Rheumatoid Synovium Research Articles

Osteoclasts in RA: Diverse origins and functions

Osteoclasts were recognized in the late 1990s as the cells responsible for generalized and focal bone loss in rheumatoid arthritis (RA). Concepts about osteoclast biology have changed radically based on recent evidence of considerable diversity in both the origins and the functions of osteoclasts. In addition, the role for osteoclasts is not confined to bone resorption but may also include active contributions to inflammatory and autoimmune responses. Thus, in RA, osteoclast progenitors may arise from both circulating cells and cells developed within the rheumatoid synovium or subchondral bone. Within the inflamed synovium, osteoclasts are activated by factors such as cytokines, immune complexes, or activators of the toll-like receptors, which are not found in healthy bone tissue. Finally, recent data suggest that osteoclasts may be capable of antigen presentation to T cells via major histocompatibility complex class I and class II molecules. Confirmation of this suggestion by future studies would indicate that osteoclasts might be involved not only in bone resorption, but also in autoimmune responses and antigen presentation. These data highlight the considerable complexity of interactions between bone tissue and the immune system. Research into these interactions may identify new targets for treatments against the bone abnormalities associated with chronic inflammatory disease.

Tumor Necrosis Factor Receptor Type I Expression of CD4+ T Cells in Rheumatoid Arthritis Enables Them to Follow Tumor Necrosis Factor Gradients Into the Rheumatoid Synovium

The cytokine tumor necrosis factor (TNF) plays a central role in the pathogenesis of rheumatoid arthritis (RA), but its disease-specific effector mechanisms have not been fully elucidated. This study was undertaken to investigate the role of TNF in T cell accumulation and migration in the synovitic joints of RA patients. Vital tissue sections from rheumatoid synovium were generated using a horizontally oscillating microtome and were coincubated with fluorescence-labeled CD4+ T cells. Migration was detected by fluorescence and confocal microscopy. Migrating T cells were recovered from the tissue and analyzed for phenotype. Chemotaxis of CD4+ T cells from RA patients in response to increasing concentrations of TNF was analyzed in Transwell experiments. CD4+ T cells from RA patients migrated into the tissue sections in significantly higher numbers than T cells from healthy controls. Migrating CD4+ T cells differed from nonmigrating ones in their increased expression of TNF receptor type I (TNFRI), which was expressed on a fraction of circulating CD4+ T cells from RA patients, but not from controls. CD4+ T cells from the peripheral blood of RA patients were also found to migrate along TNF concentration gradients ex vivo. Accordingly, blockade of either TNF or TNFRI nearly abrogated in vitro T cell migration in synovial tissue. Our findings indicate that the interaction of TNF with TNFRI is pivotal for T cell migration in synovial tissue in vitro, and thereby suggest a relevant role of the cytokine for in vivo T cell trafficking to synovitic joints.

Suppression of murine collagen-induced arthritis by vaccination of synovial vascular endothelial cells

AimsEndothelial cells (ECs) lining the lumina of blood vessels are involved in leukocyte extravasation underlying inflammatory states, such as rheumatoid arthritis (RA). The rheumatoid pannus, the site of inflammation and joint destruction in the rheumatoid synovium, relies on the development of neovascular vessels to sustain its growth. We studied a method to selectively target and destroy new synovial blood vessels by vaccination with synovial EC. Main methodsCollagen-induced arthritis (CIA) mice were vaccinated with tumor necrosis factor (TNF)-alpha-stimulated EC (TNF-EC) antigen with an incomplete adjuvant. TNF-EC was used as a model of EC in synovial tissue on RA. Key findingsArthritis was significantly decreased in TNF-EC vaccinated mice compared with non-vaccinated mice based on the arthritis score. Moreover, the TNF-EC vaccine suppressed bone erosion, hyperplasia of the synovium and expression of neovascular vessel as shown by hematoxylin–eosin staining, X-ray analysis and immunohistochemical. SignificanceVaccine therapy against vascular EC in synovial tissue may provide a novel approach to the treatment of RA.

Activation of Melanocortin Receptor 3 as a new strategy to control experimental and rheumatoid arthritis

Study ObjectiveThe anti‐inflammatory properties of melanocortin peptides have long been established, though their main molecular target(s) is still elusive. Two receptors (MC1R and MC3R) have been suggested as their main target, but the patho‐physiological impact of this important homeostatic anti‐inflammatory system is unclear: we focused here on experimental and rheumatoid (RA) arthritis.MethodsMouse Studies (1) Arthritis was induced by K/BxN serum transfer (200 μl) into wild type (WT), recessive yellow e/e (Mc1r inactive mutant) and Mc3r−/− mice and disease profiles monitored. (2) Gene expression analysis was performed in arthritic paw joints using qPCR. (3) Daily treatment with vehicle or D[Trp8]‐γ‐MSH (MC3R agonist) was given to WT and Mc3r−/− mice (20 μg) induced with K/BxN arthritis and disease profiles assessed. Human Studies qPCR was used to analyse MCR and/or pro‐inflammatory cytokine mRNA levels in RA synovium transplanted into SCID mice treated with D[Trp8]‐γ‐MSH.ResultsUpon K/BxN serum transfer, Mc3r−/− mice displayed a marked disease exacerbation whereas no different profile of disease development was displayed by the Mc1r mutant and WT mice. This heightened presence of disease in Mc3r−/− null mice was confirmed by histological analysis of paw joints. At peak of arthritic profile (D6), mRNA analysis revealed up‐regulation of Rankl, Mcp‐1, Nos2 and Il‐1β, Il‐6 in Mc3r−/− mice compared to WT. Treatment of arthritic mice with D[Trp8]‐γ‐MSH significantly attenuated disease. In another preclinical model, stimulation of human MC3R with D[Trp8]‐γ‐MSH inhibited human IL‐1β, IL‐6, TNF‐α and RANKL gene expression in RA synovium transplanted into SCID mice.ConclusionsThese findings identify MC3R‐mediated signaling as a novel anti‐inflammatory pathway for innovative therapeutic targeting in RA.

KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization

Hyaluronan (HA) has an extraordinarily high turnover in physiological tissues, and HA degradation is accelerated in inflammatory and neoplastic diseases. CD44 (a cell surface receptor) and two hyaluronidases (HYAL1 and HYAL2) are thought to be responsible for HA binding and degradation; however, the role of these molecules in HA catabolism remains controversial. Here we show that KIAA1199, a deafness gene of unknown function, plays a central role in HA binding and depolymerization that is independent of CD44 and HYAL enzymes. The specific binding of KIAA1199 to HA was demonstrated in glycosaminoglycan-binding assays. We found that knockdown of KIAA1199 abolished HA degradation by human skin fibroblasts and that transfection of KIAA1199 cDNA into cells conferred the ability to catabolize HA in an endo-β-N-acetylglucosaminidase-dependent manner via the clathrin-coated pit pathway. Enhanced degradation of HA in synovial fibroblasts from patients with osteoarthritis or rheumatoid arthritis was correlated with increased levels of KIAA1199 expression and was abrogated by knockdown of KIAA1199. The level of KIAA1199 expression in uninflamed synovium was less than in osteoarthritic or rheumatoid synovium. These data suggest that KIAA1199 is a unique hyaladherin with a key role in HA catabolism in the dermis of the skin and arthritic synovium.

Tissue factor expression in rheumatoid synovium: a potential role in pannus invasion of rheumatoid arthritis

Angiogenesis, as well as pannus formation within the joint, plays an important role in the erosion of articular cartilage and bone in the pathological process of rheumatoid arthritis (RA). Tissue factor (TF), an essential initiator of the extrinsic pathway of blood coagulation, is also involved in the angiogenesis and the pannus formation of RA progression. In the present study, we used immunofluorescence and confocal scanning methods to characterize TF immunolocalization in RA synovium. We showed that positive staining of TF could be immunolocalized in synoviocytes, CD19+ B cells and CD68+ macrophages, whereas weak or negative staining of tissue factor could be found in CD34+ endothelial cells of neo-vessels, CD3+ T cells and CD14+ monocytes in RA synovium tissues. Our study demonstrates a detailed local expression of TF in the rheumatoid synovium, and supports the notion that TF, expressed not only by the synoviocytes themselves, but also the infiltrating CD19+ B cells and CD68+ macrophages, is involved in the pannus invasion in the progression of rheumatoid arthritis.

Serum amyloid A (SAA) induces pentraxin 3 (PTX3) production in rheumatoid synoviocytes

Objective: Pentraxin 3 (PTX3) is an acute-phase reactant that is involved in amplification of the inflammatory response and innate immunity. In the present study, we evaluated the relationship between PTX3 and serum amyloid A (SAA), another acute-phase reactant, in rheumatoid synoviocytes.Methods: PTX3 mRNA expression was examined by reverse transcription polymerase chain reaction, and PTX3 protein was measured by enzyme-linked immunosorbent assay.Results: SAA induced PTX3 mRNA and PTX3 protein expression in rheumatoid synoviocytes. SAA-induced PTX3 expression was attenuated when rheumatoid synoviocytes were nucleofected with N-formyl peptide receptor ligand-1 (FPRL-1)-specific siRNA, suggesting the involvement of FPRL-1. Furthermore, SAA-induced PTX3 expression was inhibited by NF-κB or mitogen-activated protein kinase-specific inhibitors. Neither soluble TNF receptor (etanercept) nor recombinant IL-1 receptor antagonist affected PTX3 production by SAA-stimulated synoviocytes, suggesting that SAA directly induces PTX3.Conclusion: Our data suggest that SAA plays a role in the proinflammatory and immune responses in rheumatoid synovium by inducing PTX3. We provide the first evidence that the acute-phase reactant SAA, which is produced systemically by hepatocytes, perpetuates the rheumatoid inflammatory processes by inducing another proinflammatory molecule, PTX3, locally in rheumatoid synovial tissues.

Irreversible Heavy Chain Transfer to Hyaluronan Oligosaccharides by Tumor Necrosis Factor-stimulated Gene-6

The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).

The rheumatoid arthritis synovial fluid citrullinome reveals novel citrullinated epitopes in apolipoprotein E, myeloid nuclear differentiation antigen, and β‐actin

To generate a catalog of citrullinated proteins that are present in the synovia of patients with rheumatoid arthritis (RA) and to elucidate their relevance for the anti-citrullinated protein antibody response in RA. Polypeptides isolated from the synovial fluid of patients with RA were identified by mass spectrometry. Three proteins (apolipoprotein E [Apo E], myeloid nuclear differentiation antigen [MNDA], and β-actin) were studied in more detail, using immunoprecipitation and Western blotting. The presence of autoantibodies to synthetic peptides derived from these proteins in sera from patients with RA, sera from patients with other diseases, and sera from healthy control subjects was studied by enzyme-linked immunosorbent assay (ELISA). RA synovial fluid samples displayed several distinct patterns of citrullinated proteins. Using mass spectrometry, (fragments of) 192 proteins were identified, including 53 citrullinated proteins, some of which contained multiple citrullinated residues. In addition to previously reported citrullinated proteins in RA synovia (e.g., vimentin and fibrinogen), a series of novel citrullinated proteins, including Apo E, MNDA, β-actin, and cyclophilin A, was identified. Immunoprecipitation experiments confirmed the citrullination of Apo E and MNDA. ELISAs demonstrated the presence of autoreactive citrullinated epitopes in Apo E, MNDA, and β-actin. Synovial fluid samples from the inflamed joints of patients with RA contain many citrullinated proteins. Citrullinated Apo E, MNDA, and β-actin are novel antigens identified in RA synovial fluid, and only a limited number of their citrullinated epitopes are targeted by the immune system in RA.

The novel cytokine interleukin-36α is expressed in psoriatic and rheumatoid arthritis synovium

BackgroundInterleukin (IL)-36α is a recently described member of the IL-1 cytokine family with pro-inflammatory and clearly pathogenic properties in psoriasis.ObjectiveTo determine the IL-36α expression in psoriatic arthritis (PsA) compared to...

The interferon signature and STAT1 expression in rheumatoid arthritis synovial fluid macrophages are induced by tumor necrosis factor α and counter‐regulated by the synovial fluid microenvironment

Type I interferons (IFNs) have emerged as potential activators of the IFN signature and elevated STAT-1 expression in rheumatoid arthritis (RA) synovium, but mechanisms that induce synovial IFN expression are unknown. Recently, tumor necrosis factor α (TNFα) was shown to induce a delayed IFN response in macrophages. We undertook this study to test whether TNFα, classically thought to activate inflammatory NF-κB target genes in RA, also contributes to the "IFN signature" in RA synovial macrophages. Synovial fluid (SF) macrophages purified from 24 patients with RA and 18 patients with spondylarthritides (SpA) were lysed immediately after isolation or were cultured ex vivo in the absence or presence of blockade of endogenous type I IFN or TNFα. Expression of IFN-inducible target genes was measured by quantitative reverse transcription-polymerase chain reaction, and expression of their corresponding proteins was measured by enzyme-linked immunosorbent assay. Expression of an IFN signature and STAT1 in RA synovial macrophages was suppressed when type I IFNs or TNFα were blocked, whereas TNFα blockade did not affect expression of IFN response genes or STAT1 in SpA synovial macrophages. RA SF suppressed the IFN signature in RA synovial macrophages and in TNFα-, IFNα-, and IFNβ-stimulated control macrophages. Type I IFNs suppressed expression of IL8 and MMP9 in RA synovial macrophages and in TNFα-stimulated control macrophages. Our findings identify a new function of TNFα in RA synovitis by implicating TNFα as a major inducer of the RA synovial IFN response. The results suggest that the expression of IFN response genes in RA synovium is regulated by interplay between TNFα and opposing homeostatic factors expressed in the synovial microenvironment.

P097 Contact with stimulated T cells up-regulates expression of peptidylarginine deiminase 2 and 4 by human monocytes

Introduction The antigenic targets of the rheumatoid arthritis (RA)-associated autoantibodies to “citrullinated” proteins are generated following citrullination by a peptidylarginine deiminase (PAD). Of the five PAD isotypes, two—PAD2 and PAD4—are expressed in RA synovial tissue. Within this tissue, the activation of macrophages and fibroblasts mediated by T-cell contact or driven by cytokines plays a prominent part in the pathogenesis. We set to determine if cytokine stimulation and contact with T cells could play a role in PAD expression by peripheral blood monocytes and fibroblastic synoviocytes. Methods Human monocytes and T lymphocytes were derived from the blood of healthy donors. HUT-78 cells and T lymphocytes werestimulated with PHA and PMA. Human synovial fibroblasts were isolated after surgical synoviectomy. The expression of PAD was determined by real-time PCR and immunoblot. Results In monocytes, the PADI2 and PADI4 mRNAs were transiently up-regulated by contact with stimulated HUT-78 and/or T lymphocytes. Positive modulation of the PAD2 and PAD4 proteins were also observed upon contact with stimulated HUT-78 T cells. Stimulation by IL-1b or IFN-b did not modify the PADI2 and PADI4 mRNAs but enhanced PAD4 protein expression. No isotype of PAD was detected at mRNA or protein level in resting or stimulated synovial fibroblasts. Conclusion Contact of stimulated T cells with monocyte-macrophages or cytokine-activated monocyte-macrophages constitutes a highly likely source of PAD2 and PAD4 which are observed in inflamed synovial tissues. In contrast, it is most unlikely that fibroblastic synoviocytes contribute to PAD expression in rheumatoid synovial membranes.

Paper 133: Expression of Adam15 in Rheumatoid Synovium and Its Regulation by Vascular Endothelial Growth Factor in Rheumatoid Synovial Fibriblasts

Paper 133: Expression of Adam15 in Rheumatoid Synovium and Its Regulation by Vascular Endothelial Growth Factor in Rheumatoid Synovial Fibriblasts

Targeted delivery of cytokine therapy to rheumatoid tissue by a synovial targeting peptide

ObjectivesThe synovial endothelium targeting peptide (SyETP) CKSTHDRLC has been identified previously and was shown to preferentially localise to synovial xenografts in the human/severe combined immunodeficient (SCID) mouse chimera model of...

Ligation of TLR7 by rheumatoid arthritis synovial fluid single strand RNA induces transcription of TNFα in monocytes

ObjectiveThe aim of the study was to characterise the expression, regulation and pathogenic role of toll-like receptor 7 (TLR7) and TLR8 in rheumatoid arthritis (RA).MethodsExpression of TLR7 and TLR8 was...

Contact with stimulated T cells up-regulates expression of peptidylarginine deiminase 2 and 4 by human monocytes

The antigenic targets of the rheumatoid arthritis (RA)-associated autoantibodies to "citrullinated" proteins are generated following citrullination by a peptidylarginine deiminase (PAD). Of the five PAD isotypes, two - PAD2 and PAD4 - are expressed in RA synovial tissue. Within this tissue, the activation of macrophages and fibroblasts mediated by T-cell contact or driven by cytokines plays a prominent part in the pathogenesis. We wanted to determine whether cytokine stimulation and contact with T cells could play a role in PAD expression by peripheral blood monocytes and fibroblastic synoviocytes. Human monocytes and T lymphocytes were derived from the blood of healthy donors. HUT-78 cells and T lymphocytes were stimulated with PHA and PMA. Human synovial fibroblasts were isolated after surgical synoviectomy. The expression of PAD was determined by real-time PCR and immunoblot. In monocytes, the PADI2 and PADI4 mRNAs were transiently up-regulated by contact with stimulated HUT-78 and/or T lymphocytes. Positive modulation of the PAD2 and PAD4 proteins were also observed upon contact with stimulated HUT-78 T cells. Stimulation by IL-1β or IFN-β did not modify the PADI2 and PADI4 mRNAs, but enhanced PAD4 protein expression. No isotype of PAD was detected at the mRNA or protein level in resting or stimulated synovial fibroblasts. Contact between stimulated T cells and monocyte-macrophages or cytokine-activated monocyte-macrophages constitutes a highly likely source of PAD2 and PAD4, which are observed in inflamed synovial tissues. In contrast, it is most unlikely that fibroblastic synoviocytes contribute to PAD expression in rheumatoid synovial membranes.

T cell lessons from the rheumatoid arthritis synovium SCID mouse model: CD3‐rich synovium lacks response to CTLA‐4ig but is successfully treated by interleukin‐17 neutralization

To provide an intermediate step between classic arthritis models and clinical trials, the rheumatoid arthritis (RA) synovium SCID mouse model is a valuable tool for use during preclinical research. We undertook this study to investigate the validity of this humanized mouse model using anti-tumor necrosis factor (anti-TNF) and anti-interleukin-1 (anti-IL-1) treatment and to investigate the direct effect of T cells- and B cell-related therapies on the transplanted RA synovial tissue. CB17/SCID mice were engrafted with human RA synovial tissue and systemically treated with anti-TNF, anti-IL-1, anti-IL-17, CTLA-4Ig, anti-CD20, or isotype control antibodies. Validation of the model with anti-TNF treatment significantly reduced serum cytokine levels and decreased histologic inflammation, whereas anti-IL-1 therapy did not show any effect on the RA synovial grafts. In mice engrafted with B cell-rich synovial tissue, anti-CD20 treatment showed clear therapeutic effects. Surprisingly, CTLA-4Ig treatment did not show any effects in this transplantation model, despite prescreening of the synovial tissue for the presence of CD3+ T cells and the costimulatory molecules CD80 and CD86. In contrast, great therapeutic potential was observed for anti-IL-17 treatment, but only when CD3+ T cells were abundantly present in the RA synovial tissue. This human RA synovium SCID mouse model enabled us to show that CTLA-4Ig lacks direct effects on T cell activation processes in the synovial tissue. Further evidence was obtained that IL-17 might indeed be an interesting therapeutic target in RA patients with CD3-rich synovial tissue. Further characterization of the RA patients' individual synovial profiles is of great importance for achieving tailored therapy.

Epirubicin potentiates recombinant adeno‐associated virus type 2/5–mediated TRAIL expression in fibroblast‐like synoviocytes and augments the antiarthritic effects of rAAV2/5‐TRAIL

Synovial cells in rheumatoid synovium display abnormal proliferation, which leads to joint destruction. TRAIL has been described as a proapoptotic factor in fibroblast-like synoviocytes (FLS). This study was undertaken to investigate the functions of rAAV2/5-TRAIL in human FLS and in arthritic mice. Primary human FLS were infected with rAAV2/5-TRAIL in the presence or absence of epirubicin. Transgene expression was monitored by both enzyme-linked immunosorbent assay and flow cytometry. The disease-modulating activity of epirubicin plus rAAV2/5-TRAIL was investigated in mice with collagen-induced arthritis (CIA). Subtoxic doses of epirubicin potentiated rAAV2/5-mediated TRAIL expression in FLS and simultaneously enhanced the sensitivity of FLS to TRAIL. Epirubicin treatment up-regulated death receptor 4 (DR-4) and DR-5 expression and down-regulated FLIP expression, thereby enhancing the activation of procaspase 3, procaspase 8, and procaspase 9. An in vivo study showed that the combination of rAAV2/5-TRAIL gene therapy and epirubicin chemotherapy provided augmented antiarthritic effects in a mouse model of CIA. The intraarticular injection of rAAV2/5-TRAIL combined with epirubicin treatment significantly reduced the severity and incidence of CIA and joint swelling in the animals. Histologic evaluations revealed that inflammatory cell infiltration, cartilage destruction, and bone erosion were significantly reduced in the joints of the mice receiving the synthetic treatment. Results of a viral genome copy number assay indicated that epirubicin dramatically augmented the expression of rAAV2/5-TRAIL without altering its tissue distribution. These results suggest that epirubicin enhances the antiarthritic effect of rAAV2/5-TRAIL and that combination treatment might be an important therapeutic alternative, with practical significance for rheumatoid arthritis.

Serum amyloid A (SAA) induces pentraxin 3 (PTX3) production in rheumatoid synoviocytes

Pentraxin 3 (PTX3) is an acute-phase reactant that is involved in amplification of the inflammatory response and innate immunity. In the present study, we evaluated the relationship between PTX3 and serum amyloid A (SAA), another acute-phase reactant, in rheumatoid synoviocytes. PTX3 mRNA expression was examined by reverse transcription polymerase chain reaction, and PTX3 protein was measured by enzyme-linked immunosorbent assay. SAA induced PTX3 mRNA and PTX3 protein expression in rheumatoid synoviocytes. SAA-induced PTX3 expression was attenuated when rheumatoid synoviocytes were nucleofected with N-formyl peptide receptor ligand-1 (FPRL-1)-specific siRNA, suggesting the involvement of FPRL-1. Furthermore, SAA-induced PTX3 expression was inhibited by NF-κB or mitogen-activated protein kinase-specific inhibitors. Neither soluble TNF receptor (etanercept) nor recombinant IL-1 receptor antagonist affected PTX3 production by SAA-stimulated synoviocytes, suggesting that SAA directly induces PTX3. Our data suggest that SAA plays a role in the proinflammatory and immune responses in rheumatoid synovium by inducing PTX3. We provide the first evidence that the acute-phase reactant SAA, which is produced systemically by hepatocytes, perpetuates the rheumatoid inflammatory processes by inducing another proinflammatory molecule, PTX3, locally in rheumatoid synovial tissues.

PI3 Kinase δ Is a Key Regulator of Synoviocyte Function in Rheumatoid Arthritis

Class I phosphoinositide 3 kinase (PI3K) δ is a promising therapeutic target for rheumatoid arthritis (RA) because of its contribution to leukocyte biology. However, its contribution in fibroblasts has not been studied as a mechanism that contributes to efficacy. We investigated the expression and function of PI3Kδ in synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that PI3Kδ is highly expressed in RA synovium, especially in the synovial lining. Using quantitative PCR and Western blot analysis, we found that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis (OA) synovium. PI3Kδ was also expressed in cultured FLS, along with PI3Kα and PI3Kβ, whereas PI3Kγ was not detectable. PI3Kδ mRNA expression was selectively induced by inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) but not by growth factors platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ). The use of inhibitors that block individual PI3K isoforms, including the novel selective PI3Kδ inhibitor INK007, showed that PI3Kδ is required for PDGF- and TNF-induced Akt activation. PI3Kδ inhibition also diminished PDGF-mediated synoviocyte growth and sensitized cells to H(2)O(2)-induced apoptosis. These data are the first documentation of increased PI3Kδ expression in both RA synovium and cultured synoviocytes. Furthermore, these are the first data demonstrating that PI3Kδ is a major regulator of PDGF-mediated fibroblast growth and survival via Akt. Thus, targeting PI3Kδ in RA could modulate synoviocyte function via anti-inflammatory and disease-altering mechanisms.