Kleinnijenhuis J, Maass CN, Rensink AA, Otte-Höller I, David G, van den Heuvel LP, Wesseling P, de Waal RM, Verbeek MM: Accumulation of heparan sulfate proteoglycans in cerebellar senile plaques. Neurobiol Aging. 2002, 23: 537-545.View ArticlePubMedGoogle Scholar
- Pfander D, Swoboda B, Kirsch T: Expression of early and late differentiation markers (proliferating cell nuclear antigen, syndecan-3, annexin VI, and alkaline phosphatase) by human osteoarthritic chondrocytes. Am J Pathol. 2001, 159: 1777-1783.PubMed CentralView ArticlePubMedGoogle Scholar
- Kirsch T, Koyama E, Liu M, Golub EE, Pacifici M: Syndecan-3 is a selective regulator of chondrocyte proliferation. J Biol Chem. 2002, 277: 42171-42177.View ArticlePubMedGoogle Scholar
- Kosher RA: Syndecan-3 in limb skeletal development. Microsc Res Tech. 1998, 43: 123-130.View ArticlePubMedGoogle Scholar
- Casar JC, Cabello-Verrugio C, Olguin H, Aldunate R, Inestrosa NC, Brandan E: Heparan sulfate proteoglycans are increased during skeletal muscle regeneration: requirement of syndecan-3 for successful fiber formation. J Cell Sci. 2004, 117: 73-84.View ArticlePubMedGoogle Scholar
- Roskams T, Moshage H, De Vos R, Guido D, Yap P, Desmet V: Heparan sulfate proteoglycan expression in normal human liver. Hepatology. 1995, 21: 950-958.View ArticlePubMedGoogle Scholar
- Bobardt MD, Saphire AC, Hung HC: Syndecan captures, protects, and transmits HIV to T lymphocytes. Immunity. 2003, 18: 27-39.View ArticlePubMedGoogle Scholar
- Pruenster M, Mudde L, Bombosi P, Dimitrova S, Zsak M, Middleton J, Richmond A, Graham GJ, Segerer S, Nibbs RJ, Rot A: The Duffy antigen receptor for chemokines transports chemokines and supports their promigratory activity. Nat Immunol. 2009, 10: 101-108.PubMed CentralView ArticlePubMedGoogle Scholar
- Hol J, Wilhelmsen L, Haraldsen G: The murine IL-8 homologues KC, MIP-2, and LIX are found in endothelial cytoplasmic granules but not in Weibel-Palade bodies. J Leukoc Biol. 2010, 87: 501-508.View ArticlePubMedGoogle Scholar
- Nolan SL, Kalia N, Nash GB, Kamel D, Heeringa P, Savage CO: Mechanisms of ANCA-mediated leukocyte-endothelial cell interactions in vivo. J Am Soc Nephrol. 2008, 19: 973-984.PubMed CentralView ArticlePubMedGoogle Scholar
- Nowell MA, Richards PJ, Horiuchi S, Yamamoto N, Rose-John S, Topley N, Williams AS, Jones SA: Soluble IL-6 receptor governs IL-6 activity in experimental arthritis: blockade of arthritis severity by soluble glycoprotein 130. J Immunol. 2003, 171: 3202-3209.View ArticlePubMedGoogle Scholar
- Cartwright A, King S, Middleton J, Kehoe O: Is chemokine receptor CCR9 required for synovitis in rheumatoid arthritis? Deficiency of CCR9 in a murine model of antigen-induced arthritis. Open J Rheumatol Autoimmune Dis. 2012, 2: 77-84.View ArticleGoogle Scholar
- Baekkevold ES, Yamanaka T, Palframan RT, Carlsen HS, Reinholt FP, von Andrian UH, Brandtzaeg P, Haraldsen G: The Ccr7 ligand ELC (Ccl19) is transcytosed in high endothelial venules and mediates T cell recruitment. J Exp Med. 2001, 193: 1105-1112.PubMed CentralView ArticlePubMedGoogle Scholar
- Tanaka Y, Fujii K, Hübscher S, Aso M, Takazawa A, Saito K, Ota T, Eto S: Heparan sulfate proteoglycan on endothelium efficiently induces integrin-mediated T cell adhesion by immobilizing chemokines in patients with rheumatoid synovitis. Arthritis Rheum. 1998, 41: 1365-1377.View ArticlePubMedGoogle Scholar
- oude Egbrink MG, Janssen GH, Ookawa K, Slaaf DW, Reneman RS, Wehrens XH, Maaijwee KJ, Ohshima N, Struijker Boudier HA, Tangelder GJ: Especially polymorphonuclear leukocytes, but also monomorphonuclear leukocytes, roll spontaneously in venules of intact rat skin: involvement of E-selectin. J Invest Dermatol. 2002, 118: 323-326.View ArticlePubMedGoogle Scholar
- Weninger W, Ulfman LH, Cheng G, Souchkova N, Quackenbush EJ, Lowe JB, von Andrian UH: Specialized contributions by alpha(1,3)-fucosyltransferase-IV and FucT-VII during leukocyte rolling in dermal microvessels. Immunity. 2000, 12: 665-676.View ArticlePubMedGoogle Scholar
- Janssen GH, Tangelder GJ, Oude Egbrink MG, Reneman RS: Spontaneous leukocyte rolling in venules in untraumatized skin of conscious and anesthetized animals. Am J Physiol. 1994, 267: H1199-H1204.PubMedGoogle Scholar
- Barker JN: Adhesion molecules in cutaneous inflammation. Ciba Found Symp. 1995, 189: 91-101.PubMedGoogle Scholar
- Gardner L, Wilson C, Patterson AM, Bresnihan B, FitzGerald O, Stone MA, Ashton BA, Middleton J: Temporal expression pattern of Duffy antigen in rheumatoid arthritis: up-regulation in early disease. Arthritis Rheum. 2006, 54: 2022-2026.View ArticlePubMedGoogle Scholar
- Springer TA: Adhesion receptors of the immune system. Nature. 1990, 346: 425-434.View ArticlePubMedGoogle Scholar
- Götte M, Joussen AM, Klein C, Andre P, Wagner DD, Hinkes MT, Kirchhof B, Adamis AP, Bernfield M: Role of syndecan-1 in leukocyte-endothelial interactions in the ocular vasculature. Invest Ophthalmol Vis Sci. 2002, 43: 1135-1141.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.