Sharon Veenbergen

Tetraspanins in the immune response against cancer

The role of the immune system in the defense against cancer, a process termed tumor immunosurveillance, has been extensively studied. Evidence is accumulating that the molecular organization of proteins and lipids in the plasma membrane of immune cells is of critical importance. Tetraspanin proteins are expressed in the plasma membrane of all mammalian cells and play an important role in the spatial organization of partner molecules into tetraspanin-enriched microdomains. It is now well established that tetraspanins interact with one another as well as with a diverse array of key leukocyte proteins, including immune receptors, integrins, and signaling molecules. These tetraspanin-partner protein interactions control several fundamental cellular processes, which in immune cells involve antigen presentation, motility, proliferation and antibody production. We propose that differences in the tetraspanin microdomain composition account for the abilities of individual tetraspanins to either promote or suppress immune responses. In this review, we discuss novel insights into tetraspanin function in immune cells, and describe how this may control anti-tumor immunity.

Splenic suppressor of cytokine signaling 3 transgene expression affects T cell responses and prevents development of collagen‐induced arthritis

OBJECTIVE Members of the suppressor of cytokine signaling (SOCS) family are key negative intracellular regulators of cytokine and growth factor responses, including those that regulate immune responses in autoimmune disorders, such as rheumatoid arthritis (RA). The aim of this study was to investigate modulation of T cell immunity for the treatment of experimental arthritis, via enhanced expression of SOCS-3 in splenic antigen-presenting cells (APCs) obtained after intravenous injection of adenovirus encoding SOCS-3. METHODS DBA/1 mice were immunized with type II collagen, and adenovirus vectors were administered by intravenous injection before the clinical onset of collagen-induced arthritis (CIA). Splenic cellular responses were analyzed by measuring cytokine production, using Luminex multi-analyte technology. Th cell populations were analyzed by flow cytometry. RESULTS Systemic delivery of adenovirus encoding SOCS-3 resulted in enhanced transgene expression in splenic APCs, which led to decreased production of interleukin-23 (IL-23), IL-6, and tumor necrosis factor alpha, but significantly higher production of antiinflammatory IL-10, by these cells. Fluorescence-activated cell sorting analysis showed increased numbers of splenic CD4+ T cells after SOCS-3 treatment. In the presence of SOCS-3-transduced APCs, however, purified splenic CD3+ T cells showed reduced antigen-specific proliferation and a significant reduction in the production of interferon-gamma (-43%), IL-4 (-41%), and IL-17 (-70%). Interestingly, the altered splenic cellular responses were accompanied by a protective effect on CIA development, and histologic analysis of knee joints showed reduced joint inflammation and connective tissue destruction. CONCLUSION This study demonstrates effective prevention of CIA after intravenously induced overexpression of SOCS-3; this is probably caused by the generation of tolerogenic APCs, which have an inhibitory effect on Th1, Th2, and especially, Th17 cell activity.

The natural soluble form of IL-18 receptor beta exacerbates collagen-induced arthritis via modulation of T-cell immune responses.

Objective: IL-18 is a pluripotent cytokine that has been implicated in the development of rheumatoid arthritis. A soluble form of the IL-18 receptor accessory protein (sIL-18Rβ) with unknown function has recently been identified. This study examined the ability of sIL-18Rβ to inhibit IL-18 biological activities and to modulate immune responses during collagen-induced arthritis (CIA). Methods: Adenoviruses encoding sIL-18Rβ were administered intravenously in type II collagen-immunised DBA/1 mice. Humoral responses were analysed by determining anti-bovine collagen type II (BCII) antibody levels by ELISA. Cytokine production by splenic T cells and cytokine levels in serum were measured by Luminex multi-analyte technology. CD4+CD25+Foxp3+ regulatory T cells (Treg) were measured by flow cytometry. Results: Intravenous delivery of Ad5.sIL-18Rβ in collagen-immunised mice led to enhanced transgene expression in splenic antigen-presenting cells (APC). A co-culture of these sIL-18Rβ-transduced APC with purified splenic CD3+ T cells led to a marked inhibition of IL-18-induced IFNγ, IL-4 and IL-17 production by CD3+ T cells. Remarkably, systemic treatment with Ad5.sIL-18Rβ caused an exacerbation of arthritis, and histological evaluation of knee joints showed increased cartilage and bone erosion. No significant differences were observed in anti-BCII antibodies, but the aggravation was accompanied by decreased IFNγ (−30%) and IL-4 (−44%) and increased IL-17 (+84%) production by splenic CD3+ T cells. In addition, reduced circulating levels of CD4+CD25+Foxp3+ Treg and anti-inflammatory IL-10 were shown. Conclusion: This study identifies sIL-18Rβ as a novel IL-18 inhibitor, which promotes CIA after intravenous overexpression by affecting Treg levels and supporting a T helper type 17 response.

Enhanced suppressor of cytokine signaling 3 in arthritic cartilage dysregulates human chondrocyte function

OBJECTIVE To determine the expression of suppressor of cytokine signaling 3 (SOCS-3) in human articular chondrocytes and its functional consequences. METHODS Chondrocytes were isolated from the cartilage of patients with osteoarthritis (OA), patients with rheumatoid arthritis (RA), and trauma patients and from the healthy cartilage of patients with a femoral neck fracture. The human chondrocyte cell line G6 and primary bovine chondrocytes were used in validation experiments. SOCS-3 messenger RNA (mRNA) expression was measured by quantitative polymerase chain reaction, and SOCS-3 protein levels were determined by Western blotting and immunohistochemical analysis. To ascertain the role of SOCS-3 in the chondrocyte response to interleukin-1β (IL-1β) or lipopolysaccharide (LPS), the expression of SOCS3 was either reduced by small interfering RNA or enhanced by viral transduction. RESULTS The expression of SOCS-3 mRNA (but not that of SOCS-1 mRNA) was significantly enhanced in chondrocytes obtained from OA cartilage (mean ± SD ΔC(t) 3.4 ± 1.0) and RA cartilage (ΔC(t) 3.4 ± 1.4) compared with cartilage obtained from patients with femoral neck fracture (ΔC(t) 5.3 ± 1.2). The expression of SOCS3 correlated significantly with that of other genes known to be expressed in arthritic chondrocytes, such as MMP13 (r = 0.743), ADAMTS4 (r = 0.779), and ADAMTS5 (r = 0.647), and an inverse relationship was observed with COL2A1 (r = -0.561). Up-regulation of SOCS-3 by IL-1 in G6 chondrocytes and its spontaneous expression in OA chondrocytes were reduced by mithramycin, a specific inhibitor of transcription factor Sp-1. Overexpression of SOCS-3 in bovine chondrocytes reduced IL-1- and LPS-induced nitric oxide production and insulin-like growth factor 1-induced proteoglycan synthesis. Interestingly, a similar impairment of function was observed in OA chondrocytes, which was partially restored by SOCS-3 gene knockdown. CONCLUSION This study demonstrated that both SOCS-3 mRNA and SOCS-3 protein are expressed in human arthritic chondrocytes and affect cellular responses involved in cartilage pathology.

A crucial role for tumor necrosis factor receptor 1 in synovial lining cells and the reticuloendothelial system in mediating experimental arthritis.

IntroductionRheumatoid arthritis (RA) is an autoimmune inflammatory disease that mainly affects synovial joints. Biologics directed against tumor-necrosis-factor (TNF)-α are efficacious in the treatment of RA. However, the role of TNF receptor-1 (TNFR1) in mediating the TNFα effects in RA has not been elucidated and conflicting data exist in experimental arthritis models. The objective is to investigate the role of TNFR1 in the synovial lining cells (SLC) and the reticuloendothelial system (RES) during experimental arthritis.MethodsThird generation of adenovirus serotype 5 were either injected locally in the knee joint cavity or systemically by intravenous injection into the retro-orbital venous sinus to specifically target SLC and RES, respectively. Transduction of organs was detected by immunohistochemistry of the eGFP transgene. An adenoviral vector containing a short hairpin (sh) RNA directed against TNFR1 (HpTNFR1) was constructed and functionally evaluated in vitro using a nuclear factor-kappaB (NF-κB) reporter assay and in vivo in streptococcal cell wall-induced arthritis (SCW) and collagen-induced arthritis (CIA). Adenoviruses were administered before onset of CIA, and the effect of TNFR1 targeting on the clinical development of arthritis, histology, quantitative polymerase chain reaction (qPCR), cytokine analyses and T-cell assays was evaluated.ResultsSystemic delivery of Ad5.CMV-eGFP predominantly transduced the RES in liver and spleen. Local delivery transduced the synovium and not the RES in liver, spleen and draining lymph nodes. In vitro, HpTNFR1 reduced the TNFR1 mRNA expression by three-fold resulting in a 70% reduction of TNFα-induced NF-κB activation. Local treatment with HpTNFR1 markedly reduced mRNA and protein levels of interleukin (IL)-1β and IL-6 in SLC during SCW arthritis and ameliorated CIA. Systemic targeting of TNFR1 in RES of liver and spleen by systemic delivery of Ad5 virus encoding for a small hairpin RNA against TNFR1 markedly ameliorated CIA and simultaneously reduced the mRNA expression of IL-1β, IL-6 and Saa1 (75%), in the liver and that of Th1/2/17-specific transcription factors T-bet, GATA-3 and RORγT in the spleen. Flow cytometry confirmed that HpTNFR1 reduced the numbers of interferon (IFN)γ (Th1)-, IL-4 (Th2)- and IL-17 (Th17)-producing cells in spleen.ConclusionsTNFR1-mediated signaling in both synovial lining cells and the reticuloendothelial system independently played a major pro-inflammatory and immunoregulatory role in the development of experimental arthritis.

A pivotal role for antigen-presenting cells overexpressing SOCS3 in controlling invariant NKT cell responses during collagen-induced arthritis

Objective Suppressor of cytokine signalling (SOCS) proteins constitute a class of intracellular proteins that are key physiological regulators of immune cell function. It has previously been shown that antigen-presenting cells (APCs) overexpressing SOCS3 steer T helper immune responses and protect against experimental arthritis. A study was undertaken to investigate the contribution of SOCS3 in regulating invariant natural killer T (iNKT) cell responses during collagen-induced arthritis (CIA). Methods DBA/1 mice were immunised with type II collagen and adenoviruses encoding SOCS3 were administered intravenously before the clinical onset of arthritis. Murine APCs overexpressing SOCS3 were co-cultured with an iNKT cell hybridoma and interleukin 2 (IL-2) release was measured by Luminex multi-analyte technology. The frequency and activation of primary iNKT cells was assessed by flow cytometry. Murine APCs were analysed for cytokine and CD1d expression following viral SOCS3 gene transfer. Results Viral overexpression of SOCS3 in APCs resulted in reduced activation of the iNKT cell hybridoma. Importantly, during initiation of CIA, adenovirus-mediated overexpression of SOCS3 in hepatic and splenic APCs inhibited iNKT cell expansion in both organs. The iNKT cell population from SOCS3-treated mice showed low expression of the early activation marker CD69 and primary liver iNKT cells produced less interferon γ and IL-4 upon α-galactosylceramide stimulation. No differences in CD1d surface expression were observed, but SOCS3-transduced APCs produced decreased levels of proinflammatory cytokines and increased levels of IL-10. Conclusion These results demonstrate a critical role for SOCS3 in controlling the immunostimulatory capacities of APCs, which has direct implications for the effector function of iNKT cells during arthritis.

Tetraspanin CD37 protects against the development of B cell lymphoma

Worldwide, B cell non-Hodgkin lymphoma is the most common hematological malignancy and represents a substantial clinical problem. The molecular events that lead to B cell lymphoma are only partially defined. Here, we have provided evidence that deficiency of tetraspanin superfamily member CD37, which is important for B cell function, induces the development of B cell lymphoma. Mice lacking CD37 developed germinal center-derived B cell lymphoma in lymph nodes and spleens with a higher incidence than Bcl2 transgenic mice. We discovered that CD37 interacts with suppressor of cytokine signaling 3 (SOCS3); therefore, absence of CD37 drives tumor development through constitutive activation of the IL-6 signaling pathway. Moreover, animals deficient for both Cd37 and Il6 were fully protected against lymphoma development, confirming the involvement of the IL-6 pathway in driving tumorigenesis. Loss of CD37 on neoplastic cells in patients with diffuse large B cell lymphoma (DLBCL) directly correlated with activation of the IL-6 signaling pathway and with worse progression-free and overall survival. Together, this study identifies CD37 as a tumor suppressor that directly protects against B cell lymphomagenesis and provides a strong rationale for blocking the IL-6 pathway in patients with CD37- B cell malignancies as a possible therapeutic intervention.

Targeting Growth Factors in Arthritis: A Rational for Restoring the IGF-I Response in Chondrocytes

The role of growth factors in the pathogenesis of arthritis has received considerably less attention as compared to proinflammatory cytokines in recent years. This is understandable as it is evident that current anti-tumor necrosis factor-α (TNFα) therapy is efficacious in at least in 60% of the rheumatoid arthritis (RA) patients and when given in combination with methotrexate stops radiological progression of bone erosion. Other proinflammatory cytokines, such as interleukin-1 (IL-1) and IL-17 have also been implicated in the dysregulation of bone and cartilage remodelling characteristic of rheumatoid arthritis (RA) and/or osteoarthritis (OA). The direct effects of anti-cytokine treatment on cartilage pathology are not that easy to determine in humans and it remains to be seen if amelioration of cartilage destruction or better cartilage repair has been accomplished with anti-TNF therapy. A complicating factor is that chondrocytes in RA and OA become non-responsive to insulin-like growth factor-I (IGF-I). IGF-I is the main growth factor for stimulation of chondrocyte proteoglycan and collagen type II synthesis in order to maintain the cartilage matrix integrity. This article reviews our current understanding of the role of IGF-I on cartilage extracellular matrix production and the mechanisms underlying the cause of IGF-I non-responsiveness, and in particular the inhibition of IGF-receptor signalling by suppressor of cytokine signalling (SOCS) proteins. The epigenetic and cytokine-dependent regulation of SOCS expression may give an explanation for the loss of IGF-I signalling in chondrocytes in ageing-related diseases such as OA and RA. Whether IGF-1 is replaceable by other growth factors to maintain the cartilage extracellular matrix will be discussed. It is concluded that in addition to the current use of anti-cytokine therapy in arthritis the IGF-I response of chondrocytes must be restored in order to achieve cartilage matrix repair.

THU0022 Enhanced expression of suppressor of cytokine signaling 3 in arthritic cartilage dysregulates human chondrocyte function

Background The intracellular suppressor of cytokine signaling (SOCS)-proteins are inducible and negative regulators in receptor signaling pathways of several cytokines, toll-like receptor ligands and some growth factors. We previously have shown in mice that SOCS3 is expressed in chondrocytes during arthritis [1]. Furthermore, SOCS3 overexpression inhibits the insulin-like growth factor (IGF)-1 response, the main anabolic factor for chondrocytes. Objectives To determine the expression and functional consequnces of SOCS3 in human articular chondrocytes. Methods Chondrocytes were isolated from articular cartilage of patients undergoing surgical joint replacement. The human immortalized chondrocyte cell line G6, human mesenchymal stem cell (MSC)-differentiated chondrocytes, and primary bovine chondrocytes were used for comparison. SOCS3 mRNA and protein levels were measured by quantitative PCR, western blotting and immunohistochemistry, respectively. Regulation of SOCS3 expression was examined following incubation with different cytokines and Toll-like receptor (TLR) agonists. To determine the effect of SOCS3 on the chondrocyte response to various stimuli, SOCS3 was either reduced by an inhibitor of SP1 (Mithramycin) or with short interference RNA, and enhanced by adenoviral transduction. Results The expression of SOCS3 was significantly enhanced in chondrocytes obtained from cartilage of osteoarthritis (OA) (ΔCt 3.4±1.0, n=18 patients) and rheumatoid arthritis (RA) (ΔCt 3.4±1.4, n=6) as compared to and healthy cartilage from fractures of neck of femur (NOF) patients (ΔCt 5.3±1.2, n=8). The expression of SOCS3 correlated markedly with other genes known to be expressed in arthritic chondrocytes such as RUNX2 (r=0.341), MMP13 (r=0.511), ADAMTS4 (r=0.779), and ADAMTS5 (r=0.647). No correlation was found with aggrecan expression and an inverse relationship was found with the collagen-type II gene Col2A1 (r=0.577). Western blots and imunohistochemisty confirmed the enhanced expression of SOCS3 at the protein level in arthritic cartilage. The expression of SOCS1 in chondrocytes was low and similar between the different patient groups. The expression of SOCS3 in the immortalized human chondrocyte cell-line (G6) and MSC-derived chondrocytes could be enhanced by interleukin-1 and conditioned medium of OA synovium explants. This was dependent on the transcription factor SP1 as the specific inhibitor mithramycin prevented SOCS3 upregulation in these cells. Forced expression of SOCS3 in bovine chondrocytes impairs several aspects of chondrocyte function, including nitiric oxide production and proteoglycan synthesis. Interestingly, a similar impairment of function was found in OA chondrocytes and knockdown of SOCS3 in these chondrocytes partially restored human chondrocyte function. Conclusions This study demonstrates that SOCS3 is highly expressed in human articular chondrocytes and affects cellular responses, which may have important implications for cartilage pathology in humans. References Smeets RL, Veenbergen S, Arntz OJ, Bennink MB, Joosten LA, van den Berg WB, van de Loo FA. A novel role for suppressor of cytokine signaling 3 in cartilage destruction via induction of chondrocyte desensitization toward insulin-like growth factor. Arthritis Rheum. 2006;54(5):1518-28. Disclosure of Interest None Declared