Peihua Wu

Analysis of IL-17 + cells in facet joints of patients with spondyloarthritis suggests that the innate immune pathway might be of greater relevance than the Th17-mediated adaptive immune response

IntroductionIn this study, we analysed the number of IL-17+ cells in facet joints, in the peripheral blood (PB) and synovial fluid (SF) of spondyloarthritis (SpA) patients and compared these results with those of patients with other rheumatic diseases and controls.MethodsImmunohistochemical analysis of IL-17+ cells was performed in facet joints of 33 ankylosing spondylitis (AS) patients and compared with data from 20 osteoarthritis (OA) patients. The frequency of IL-17+CD4+ T cells in PB and SF of SpA patients (PB n = 30, SF n = 11), rheumatoid arthritis (RA) patients (PB n = 14, SF n = 7), OA patients (PB n = 10) and healthy controls (PB n = 12) was analysed after stimulation with Staphylococcus aureus Enterotoxin B and phorbol 12-myristate 13-acetate/ionomycin and quantified by flow cytometry.ResultsIn AS facet joints, the frequency of IL-17-secreting cells was significantly higher than in samples obtained from OA patients (P < 0.001), with a slight predominance of IL-17+ cells among the mononuclear cells (61.5% ± 14.9%) compared to cells with polysegmental nuclei. Immunofluorescence microscopy revealed that the majority of IL-17+ cells were myeloperoxidase-positive (35.84 ± 13.06/high-power field (HPF) and CD15+ neutrophils (24.25 ± 10.36/HPF), while CD3+ T cells (0.51 ± 0.49/HPF) and AA-1+ mast cells (2.28 ± 1.96/HPF) were less often IL-17-positive. The frequency of IL-17+CD4+ T cells in the PB and SF of SpA patients did not differ significantly compared to RA patients, OA patients or healthy controls.ConclusionsOur data suggest an important role for IL-17 in the inflammatory processes in AS. However, the innate immune pathway might be of greater relevance than the Th17-mediated adaptive immune response.

TNF signaling drives myeloid-derived suppressor cell accumulation

TNF, an inflammatory cytokine that is enriched in the tumor microenvironment, promotes tumor growth and subverts innate immune responses to cancer cells. We previously reported that tumors implanted in TNF receptor-deficient (Tnfr-/-) mice are spontaneously rejected; however, the molecular mechanisms underlying this rejection are unclear. Here we report that TNF signaling drives the peripheral accumulation of myeloid-derived suppressor cells (MDSCs). MDSCs expand extensively during inflammation and tumor progression in mice and humans and can enhance tumor growth by repressing T cell-mediated antitumor responses. Peripheral accumulation of MDSCs was drastically impaired in Tnfr-/- mice. Signaling of TNFR-2, but not TNFR-1, promoted MDSC survival through upregulation of cellular FLICE-inhibitory protein (c-FLIP) and inhibition of caspase-8 activity. Loss of TNFRs impaired the induction of MDSCs from bone marrow cells, but this could be reversed by treatment with caspase inhibitors. These results demonstrate that TNFR-2 signaling promotes MDSC survival and accumulation and helps tumor cells evade the immune system.

Chlamydia pneumoniae--a new causative agent of reactive arthritis and undifferentiated oligoarthritis.

OBJECTIVE--To examine whether reactive arthritis (ReA) known to occur after a urogenital infection with Chlamydia trachomatis can also follow an infection with Chlamydia pneumoniae, a recently described species of Chlamydiae that is a common cause of respiratory tract infections. METHODS--Specific antibodies (microimmunofluorescence test) and lymphocyte proliferation to C trachomatis and C pneumoniae in paired samples of peripheral blood and synovial fluid were investigated in 70 patients with either reactive arthritis (ReA) or undifferentiated oligoarthritis (UOA). RESULTS--Five patients with acute ReA after an infection with C pneumoniae are reported. Three had a symptomatic preceding upper respiratory tract infection and two had no such symptoms. In all patients a C pneumoniae-specific lymphocyte proliferation in synovial fluid and a high specific antibody titre suggesting an acute infection was found. CONCLUSION--C pneumoniae needs to be considered a new important cause of reactive arthritis.

A Converse 4-1BB and CD40 Ligand Expression Pattern Delineates Activated Regulatory T Cells (Treg) and Conventional T Cells Enabling Direct Isolation of Alloantigen-Reactive Natural Foxp3+ Treg

Natural regulatory T cells (nTreg) play a central role in the induction and maintenance of immunological tolerance. Experimental transplant models and recent clinical trials demonstrate that nTreg can control alloreactivity. To upgrade Treg-based cell therapies to a selective suppression of undesired immune reactions, only the transfer of Ag-specific nTreg represents the appropriate therapeutic option. However, Ag-specific nTreg are present at extremely low frequencies in the periphery, and so far appropriate surface markers for their precise identification are missing. In this study, we demonstrate that activated nTreg and activated conventional T cells differ in their 4-1BB and CD40 ligand (CD40L) expression signatures, allowing a clear dissection from each other. Based on the expression of 4-1BB and absence of CD40L expression, human alloantigen-reactive Foxp3+ nTreg can be directly isolated from MLR cultures with high purity. Alloantigen-reactive 4-1BB+CD40L− nTreg were characterized by a completely demethylated Treg-specific demethylated region and showed alloantigen-specific suppressive properties superior to polyclonal Treg. Importantly, isolated 4-1BB+CD40L− nTreg maintain the nTreg phenotype and alloantigen-reactivity after in vitro expansion. Our results offer the possibility to simultaneously analyze Ag-specific nTreg and conventional T cells, and to establish cellular therapies with Ag-specific nTreg aiming at a specific inhibition of unwanted immunity.

Cytokine-induced human IFN-γ–secreting effector-memory Th cells in chronic autoimmune inflammation

T-helper (Th) cells activated by cytokines in the absence of T-cell receptor ligation are suspected to participate in inflammatory processes by production of interferon-gamma (IFN-gamma). Still, the relevance of such a mechanism has not been addressed in humans. Here we demonstrate that a subset of human effector-memory Th cells expressing functional interleukin-12R (IL-12R), IL-18Ralpha, and CCR5 ex vivo can be induced to secrete IFN-gamma by cytokines signaling via the IL-2R common gamma-chain in combination with IL-12 and IL-18. Cytokine-driven IFN-gamma production depends on JAK3- and p38 mitogen-activated kinase signals and is sensitive to suppression by CD25(++) regulatory T cells. Contrary to IFN-gamma(+) Th cells induced upon antigen-specific stimulation, their cytokine-activated counterparts characteristically lack expression of costimulator 4-1BB (CD137). Strikingly, the majority of Th cells infiltrating inflamed joints of rheumatoid arthritis patients is equipped with receptors prerequisite for cytokine-induced IFN-gamma secretion. Among these cells, we detected a substantial fraction that secretes IFN-gamma directly ex vivo but lacks 4-1BB expression, indicating that cytokine-induced IFN-gamma(+) Th cells operate in autoimmune inflammation. Our data provide a rationale for how human effector-memory Thcells can participate in perpetuating inflammatory processes in autoimmunity even in the absence of T-cell receptor ligation.

IL-10 is excluded from the functional cytokine memory of human CD4+ memory T lymphocytes

Epigenetic modifications, including DNA methylation, profoundly influence gene expression of CD4+ Th-specific cells thereby shaping memory Th cell function. We demonstrate here a correlation between a lacking fixed potential of human memory Th cells to re-express the immunoregulatory cytokine gene IL10 and its DNA methylation status. Memory Th cells secreting IL-10 or IFN-γ were directly isolated ex vivo from peripheral blood of healthy volunteers, and the DNA methylation status of IL10 and IFNG was assessed. Limited difference in methylation was found for the IL10 gene locus in IL-10-secreting Th cells, as compared with Th cells not secreting IL-10 isolated directly ex vivo or from in vitro-established human Th1 and Th2 clones. In contrast, in IFN-γ+ memory Th cells the promoter of the IFNG gene was hypomethylated, as compared with IFN-γ-nonsecreting memory Th cells. In accordance with the lack of epigenetic memory, almost 90% of ex vivo-isolated IL-10-secreting Th cells lacked a functional memory for IL-10 re-expression after restimulation. Our data indicate that IL10 does not become epigenetically marked in human memory Th cells unlike effector cytokine genes such as IFNG. The exclusion of IL-10, but not effector cytokines, from the functional memory of human CD4+ T lymphocytes ex vivo may reflect the need for appropriate regulation of IL-10 secretion, due to its potent immunoregulatory potential.

Identification of Novel Human Aggrecan T Cell Epitopes in HLA-B27 Transgenic Mice Associated with Spondyloarthropathy

The pathology of ankylosing spondylitis, reactive arthritis, and other spondyloarthropathies (SpA) is closely associated with the human leukocyte class I Ag HLA-B27. A characteristic finding in SpA is inflammation of cartilage structures of the joint, in particular at the site of ligament/tendon and bone junction (enthesitis). In this study, we investigated the role of CD8+ T cells in response to the cartilage proteoglycan aggrecan as a potential candidate autoantigen in BALB/c-B27 transgenic mice. We identified four new HLA-B27-restricted nonamer peptides, one of them (no. 67) with a particularly strong T cell immunogenicity. Peptide no. 67 immunization was capable of stimulating HLA-B27-restricted, CD8+ T cells in BALB/c-B27 transgenic animals, but not in wild-type BALB/c mice. The peptide was specifically recognized on P815-B27 transfectants by HLA-B27-restricted CTLs, which were also detectable by HLA tetramer staining ex vivo as well as in situ. Most importantly, analysis of the joints from peptide no. 67-immunized mice induced typical histological signs of SpA. Our data indicate that HLA-B27-restricted epitopes derived from human aggrecan are involved in the induction of inflammation (tenosynovitis), underlining the importance of HLA-B27 in the pathogenesis of SpA.

Treatment of spondyloarthropathies with antibodies against tumour necrosis factor α: first clinical and laboratory experiences

Drug treatment of patients with spondyloarthropathies (SpA), especially ankylosing spondylitis (AS) has limited capacity.1 Pain but not disease activity can be reduced by non-steroidal anti-inflammatory drugs (NSAIDs), in severe cases very high doses are needed.2 By examination of sacroiliac biopsy specimens we have shown that, in correlation to disease activity assessed by magnetic resonance imaging (MRI), T cells and macrophages3 and tumour necrosis factor α (TNFα) mRNA4 and protein (fig 1) but no bacterial DNA5 is present in these joints that are pathognomonically involved in AS.6 Furthermore, anti-TNFα monoclonal antibodies (mAb) have recently been shown to be efficacious in Crohns disease7—a disease that is strongly linked to AS because more than 60% of AS patients have clinically often silent gut lesions resembling Crohns colitis.8 Furthermore, in another chronic inflammatory rheumatic disease, rheumatoid arthritis (RA), anti-TNF treatment has proved efficacious and even seems to prevent joint damage.9 Figure 1 Immunohistological examination of a sacroiliac biopsy specimen obtained by computed tomography guided biopsy of a 23 year old patient with ankylosing spondylitis, four years disease duration, with inflammatory back pain grade 6 on a visual analogue scale (0–10). The red staining indicates a mononuclear cell positive for TNFα (APAAP staining technique). TNFα is a cytokine that is mainly produced by monocytes and macrophages and to a lesser degree by T cells. There are two specific receptors, a 55 kDa and a 75 kDa present on many cell types. TNFα mediates inflammatory and immunoregulatory activities. Effects on cells such as lymphocyte activation and fibroblast proliferation, on mediators such as other cytokines like interleukin 1(IL1), IL6 and IL8, chemokines, prostaglandins, metalloproteinases, on the vasculature by promoting angiogenesis and on upregulation of adhesion molecules and transendothelial migration of leucocytes are well described. In in vitro and in …

Aetiological role of bacteria associated with reactive arthritis in pauciarticular juvenile chronic arthritis.

BACKGROUND: The cause of juvenile chronic arthritis (JCA) is unknown. Pauciarticular JCA is the most common subtype and can be subdivided into early (type I) and late onset (type II) forms, the latter clinically resembling reactive arthritis. METHODS: The cellular immune responses to bacteria associated with reactive arthritis in blood and synovial fluid from 39 children with pauciarticular JCA, three children with classical reactive arthritis, and two children with psoriatic arthritis were examined. Specific titres of antibodies to bacteria in serum samples were measured in all patients. RESULTS: A bacteria specific synovial cellular immune response was found in two of three (67%) patients with reactive arthritis and 14 of 28 (50%) patients with pauciarticular JCA type II but only in one of 11 (9%) patients with pauciarticular JCA type I and none in patients with psoriatic arthritis. Six patients responded specifically to Chlamydia trachomatis and 11 to Yersinia enterocolitica. Antigen specific lymphocyte proliferation correlated poorly with the specific antibody response. CONCLUSIONS: These findings suggest that bacteria with associated reactive arthritis may have a causative role in pauciarticular JCA type II but not in JCA type I.

Loss of methylation at the IFNG promoter and CNS-1 is associated with the development of functional IFN-γ memory in human CD4(+) T lymphocytes.

Cytokine memory for IFN‐γ production by effector/memory Th1 cells plays a key role in both protective and pathological immune responses. To understand the epigenetic mechanism determining the ontogeny of effector/memory Th1 cells characterized by stable effector functions, we identified a T‐cell‐specific methylation pattern at the IFNG promoter and CNS‐1 in ex vivo effector/memory Th1 cells, and investigated methylation dynamics of these regions during the development of effector/memory Th1 cells. During Th1 differentiation, demethylation occurred at both the promoter and CNS‐1 regions of IFNG as early as 16 h, and this process was independent of cell proliferation and DNA synthesis. Using an IFN‐γ capture assay, we found early IFN‐γ‐producing cells from 2‐day differentiating cultures acquired “permissive” levels of demethylation and developed into effector/memory Th1 cells undergoing progressive demethylation at the IFNG promoter and CNS‐1 when induced by IL‐12. Methylation levels of these regions in effector/memory Th1 cells of peripheral blood from rheumatoid arthritis patients correlated inversely with reduced frequencies of IFN‐γ‐producers, coincident with recruitment of effector/memory Th1 cells to the site of inflammation. Thus, after termination of TCR stimulation, IL‐12 signaling potentiates the stable functional IFN‐γ memory in effector/memory Th1 cells characterized by hypomethylation at the IFNG promoter and CNS‐1.