Synovial Lining Macrophages Research Articles

Quantification of macrophage activity in knee synovial tissue using [18F]FEPPA positron emission tomography

ObjectiveInflammation in knee osteoarthritis (OA) is mediated primarily by synovial tissue macrophages, but non-invasive measurement of macrophage activity in vivo is a challenge. Activated macrophages markedly increase expression of the translocator protein (TSPO). In the context of other diseases TSPO-PET using the [18F]FEPPA tracer (binding to TSPO) has been reported to be an effective method for imaging macrophages in vivo. The goal of this study was to validate the use of [18F]FEPPA PET radiotracer to accurately measure macrophage activation in knee synovial tissue. DesignTen participants with late-stage OA scheduled for knee replacement surgery were recruited. Prior to surgery, 5 of the participants underwent a clinical [18F]FEPPA PET/MRI scan and the standard uptake value (SUV) in the suprapatellar synovial region was calculated. Suprapatellar synovial tissue samples were collected from all participants at the time of surgery and were imaged ex vivo with [18F]FEPPA autoradiography. Tracer uptake was compared to TSPO antibody immunostaining in serial tissue sections. The correlation between the [18F]FEPPA uptake and gold standard TSPO fluorescent intensity was measured. RESULTSThe autoradiography [18F]FEPPA signal in the synovial lining was correlated with the TSPO signal in the. lining macrophages measured by immunohistochemistry (r = 0.81, p = 0.0040, CI [0.37, 0.95]). Similarly, PET/MRI scans demonstrated similar correlation between SUV in synovial tissues in vivo and in situ TSPO signal in lining macrophages {r = 0.90, p = 0.083, CI [0.086, 0.99]) Conclusion[18F]FEPPA uptake in knee synovial tissue is strongly correlated to the expression of TSPO in synovial lining macrophages, suggesting that clinical [18F]FEPPA PET/MRI may be a valid measure of true macrophage activity in synovial tissues in knee OA. [18F]FEPPA may be an effective tool to assess macrophage activation both ex vivo and in vivo, and should be investigated further to assess its performance and measurement properties in different clinical contexts of knee OA including disease states and responses to treatment.

AXL Expression on Homeostatic Resident Liver Macrophages Is Reduced in Cirrhosis Following GAS6 Production by Hepatic Stellate Cells

AXL and MERTK expression on circulating monocytes modulated immune responses in patients with cirrhosis (CD14+HLA-DR+AXL+) and acute-on-chronic liver failure (CD14+MERTK+). AXL expression involved enhanced efferocytosis, sustained phagocytosis, but reduced tumor necrosis factor-α/interleukin-6 production and T-cell activation, suggesting a homeostatic function. Axl was expressed on murine airway in tissues contacting the external environment, but not interstitial lung- and tissue-resident synovial lining macrophages. We assessed AXL expression on tissue macrophages in patients with cirrhosis. Using multiplexed immunofluorescence we compared AXL expression in liver biopsies in cirrhosis (n= 22), chronic liver disease (n= 8), non-cirrhotic portal hypertension (n= 4), and healthy controls (n= 4). Phenotype and function of isolated primary human liver macrophages were characterized by flow cytometry (cirrhosis, n= 11; control, n= 14) exvivo. Also, AXL expression was assessed on peritoneal (n= 29) and gut macrophages (n= 16) from cirrhotic patients. Regulation of AXL expression was analyzed invitro and exvivo using primary hepatic stellate cells (HSCs), LX-2 cells, and GAS6 in co-culture experiments. AXL was expressed on resident (CD68+) but not tissue-infiltrating (MAC387+) liver macrophages, hepatocytes, HSCs, or sinusoidal endothelial cells. Prevalence of hepatic CD68+AXL+ cells significantly decreased with cirrhosis progression: (healthy, 90.2%; Child-Pugh A, 76.1%; Child-Pugh B, 64.5%; and Child-Pugh C, 18.7%; all P < .05) and negatively correlated with Model for End-Stage Liver Disease and C-reactive protein (all P < .05). AXL-expressing hepatic macrophages were CD68highHLA-DRhighCD16highCD206high. AXL expression also decreased on gut and peritoneal macrophages from cirrhotic patients but increased in regional lymph nodes. GAS6, enriched in the cirrhotic liver, appeared to be secreted by HSCs and down-regulate AXL invitro. Decreased AXL expression on resident liver macrophages in advanced cirrhosis, potentially in response to activated HSCs-secreted GAS6, suggests a role for AXL in the regulation of hepatic immune homeostasis.

There is a correlation between histopathological findings of joint capsule and synovium, and postoperative clinical outcomes and treatment in patients with isolated type II superior labrum anterior posterior lesions.

Objectives This study aims to histologically examine the joint capsule and synovium to determine the correlation between histopathological findings and postoperative clinical outcomes in patients with isolated type II superior labrum anterior posterior (SLAP) lesions.Patients and methods Thirty-eight patients (24 males, 14 females; mean age 53.2±6.6 years; range, 45 to 67 years) who underwent arthroscopic treatment of type II SLAP lesions between June 2017 and September 2018 were evaluated prospectively. Visual analog scale (VAS), Simple Shoulder Test (SST), and American Shoulder and Elbow Surgeons (ASES) scores of all patients were recorded preoperatively, and at 6th and 12th months postoperatively. Biceps tenotomy was applied as arthroscopic surgical treatment in all patients. Biopsy materials obtained from rotator interval joint capsule and synovium during the arthroscopy were evaluated histopathologically. The density of the vessels in the specimens was defined as low, medium, and high by the pathologist. The patients with medium or low vessel density in specimens were group 1 (n=14) and those with high vessel density group 2 (n=24).Results In group 2, preoperative VAS score was significantly higher. There was no difference between the scores of the groups except for the sixth month SST score which was significantly higher in group 1. Histopathological evaluation revealed that the number of lymphocytes, fibroblasts, mast cells, myofibroblast, synovial lining cells, macrophages, and amount of collagen in connective tissue were significantly higher in group 2. In five patients of group 2, the rehabilitation program was interrupted due to pain and difficulty in gaining a range of motion during the first four weeks postoperatively. Four of these patients recovered with medication and long-duration physiotherapy. Shoulder stiffness developed in one patient who required arthroscopic release and further rehabilitation.ConclusionThere is a correlation between histopathological findings of joint capsule and synovium, and postoperative clinical outcomes and treatment in patients with isolated type II SLAP lesions. Almost 20% of patients who had pathologic histological findings in joint capsule and synovium needed pain control and long-duration rehabilitation program after arthroscopic surgery for better shoulder function recovery and prevention of shoulder stiffness.

Immunohistochemical Analysis of Inflammatory Rheumatoid Synovial Tissues Using Anti-Human Podoplanin Monoclonal Antibody Panel.

Podoplanin (PDPN) is a transmembrane sialoglycoprotein, which is expressed in several normal tissues and malignant tumors. Although PDPN expression in rheumatoid arthritis (RA) has been reported, the role of PDPN in RA and other arthritic conditions has not been fully elucidated. In this study, we examined PDPN expression in inflammatory synovial tissues using an anti-human PDPN (hPDPN) monoclonal antibody (mAb) panel to select the most useful one for evaluation of synovitis. Synovial tissue samples were obtained from 11 RA patients and 9 osteoarthritis (OA) patients undergoing joint surgery. PDPN-positive cells were immunostained by a panel of PDPN mAbs (NZ-1, LpMab-3, LpMab-7, LpMab-10, LpMab-12, LpMab-13, and LpMab-17), followed by cell grading of inflammation and cell counting of PDPN-positivity by a quantitative analyzer. Immunohistochemistry showed that PDPN was markedly expressed in both macrophage-like type A and fibroblast-like type B lining cells of the hyperplastic synovial lining cell layer, and macrophages and fibroblasts in the stroma of RA. Among anti-PDPN mAbs, LpMab-12 showed the highest score. In inflammatory OA synovium, PDPN expression was also detectable. Although LpMab-12 also showed the highest score in OA, the difference was not statistically significant. The inflammatory synovitis score of RA was significantly higher than that of OA. PDPN was expressed in inflammatory lining cells and sublining stroma of RA and OA synovium. In the seven anti-hPDPN antibodies examined, LpMab-12 was the most stainable antibody for PDPN in RA synovitis. Thus, LpMab-12 for PDPN has a possible and promising specific biomarker for evaluating synovitis in RA and inflammatory OA.

OP0024 Synovial Activation Drives Efficacy of Anti-Inflammatory Effects of Adipose-Derived Stem Cells in Experimental OA Which is Reflected by Serum Levels of S100a8/A9

BackgroundSynovitis is evident in a substantial subpopulation of patients with early osteoarthritis (OA). Recently we have shown that adipose derived stem cells (ASC) inhibit synovitis and joint destruction after local...

FRI0044 Cholesterol accumulation by low density lipoprotein receptor deficiency or a cholesterol-rich diet enhances osteophyte formation during experimental osteoarthritis by activating transforming growth factor-beta

BackgroundSynovial macrophages have previously shown to play a significant role in the etiopathology of experimental collagenase-induced osteoarthritis (OA)1. In addition to production of the pro-inflammatory protein S100A8/9 in early OA2,...

The transient receptor potential vanilloid 4 ion channel regulates the biosynthetic response of chondrocytes to dynamic loading

The transient receptor potential vanilloid 4 ion channel regulates the biosynthetic response of chondrocytes to dynamic loading

Liposomal Targeting of Prednisolone Phosphate to Synovial Lining Macrophages during Experimental Arthritis Inhibits M1 Activation but Does Not Favor M2 Differentiation

BackgroundTo determine the effects of liposomal targeting of prednisolone phosphate (Lip-PLP) to synovial lining macrophages on M1 and M2 polarization in vitro and during experimental arthritis.Material and MethodsExperimental arthritis (antigen and immune complex induced) was elicited in mice and prednisolone containing liposomes were given systemically. Synovium was investigated using microarray analysis, RT-PCR and histology. Bone–marrow macrophages were stimulated towards M1 using LPS and IFNγ before treatment by PLP-liposomes. M1 and M2 markers were determined using RT-PCR.ResultsMicroarray analysis of biopsies of inflamed synovium during antigen induced arthritis (AIA) showed an increased M1 signature characterized by upregulation of IL-1β, IL-6 and FcγRI starting from day 1 and lasting up until day 7 after arthritis induction. The M2 signature remained low throughout the 7 day course of arthritis. Treatment of AIA with intravenously delivered Lip-PLP strongly suppressed joint swelling and synovial infiltration whereas colloidal gold containing liposomes exclusively targeted the macrophages within the inflamed synovial intima layer. In vitro studies showed that Lip-PLP phagocytosed by M1 macrophages resulted in a suppression of the M1 phenotype and induction of M2 markers (IL-10, TGF-β, IL-1RII, CD163, CD206 and Ym1). In vivo, Lip-PLP treatment strongly suppressed M1 markers (TNF-α, IL-1β, IL-6, IL-12p40, iNOS, FcγRI, Ciita and CD86) after local M1 activation of lining macrophages with LPS and IFN-γ and during experimental AIA and immune complex arthritis (ICA). In contrast, M2 markers were not significantly upregulated in antigen-induced arthritis and down regulated in immune complex arthritis.ConclusionThis study clearly shows that systemic treatment with PLP-liposomes selectively targets synovial lining macrophages and inhibits M1 activation. In contrast to in vitro findings, PLP-liposomes do not cause a shift of synovial lining macrophages towards M2.

Antiinflammatory and chondroprotective effects of intraarticular injection of adipose‐derived stem cells in experimental osteoarthritis

In experimental collagenase-induced osteoarthritis (OA) in the mouse, synovial lining macrophages are crucial in mediating joint destruction. It was recently shown that adipose-derived stem cells (ASCs) express immunosuppressive characteristics. This study was undertaken to explore the effect of intraarticular injection of ASCs on synovial lining thickness and its relation to joint pathology in experimental mouse OA. ASCs were isolated from fat surrounding the inguinal lymph nodes and cultured for 2 weeks. Experimental OA was induced by injection of collagenase into the knee joints of C57BL/6 mice. OA phenotypes were measured within 8 weeks after induction. Histologic analysis was performed, and synovial thickening, enthesophyte formation, and cartilage destruction were measured in the knee joint. ASCs were injected into the knee joints of mice 7 days after the induction of collagenase-induced OA. On day 1, green fluorescent protein-labeled ASCs were attached to the lining layer in close contact with macrophages. Thickening of the synovial lining, formation of enthesophytes associated with medial collateral ligaments, and formation of enthesophytes associated with cruciate ligaments were significantly inhibited on day 42 after ASC treatment, by 31%, 89%, and 44%, respectively. Destruction of cartilage was inhibited on day 14 (65%) and day 42 (35%). In contrast to early treatment, injection of ASCs on day 14 after OA induction showed no significant effect on synovial activation or joint pathology on day 42. These findings indicate that a single injection of ASCs into the knee joints of mice with early-stage collagenase-induced OA inhibits synovial thickening, formation of enthesophytes associated with ligaments, and cartilage destruction.

A high cholesterol diet causes fat uptake by synovial lining macrophages and enhances joint inflammation and cartilage destruction during experimental arthritis

Backgroundand objectivesEarlier studies have shown that synovial lining macrophages are crucial in mediating joint inflammation and cartilage destruction during experimental arthritis.1 Oxidised low-density lipoprotein (ox-LDL) has been shown to activate...

Characterization of interleukin-7 and interleukin-7 receptor in the pathogenesis of rheumatoid arthritis

To characterize the expression of interleukin-7 (IL-7) and IL-7 receptor (IL-7R) in rheumatoid arthritis (RA) synovial tissue and to examine their regulation and pathogenic role in macrophages, endothelial cells, and synovial tissue fibroblasts in RA. Expression of IL-7 and IL-7R in RA and normal synovial tissue was demonstrated by immunohistochemistry. Expression and regulation of IL-7 and IL-7R in RA peripheral blood in vitro-differentiated macrophages, RA synovial tissue fibroblasts, and human microvascular endothelial cells (HMVECs) were determined by real-time reverse transcription-polymerase chain reaction and/or flow cytometry. Enzyme-linked immunosorbent assay was used to examine production of proangiogenic factors by IL-7-activated macrophages, RA fibroblasts, and endothelial cells. IL-7 and IL-7R were coexpressed on RA synovial tissue lining and sublining macrophages and endothelial cells. Expression of IL-7 and its receptor was significantly elevated in RA synovial fluid and peripheral blood macrophages as well as RA fibroblasts, compared to normal cells. Toll-like receptor 4 ligation (with lipopolysaccharide) and tumor necrosis factor α (TNFα) stimulation modulated expression of IL-7 and IL-7R on RA macrophages and HMVECs. However, in RA fibroblasts, lipopolysaccharide and TNFα activation increased expression of IL-7R only. IL-7 also mediated RA pathogenesis by inducing production of potent proangiogenic factors from macrophages and endothelial cells. We have identified, for the first time, regulators of IL-7 and IL-7R expression in RA fibroblasts, RA peripheral blood in vitro-differentiated macrophages, and endothelial cells. Our results document a novel role of IL-7 in RA angiogenesis.

Inhibition of M1 macrophage activation in favour of M2 differentiation by liposomal targeting of glucocorticoids to the synovial lining during experimental arthritis

Background and objectiveExperimental antigen-induced arthritis (AIA) is highly driven by pro-inflammatory ‘M1’ macrophages which express cytokines such as tumour necrosis factor α (TNFα), interleukin 1β (IL-1β), IL-6 and IL-12. However,...

Synovial fibroblasts self‐direct multicellular lining architecture and synthetic function in three‐dimensional organ culture

To define the intrinsic capacity of fibroblast-like synoviocytes (FLS) to establish a 3-dimensional (3-D) complex synovial lining architecture characterized by the multicellular organization of the compacted synovial lining and the elaboration of synovial fluid constituents. FLS were cultured in spherical extracellular matrix (ECM) micromasses for 3 weeks. The FLS micromass architecture was assessed histologically and compared with that of dermal fibroblast controls. Lubricin synthesis was measured via immunodetection. Basement membrane matrix and reticular fiber stains were performed to examine ECM organization. Primary human and mouse monocytes were prepared and cocultured with FLS in micromass to investigate cocompaction in the lining architecture. Cytokine stimuli were applied to determine the capacity for inflammatory architecture rearrangement. FLS, but not dermal fibroblasts, spontaneously formed a compacted lining architecture over 3 weeks in the 3-D ECM micromass organ cultures. These lining cells produced lubricin. FLS rearranged their surrounding ECM into a complex architecture resembling the synovial lining and supported the survival and cocompaction of monocyte/macrophages in the neo-lining structure. Furthermore, when stimulated by cytokines, FLS lining structures displayed features of the hyperplastic rheumatoid arthritis synovial lining. This 3-D micromass organ culture method demonstrates that many of the phenotypic characteristics of the normal and the hyperplastic synovial lining in vivo are intrinsic functions of FLS. Moreover, FLS promote survival and cocompaction of primary monocytes in a manner remarkably similar to that of synovial lining macrophages. These findings provide new insight into inherent functions of the FLS lineage and establish a powerful in vitro method for further investigation of this lineage.

Expression of mucin 3 and mucin 5AC in arthritic synovial tissue

Rheumatoid arthritis (RA) is a chronic inflammatory disease that is characterized by hypertrophy of the synovial tissue, leukocyte infiltration, angiogenesis, and ultimately joint destruction. Mucins (MUCs) are a family of heavily glycosylated proteins that protect epithelial membranes and are used as ligands for cell adhesion. MUC gene expression has been found to be altered in many cancers and inflammatory states. This study was undertaken to examine its expression in synovial tissue (ST) and role in arthritis. We performed immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction to determine expression patterns of MUC1, MUC2, MUC3, and MUC5AC in RA, osteoarthritic (OA), and normal human ST. MUC3 was expressed in synovial lining cells, macrophages, and fibroblasts. Significantly more RA (n=12) and OA (n=13) synovial lining cells expressed MUC3 than did normal synovial lining cells (n=7) (22% and 24% versus 0.4%, respectively; P<0.05). Additionally, macrophages in RA and OA ST expressed significantly more MUC3 than did macrophages in normal ST (50% and 51% versus 10%, respectively; P<0.05). MUC5AC was expressed at low levels in synovial lining cells, macrophages, and endothelial cells in RA and OA ST, and was barely expressed in normal ST. MUC1 and MUC2 proteins were not detected in ST. Messenger RNA (mRNA) for MUC3 and MUC5AC was detected in ST, and mRNA for MUC3 was detected in cultured ST fibroblasts. These data demonstrate up-regulated MUC expression by ST cells and suggest a novel role of MUC3 and MUC5AC in the pathogenesis of arthritis.

Acute CD4+ T lymphocyte–dependent interleukin‐1–driven arthritis selectively requires interleukin‐2 and interleukin‐4, joint macrophages, granulocyte–macrophage colony‐stimulating factor, interleukin‐6, and leukemia inhibitory factor

To further investigate the effects of interleukin-1 (IL-1) in immune-mediated joint inflammation, we examined the role of IL-2, Th1 interferon-gamma (IFNgamma), and Th2 (IL-4) cytokines, joint macrophages, and macrophage-derived cytokines (IL-12 p40, IL-6, leukemia inhibitory factor [LIF], oncostatin M [OSM], and granulocyte-macrophage colony-stimulating factor [GM-CSF]) in a CD4+ T lymphocyte-dependent model of acute arthritis. Methylated bovine serum albumin (mBSA)/IL-1-induced arthritis was elicited in wild-type, gene-knockout, and monoclonal antibody-treated mice. Synovial lining macrophages were selectively depleted by intraarticular injection of clodronate liposomes prior to disease induction. The severity of arthritis was assessed histologically. Mice deficient in IL-2 were almost completely protected from arthritis, and neutralization of IL-4 reduced the severity of disease. In contrast, arthritis severity and resolution appeared to be independent of IFNgamma. Synovial lining macrophage depletion markedly reduced arthritis severity. IL-6 or LIF deficiency was only modestly protective, although as previously reported, GM-CSF deficiency conferred profound disease resistance. IL-12 p40-deficient mice (which lack IL-12 and IL-23) and OSM receptor-deficient mice were susceptible to mBSA/IL-1-induced arthritis. Acute mBSA/IL-1-induced arthritis is dependent on IL-2 and IL-4, but not IFNgamma. In vivo, the Th1/Th2 paradigm may be distorted by the presence of macrophage-derived cytokines such as IL-1. Synovial lining macrophages are essential in mBSA/IL-1-induced arthritis. However, the requirement for macrophage-derived cytokines is selective; that is, IL-6, LIF, and especially GM-CSF are necessary, but IL-12, IL-23, and OSM are dispensable. IL-1 may therefore influence both adaptive and innate immune mechanisms in acute inflammatory arthritis.

MHC II+ CD45+ cells from synovium-rich tissues of normal rats: phenotype, comparison with macrophage and dendritic cell lineages and differentiation into mature dendritic cells in vitro

Synovial tissues are frequent sites of inflammatory disorders in which dendritic cells (DCs) may play an important role. This study examines potential antigen-presenting cells obtained from synovium-rich tissues (SRTs) by vascular perfusion of rat hind limbs with collagenase and further enzymatic digestion of the disarticulated hind paws in vitro. The three sub-populations of interest were: CD45+MHC IIhi, mainly CD11c+ and CD163-; CD45+MHC IIlo, mainly CD11c- and CD163+ and CD45+MHC II-, mainly CD11c- and CD163+. Expression of CD11c and CD163 correlated with ruffled cell-surface (CD11c+CD163-) and highly vacuolated cytoplasm (CD11c-CD163+), respectively. Culture of the CD45+CD163- sub-population in granulocyte macrophage colony-stimulating factor (GM-CSF) yielded CD45+MHC IIhi CD11c+CD163- cells with veiled morphology, while the large vacuolated cells that expressed CD163 resembled type A synoviocytes in both surface antigen phenotype and morphology. These results demonstrate that SRTs contain indeterminate cells that can differentiate into mature DCs in vitro in response to GM-CSF, plus mature synovial lining macrophages.

Synovial lining macrophages mediate osteophyte formation during experimental osteoarthritis

Objective In human osteoarthritis (OA), various forms of pathology are observed. Besides cartilage damage and fibrosis, neogenesis of bone, osteophyte formation, also occurs. Osteophytes are thought to limit joint movement and cause pain. The objective of this study was to investigate whether synovial macrophages are involved in osteophyte formation in experimental OA, and if they are, to study which mechanism may be involved. Design Experimental OA was induced by two intra-articular injections of collagenase on alternate days into murine knee joints. The role of synovial lining macrophages in this model was studied by selective removal of these cells prior to OA induction using clodronate liposomes. After 1 and 2 weeks, knee joints were dissected and examined (immuno)histologically. Results At days 7 and 14 after induction of OA, osteophyte formation and fibrosis were observed. Depletion of synovial macrophages resulted in spectacular reduction of osteophyte formation, 84% and 66%, respectively, at days 7 and 14. Fibrosis and synovial activation, measured by MRP8/14 expression, were also ameliorated (40–60%). In addition, production of growth factors (TGFβ, BMP-2 and BMP-4) in the lining was largely prevented but production of these growth factors in deeper layers of the synovium and the periosteum did not differ from controls. Conclusions These results indicate the synovial macrophage to be a pivotal cell in the synovium mediating osteophyte formation and other OA-related pathology, like fibrosis, during experimental OA. Production of growth factors and induction of synovial activation by these cells may play a crucial role in this event.

Crucial role of synovial lining macrophages in the promotion of transforming growth factor beta-mediated osteophyte formation.

To investigate in vivo and in vitro whether macrophages have an intermediate role in transforming growth factor beta (TGFbeta)-induced osteophyte formation. In vivo, synovial lining macrophages were selectively depleted by injection of clodronate-laden liposomes 7 days prior to injection of 20 ng or 200 ng of TGFbeta into murine knee joints 3 times, on alternate days. Total knee joint sections were obtained on day 7 after the last injection and stained with Safranin O. Production of bone morphogenetic protein 2 (BMP-2) and BMP-4 was determined by immunolocalization. The interaction between murine macrophages and mesenchymal cells (precursors with chondrogenic potential) was studied in vitro using a Transwell system in which RAW macrophages were cocultured with C3H10T1/2 mesenchymal cells. Spheroid neocartilage formation was quantified microscopically after staining with May-Grünwald-Giemsa. Triple injections of 20 ng or 200 ng of TGFbeta into normal murine knee joints induced significant osteophyte formation at the lateral and medial sites of the patella and femur on day 7 after the last injection. Strikingly, removal of synovial lining macrophages prior to TGFbeta injection resulted in a drastic reduction of osteophyte formation (by 70% and 64% after injection of 20 ng and 200 ng of TGFbeta, respectively). Synovial lining cells produced BMP-2 and BMP-4 after TGFbeta stimulation, whereas BMP-2 and BMP-4 were absent in the synovial tissue after macrophage depletion. In vitro, clustering and spheroid formation of C3H10T1/2 was induced by TGFbeta concentrations of >1 ng/ml. However, in the Transwell system, in the presence of murine macrophages, 0.5 ng/ml of TGFbeta was very effective in generating large spheroids, suggestive of macrophage-derived (co)factors. In coculture supernatants, TGFbeta concentrations were not elevated in the presence of macrophages, indicating generation of other growth factors involved in spheroid formation. These findings indicate that macrophages are crucial intermediate factors in osteophyte formation induced by TGFbeta, probably by inducing other chondrogenic signals.

Chemokine receptor expression and in vivo signaling pathways in the joints of rats with adjuvant-induced arthritis.

This study was undertaken to characterize the role of CC chemokines and their receptors in rat adjuvant-induced arthritis (AIA), a model for rheumatoid arthritis (RA). Furthermore, we investigated the signaling pathways associated with CC receptors as well as the cell type distribution of the receptors. Using TaqMan real-time reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemistry, we defined chemokine and chemokine receptor messenger RNA (mRNA) expression, CC chemokine receptor (CCR) protein activation during the disease course, CCR-associated signaling pathways, and immunopositive CCR5, phosphorylated signal transducer and activator of transcription 1 (p-STAT-1), and p-STAT-3 cells in rat AIA versus control joints. We showed significant up-regulation of CCR1, CCR2, CCR5, and macrophage inflammatory protein 1beta/CCL4 mRNA in AIA on post-adjuvant injection day 18, coincident with peak inflammation. Additionally, increases in tyrosine phosphorylation of CCR1 (days 14, 18, 21, and 24), CCR2 (days 14 and 18), and CCR5 (days 14, 18, and 21) were detected in AIA rats compared with control (nonarthritic) rats. JAK-1, STAT-1, and STAT-3 were associated with CCR1 and were highly tyrosine phosphorylated on days 14 and 18. Moreover, CCR2 was associated with JAK-2, STAT-1, and STAT-3 on day 18. The association of STAT-1 and STAT-3 with CCR5 on days 18 and 21 correlated with JAK-1 phosphorylation and binding on day 18. However, the activation of JNK was not associated with CCR5 activation in rat AIA. Immunohistochemical analysis demonstrated that the expression of CCR5, p-STAT-1, and p-STAT-3 was detected on synovial lining cells, macrophages, and endothelial cells in arthritic rat ankles on post-adjuvant injection day 18. While the majority of the CCR5 and p-STAT-1 immunostaining was on synovial lining cells and macrophages, p-STAT-3 was predominantly expressed on endothelial cells. CCR1, CCR2, and CCR5 mRNA expression and tyrosine phosphorylation increased with peak inflammation in the AIA model. CCR1, CCR2, and CCR5 tyrosine phosphorylation are associated with the JAK/STAT-1/STAT-3 pathway at different stages of rat AIA, as well as with macrophage and endothelial cell infiltration. However, their signaling activation overlaps with peak inflammation. Up-regulation and activation of CCRs may play a role in macrophage and endothelial cell infiltration in rat AIA joints in addition to activating the associated signaling pathways. The downstream intermediate signaling proteins associated with CC receptors may be used as potential tools to control inflammation in RA.

The expression and localization of β-endorphin and µ,δ-opioid receptors in synovial tissues in patient with rheumatoid arthritis and osteoarthritis

An examination of the localization and distribution of β-endorphin and opioid receptors in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Immunohistochemical staining, reverse transcription polymerase chain reaction (RT–PCR) analysis, and in situ hybridization were performed using synovial tissues obtained from RA and OA patients. Immunohistochemical staining showed that β-endorphin was strongly expressed in synovial lining cells and in a few lymphocytes and macrophages surrounding the vessels, whereas µ- and δ-opioid receptors were expressed in lymphocytes and macrophages. However, we detected the weak expression of these opioid peptides in synovial tissues of OA patients. RT–PCR analysis showed that preproopiomelanocortin (POMC) mRNA, a precursor of β-endorphin, was strongly expressed in synovial tissues of RA patients, but these PCR products of synovial tissues obtained from OA patients were weakly detected compared with those from RA patients. POMC mRNA was also expressed in synovial tissues in RA patients. In in situ hybridization, the expression of POMC mRNA was detected in macrophages, synovial lining cells, and fibroblasts in synovial tissues of RA patients as well as β-endorphin. In RA patients, β-endorphin and µ- and δ-opioid receptors are synthesized and located in synovial lining cells, lymphocytes, and macrophages surrounding the vessels in synovial tissues, and may play a role in the regulation and modulation of inflammation.