Coexpression of CD25 and CD27 identifies FoxP3+ regulatory T cells in inflamed synovia.

A better understanding of the role of CD4+CD25+ regulatory T cells in disease pathogenesis should follow from the discovery of reliable markers capable of discriminating regulatory from activated T cells. We report that the CD4+CD25+ population in synovial fluid of juvenile idiopathic arthritis (JIA) patients comprises both regulatory and effector T cells that can be distinguished by expression of CD27. CD4+CD25+CD27+ cells expressed high amounts of FoxP3 (43% of them being FoxP3+), did not produce interleukin (IL)-2, interferon-gamma, or tumor necrosis factor, and suppressed T cell proliferation in vitro, being, on a per cell basis, fourfold more potent than the corresponding peripheral blood population. In contrast, CD4+CD25+CD27- cells expressed low amounts of FoxP3, produced effector cytokines and did not suppress T cell proliferation. After in vitro activation and expansion, regulatory but not conventional T cells maintained high expression of CD27. IL-7 and IL-15 were found to be present in synovial fluid of JIA patients and, when added in vitro, abrogated the suppressive activity of regulatory T cells. Together, these results demonstrate that, when used in conjunction with CD25, CD27 is a useful marker to distinguish regulatory from effector T cells in inflamed tissues and suggest that at these sites IL-7 and IL-15 may interfere with regulatory T cell function.

Interferon-γ-dependent inhibition of B cell activation by bone marrow-derived mesenchymal stem cells in a murine model of systemic lupus erythematosus.

OBJECTIVE: Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent cells characterized by immunomodulatory properties and are therefore considered a promising tool for the treatment of immune-mediated diseases. This study was undertaken to assess the influence of murine BM-MSCs on the activation of B cells in (NZB × NZW)F(1) mice as an animal model of systemic lupus erythematosus (SLE). METHODS: We evaluated the in vitro effects of BM-MSCs on the proliferation and differentiation to plasma cells of splenic mature B cell subsets, namely follicular and marginal zone B cells isolated from (NZB × NZW)F(1) mice. Lupus mice were also treated with BM-MSCs, and serum autoantibodies, proteinuria, histologic changes in the kidney, and survival rates were monitored. RESULTS: BM-MSCs inhibited antigen-dependent proliferation and differentiation to plasma cells of follicular and marginal zone B cells in vitro. This inhibitory effect was dependent on interferon-γ (IFNγ) and was mediated by cell-to-cell contact, involving the programmed death 1 (PD-1)/PD ligand pathway. In vivo treatment with BM-MSCs did not affect the levels of anti-double-stranded DNA antibodies or proteinuria. However, a reduction in glomerular immune complex deposition, lymphocytic infiltration, and glomerular proliferation was observed. CONCLUSION: Our findings indicate that BM-MSCs affect B cell receptor-dependent activation of both follicular and marginal zone B cells from lupus mice. This inhibitory effect is IFNγ-dependent and cell contact-dependent. MSCs in vivo do not affect the production of autoantibodies, the level of proteinuria, or the mortality rates. Nonetheless, the significant improvement in histologic findings in the kidney supports the potential role of MSCs in the prevention of glomerular damage.

The tumor suppressor gene TRC8/RNF139 is disrupted by a constitutional balanced translocation t(8;22)(q24.13;q11.21) in a young girl with dysgerminoma.

BACKGROUND: RNF139/TRC8 is a potential tumor suppressor gene with similarity to PTCH, a tumor suppressor implicated in basal cell carcinomas and glioblastomas. TRC8 has the potential to act in a novel regulatory relationship linking the cholesterol/lipid biosynthetic pathway with cellular growth control and has been identified in families with hereditary renal (RCC) and thyroid cancers. Haploinsufficiency of TRC8 may facilitate development of clear cell-RCC in association with VHL mutations, and may increase risk for other tumor types. We report a paternally inherited balanced translocation t(8;22) in a proposita with dysgerminoma. METHODS: The translocation was characterized by FISH and the breakpoints cloned, sequenced, and compared. DNA isolated from normal and tumor cells was checked for abnormalities by array-CGH. Expression of genes TRC8 and TSN was tested both on dysgerminoma and in the proposita and her father. RESULTS: The breakpoints of the translocation are located within the LCR-B low copy repeat on chromosome 22q11.21, containing the palindromic AT-rich repeat (PATRR) involved in recurrent and non-recurrent translocations, and in an AT-rich sequence inside intron 1 of the TRC8 tumor-suppressor gene at 8q24.13. TRC8 was strongly underexpressed in the dysgerminoma. Translin is underexpressed in the dysgerminoma compared to normal ovary.TRC8 is a target of Translin (TSN), a posttranscriptional regulator of genes transcribed by the transcription factor CREM-tau in postmeiotic male germ cells. CONCLUSION: A role for TRC8 in dysgerminoma may relate to its interaction with Translin. We propose a model in which one copy of TRC8 is disrupted by a palindrome-mediated translocation followed by complete loss of expression through suppression, possibly mediated by miRNA.

Differential regulation of chemokine production by Fcgamma receptor engagement in human monocytes: association of CCL1 with a distinct form of M2 monocyte activation (M2b, Type 2).

CC chemokine ligand 1 (CCL1; I-309) is a CC chemokine that interacts with CC chemokine receptor 8, which is preferentially expressed in polarized T helper cell type 2 and Tc2 cells, in eosinophils, and in T regulatory cells. The present study, prompted by transcriptional profiling of human monocytes undergoing different forms of activation, was designed to characterize the production of CCL1 in monocytes compared with the production of other chemokines (CCL2, CCL22, and CCL18) differentially regulated by distinct activation signals. Lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), interleukin (IL)-1beta, tumor necrosis factor alpha, IL-4, IL-13, IL-10, IL-6, IL-18, and combinations thereof did not induce CCL1 production in monocytes, and some of these signals stimulated production of reference chemokines. Induction of CCL1 in monocytes required engagement of Fc receptor for immunoglobulin G (FcgammaR)II and exposure to IL-1beta or LPS. This combination of stimuli results in a form of M2 (M2b, Type 2) macrophage activation. FcgammaR engagement also induced CCL22 and amplified its stimulation by IL-4. In contrast, FcgammaR stimulation inhibited the IL-10- and LPS-mediated induction of CCL18. IL-10, IL-4, and IFN-gamma inhibited induction of CCL1 by FcgammaR ligation and IL-1beta. CCL1 was present in synovial fluids and macrophages in juvenile idiopathic arthritis. Thus, regulation of CCL1 in human monocytes is unique, with an obligate requirement of FcgammaR engagement and costimulation by signals (IL-1beta and LPS), which use the myeloid differentiation primary-response protein 88 adaptor protein. Thus, CCL1 is a CC chemokine with a unique pattern of regulation associated with a distinct form of M2 (Type 2, M2b) monocyte activation, which participates in macrophage-dependent regulatory circuits of innate and adaptive immunity.

Different subcellular localization of ALCAM molecules in neuroblastoma: Association with relapse.

BACKGROUND: The Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166), involved in nervous system development, has been linked to tumor progression and metastasis in several tumors. No information is available on ALCAM expression in neuroblastoma, a childhood neoplasia originating from the sympathetic nervous system. METHODS: ALCAM expression was analysed by immunofluorescence and immunohistochemistry on differentiated neuroblastoma cell lines and on archival specimens of stroma-poor, not MYCN amplified, resectable neuroblastoma tumors, respectively. RESULTS: ALCAM is variously expressed in neuroblastoma cell lines, is shed by metalloproteases and is cleaved by ADAM17/TACE in vitro. ALCAM is expressed in neuroblastoma primary tumors with diverse patterns of subcellular localization and is highly expressed in the neuropil area in a subgroup of cases. Tumor specimens showing high expression of ALCAM at the membrane of the neuroblast body or low levels in the neuropil area are associated with relapse (P=0.044 and P<0.0001, respectively). In vitro differentiated neuroblastoma cells show strong ALCAM expression on neurites, suggesting that ALCAM expression in the neuropil is related to a differentiated phenotype. CONCLUSION: Assessment of ALCAM localization by immunohistochemistry may help to identify patients who, in the absence of negative prognostic factors, are at risk of relapse and require a more careful follow-up.

The hypoxic synovial environment regulates expression of vascular endothelial growth factor and osteopontin in juvenile idiopathic arthritis.

OBJECTIVE: Synovial angiogenesis, a critical determinant of juvenile idiopathic arthritis (JIA) pathogenesis, is sustained by various mediators, including vascular endothelial growth factor (VEGF) and osteopontin (OPN). We characterized the contribution of the local hypoxic environment to VEGF and OPN production by monocytic cells recruited to the synovium in JIA. METHODS: Paired synovial fluid (SF) and peripheral blood (PB) samples were collected from 20 patients with JIA. Mononuclear cells (MC) were isolated, and monocytic cells were purified by adherence, maintained in a hypoxic environment, or subjected to reoxygenation. VEGF and OPN protein concentrations were tested in SF, plasma, and culture supernatants by ELISA, and mRNA expression was assessed in freshly purified and cultured cells by reverse transcriptase-polymerase chain reaction. Synovial tissue was obtained at synovectomy from 4 patients with JIA, and analyzed by immunohistochemistry for VEGF, OPN, CD68, and hypoxia-inducible factor-1alpha (HIF-1alpha). RESULTS: VEGF mRNA expression was increased in SFMC and SF monocytic cells compared to matched PBMC and PB monocytic cells or SF lymphocytes, correlating with significantly higher protein levels in SF relative to plasma samples. Accordingly, OPN mRNA expression in SF monocytic cells was associated with significant increase of SF protein. Immunohistochemistry revealed the presence of both factors in synovial tissues at the level of the lining and sublining layers, which colocalized with intense CD68 and HIF-1alpha staining, suggesting production by hypoxic synovial monocytic cells. VEGF and OPN expression was abrogated upon SF monocytic cell reoxygenation and maintained by exposure to prolonged hypoxia. CONCLUSION: Hypoxic synovial monocytic cells are a likely source of VEGF and OPN in JIA. These data point to a role for hypoxia in the perpetuation of synovitis in JIA.

CX3CR1 is expressed by human B lymphocytes and mediates [corrected] CX3CL1 driven chemotaxis of tonsil centrocytes.

BACKGROUND: Fractalkine/CX(3)CL1, a surface chemokine, binds to CX(3)CR1 expressed by different lymphocyte subsets. Since CX(3)CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX(3)CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX(3)CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX(3)CR1 but only germinal centre B cells were attracted by soluble CX(3)CL1 in a transwell assay. CX(3)CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX(3)CR1(+) germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX(3)CL1. ELISA assay showed that soluble CX(3)CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX(3)CL1 did not attract spleen B cells from wild type mice. OVA immunized CX(3)CR1(-/-) or CX(3)CL1(-/-) mice showed significantly decreased specific IgG production compared to wild type mice. CONCLUSION/SIGNIFICANCE: We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX(3)CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.

Phenotypic and functional characterization of switch memory B cells from patients with oligoarticular juvenile idiopathic arthritis.

INTRODUCTION: In chronic inflammatory disorders, B cells can contribute to tissue damage by autoantibody production and antigen presentation to T cells. Here, we have characterized synovial fluid and tissue B-cell subsets in patients with oligoarticular juvenile idiopathic arthritis (JIA), an issue not addressed before in detail. METHODS: B cells from synovial fluid (SF) and peripheral blood (PB) of 25 JIA patients, as well as from PB of 20 controls of comparable age, were characterized by multicolor flow cytometry. Immunoglobulin-secreting cells were detected by ELISPOT. Immunohistochemical analyses of synovial tissue from three JIA patients were performed. RESULTS: JIA SF B cells were enriched in CD27+ and CD27- switch memory B cells, but not in CD27+ IgM memory B cells, compared with patient and control PB. Plasma blasts were more abundant in SF and secreted higher amounts of IgG. Lymphoid aggregates not organized in follicle-like structures were detected in synovial tissue sections and were surrounded by CD138+ plasma cells. Finally, transitional B cells were significantly increased in JIA PB versus SF or control PB. CCR5, CCR8, CXCR2, and CXCR3 were upregulated, whereas CCR6, CCR7, and CXCR5 were downregulated on SF CD27+ and CD27- switch memory B cells compared with their circulating counterparts. SF CD27+ and CD27- switch memory B cells expressed at high levels the costimulatory molecule CD86 and the activation marker CD69. CONCLUSIONS: This study demonstrates for the first time an expansion of activated switch memory B cells and of IgG-secreting plasma blasts in the SF from oligoarticular JIA patients. Memory B cells belonged to either the CD27+or the CD27- subsets and expressed CD86, suggesting their involvement in antigen presentation to T cells. Patterns of chemokines-receptor expression on CD27+ and CD27- switch memory B cells delineated potential mechanisms for their recruitment to the inflamed joints.

Mutation-independent anaplastic lymphoma kinase overexpression in poor prognosis neuroblastoma patients.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified as a major oncogene associated with familial and sporadic neuroblastomas (NBL). Yet, a direct correlation between endogenous expression level of the ALK protein, oncogenic potential, and clinical outcome has not been established. We investigated ALK genetic mutations, protein expression/phosphorylation, and functional inhibition both in NBL-derived cell lines and in 34 localized and 48 advanced/metastatic NBL patients. ALK constitutive phosphorylation/activation was observed in high-ALK expressing cells, harboring either a mutated or a wild-type receptor. No activation was found in cell lines with low expression of wild-type ALK. After 72 hours of treatments, small molecule ALK inhibitor CEP-14083 (60 nmol/L) induced growth arrest and cell death in NBL cells overexpressing wild-type (viability: ALK(high) 12.8%, ALK(low) 73%, P = 0.0035; cell death: ALK(high) 56.4%, ALK(low) 16.2%, P = 0.0001) or mutated ALK. ALK protein expression was significantly up-regulated in advanced/metastatic compared with localized NBLs (ALK overexpressing patients: stage 1-2, 23.5%; stage 3-4, 77%; P < 0.0001). Interestingly, protein levels did not always correlate with ALK genetic alterations and/or mRNA abundance. Both mutated and wild-type ALK receptor can exert oncogenic activity in NBL cells. However, wild-type ALK receptor requires a critical threshold of expression to achieve oncogenic activation. Overexpression of either mutated or wild-type ALK defines poor prognosis patients. Alternative mechanisms other than direct mutations and/or gene amplification regulate the ALK level of expression in NBL cells. Wild-type ALK is a potential therapeutic target for advanced/metastatic NBLs.

Hypoxic synovial environment and expression of macrophage inflammatory protein 3gamma/CCL20 in juvenile idiopathic arthritis.

OBJECTIVE: Synovial inflammation is a major determinant of juvenile idiopathic arthritis (JIA) pathogenesis and is mediated by local chemokine secretion. Monocytic cells are an important source of chemokines. The purpose of this study was to investigate expression of CCL20, a macrophage inflammatory protein, in synovial fluid (SF) and SF-derived monocytic cells from JIA patients and its regulation by hypoxia, a common feature of the inflamed synovial environment. METHODS: Mononuclear cells and monocytic cells were isolated from paired SF and peripheral blood (PB) samples from JIA patients and were maintained in a hypoxic environment or subjected to reoxygenation. CCL20 concentrations in SF, PB, and culture supernatants were measured by enzyme-linked immunosorbent assay. CCL20 expression was assessed in both freshly purified and cultured cells by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha were detected in the synovial tissue and cells of JIA patients by immunohistochemistry and Western blotting. RESULTS: CCL20 concentrations were significantly higher in SF compared with PB samples (P < 0.0001). SF mononuclear cells, but not matched PB mononuclear cells, constitutively expressed CCL20 messenger RNA. The SF monocytic cell fraction produced higher amounts of CCL20 than did SF lymphocytes, and CCL20 expression was associated with HIF positivity. Reoxygenation abrogated HIF and CCL20 expression, which was maintained in SF monocytic cells exposed to prolonged hypoxia. CONCLUSION: CCL20 is released into the SF of JIA patients, and SF monocytic cells are a major source of this chemokine. The hypoxic synovial microenvironment may directly contribute to the persistent inflammation associated with JIA by increasing CCL20 production by SF monocytic cells, thus representing a potential therapeutic target.

Chemokine receptor CCR7 is expressed in muscle fibers in juvenile dermatomyositis.

To better elucidate the pathogenesis of lymphocyte recruitment of memory CD4(+) T cells in inflammatory myopathies, we studied the expression of CCR5 and CCR7 on CD4 memory T cells in muscle tissue from 11 patients with juvenile dermatomyositis, six adult patients with polymyositis, two patients with Duchenne muscular dystrophy, and two patients with spinal muscular atrophy. A prevalent infiltration of CCR5(+) effector CD4 T memory cells is observed in inflammatory myopathies. Moreover, we found a strong expression of CCR7 in perifascicular atrophic and in degenerating/regenerating muscle fibers in juvenile dermatomyositis (JDM) but not in fibers from adult polymyositis and Duchenne muscular dystrophy. The selective expression of CCR7 in JDM may open new perspectives in the understanding of the pathogenesis of inflammatory myopathies, offering a new tool for the differential diagnosis of these disorders.

Phenotypic and functional characterisation of CCR7+ and CCR7- CD4+ memory T cells homing to the joints in juvenile idiopathic arthritis.

The aim of the study was to characterise CCR7+ and CCR7- memory T cells infiltrating the inflamed joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the functional and anatomical heterogeneity of these cell subsets in relation to the expression of the inflammatory chemokine receptors CXCR3 and CCR5. Memory T cells freshly isolated from the peripheral blood and synovial fluid (SF) of 25 patients with JIA were tested for the expression of CCR7, CCR5, CXCR3 and interferon-gamma by flow cytometry. The chemotactic activity of CD4 SF memory T cells from eight patients with JIA to inflammatory (CXCL11 and CCL3) and homeostatic (CCL19, CCL21) chemokines was also evaluated. Paired serum and SF samples from 28 patients with JIA were tested for CCL21 concentrations. CCR7, CXCR3, CCR5 and CCL21 expression in synovial tissue from six patients with JIA was investigated by immunohistochemistry. Enrichment of CD4+, CCR7- memory T cells was demonstrated in SF in comparison with paired blood from patients with JIA. SF CD4+CCR7- memory T cells were enriched for CCR5+ and interferon-gamma+ cells, whereas CD4+CCR7+ memory T cells showed higher coexpression of CXCR3. Expression of CCL21 was detected in both SF and synovial membranes. SF CD4+ memory T cells displayed significant migration to both inflammatory and homeostatic chemokines. CCR7+ T cells were detected in the synovial tissue in either diffuse perivascular lymphocytic infiltrates or organised lymphoid aggregates. In synovial tissue, a large fraction of CCR7+ cells co-localised with CXCR3, especially inside lymphoid aggregates, whereas CCR5+ cells were enriched in the sublining of the superficial subintima. In conclusion, CCR7 may have a role in the synovial recruitment of memory T cells in JIA, irrespective of the pattern of lymphoid organisation. Moreover, discrete patterns of chemokine receptor expression are detected in the synovial tissue.