Chemokine receptors in the rheumatoid synovium: upregulation of CXCR5

In patients with rheumatoid arthritis (RA), chemokine and chemokine receptor interactions play a central role in the recruitment of leukocytes into inflamed joints. This study was undertaken to characterize the expression of chemokine receptors in the synovial tissue of RA and non-RA patients. RA synovia (n = 8) were obtained from knee joint replacement operations and control non-RA synovia (n = 9) were obtained from arthroscopic knee biopsies sampled from patients with recent meniscal or articular cartilage damage or degeneration. The mRNA expression of chemokine receptors and their ligands was determined using gene microarrays and PCR. The protein expression of these genes was demonstrated by single-label and double-label immunohistochemistry. Microarray analysis showed the mRNA for CXCR5 to be more abundant in RA than non-RA synovial tissue, and of the chemokine receptors studied CXCR5 showed the greatest upregulation. PCR experiments confirmed the differential expression of CXCR5. By immunohistochemistry we were able to detect CXCR5 in all RA and non-RA samples. In the RA samples the presence of CXCR5 was observed on B cells and T cells in the infiltrates but also on macrophages and endothelial cells. In the non-RA samples the presence of CXCR5 was limited to macrophages and endothelial cells. CXCR5 expression in synovial fluid macrophages and peripheral blood monocytes from RA patients was confirmed by PCR. The present study shows that CXCR5 is upregulated in RA synovial tissue and is expressed in a variety of cell types. This receptor may be involved in the recruitment and positioning of B cells, T cells and monocytes/macrophages in the RA synovium. More importantly, the increased level of CXCR5, a homeostatic chemokine receptor, in the RA synovium suggests that non-inflammatory receptor–ligand pairs might play an important role in the pathogenesis of RA.     

Expression of ID family genes in the synovia from patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by aggressive proliferation of synovial tissue leading to destruction of cartilage and bone. To identify molecules which play a crucial role for the pathogenesis, we compared mRNA expression pattern of RA synovium with that of osteoarthritis (OA), using the differential display. From the panel of differentially expressed genes, ID1 (inhibitor of differentiation 1) was considered to be particularly relevant to the pathogenesis of RA, because Id family genes have been shown to play a role in cell proliferation and angiogenesis. To examine whether the up-regulation of these genes is consistently observed in the patients with RA, mRNA levels of ID1 and ID3 in the synovial tissues from 13 patients with RA and 6 patients with OA were semi-quantitatively analyzed by RT-PCR. Mean mRNA levels of ID1 and ID3 were significantly elevated in RA synovia compared with OA by 8.6-fold (P = 0.0044) and 3.3-fold (P = 0.0085), respectively. Immunohistochemistry revealed striking staining of Id1 and Id3 in the endothelial cells, suggesting a possible role of Id in severe angiogenesis observed in RA. The expression of Id family genes in the synovium constitutes a new finding of particular interest. Their functional role as well as their contribution to the genetic susceptibility to RA requires further investigation.     

Lack of reactivity of rheumatoid arthritis synovial membrane DNA with cloned Epstein Barr virus DNA probes

Rheumatoid arthritis (RA) synovial membranes contain a 62,000 dalton (62 Kd) molecule that shares an antigenic epitope with the EBNA-1 antigen of Epstein Barr virus (EBV). This cross-reactive epitope(s) is defined by a monoclonal anti-EBNA-1 antibody (MoAb P135) and by a rabbit antibody directed against a (glycine-alanine)-containing synthetic peptide from the internal repeat region-3 (IR-3) of EBNA-1. To determine whether this 62 Kd protein may result from EBV infection of RA synovial membranes, we used cloned DNA probes from the Bam M, Bam V, and Eco D regions of EBV. These probes did not show detectable reactivity with RA synovial membrane DNA in Southern blotting or slot blotting experiments. Reconstruction experiments performed with purified EBV DNA demonstrated the ability to detect 0.03 pg of viral DNA per 20 micrograms of normal genomic DNA, or approximately 1 EBV copy per 100 cells. In contrast, we found a low but significant level of reactivity of RA synovial membrane DNA with the EBV-encoded Bam K probe that encodes the EBNA-1 antigen. However, this probe also was reactive with normal genomic DNA to a similar extent. These results suggest that the 62 Kd antigen in RA synovial lining cells is probably encoded by cellular genes similar to the IR-3 region of EBV and does not result from EBV infection of the RA synovial membrane.     

Increased frequency of EBV-specific effector memory CD8+ T cells correlates with higher viral load in rheumatoid arthritis

EBV is a candidate trigger of rheumatoid arthritis (RA). We determined both EBV-specific T cell and B cell responses and cell-associated EBV DNA copies in patients with RA and demographically matched healthy virus carriers. Patients with RA showed increased and broadened IgG responses to lytic and latent EBV-encoded Ags and 7-fold higher levels of EBV copy numbers in circulating blood cells. Additionally, patients with RA exhibited substantial expansions of CD8(+) T cells specific for pooled EBV Ags expressed during both B cell transformation and productive viral replication and the frequency of CD8(+) T cells specific for these Ags correlated with cellular EBV copy numbers. In contrast, CD4(+) T cell responses to EBV and T cell responses to human CMV Ags were unchanged, altogether arguing against a defective control of latent EBV infection in RA. Our data show that the regulation of EBV infection is perturbed in RA and suggest that increased EBV-specific effector T cell and Ab responses are driven by an elevated EBV load in RA.     

High synovial expression of the inhibitory FcgammaRIIb in rheumatoid arthritis

Activating Fc gamma receptors (FcgammaRs) have been identified as having important roles in the inflammatory joint reaction in rheumatoid arthritis (RA) and murine models of arthritis. However, the role of the inhibitory FcgammaRIIb in the regulation of the synovial inflammation in RA is less known. Here we have investigated synovial tissue from RA patients using a novel monoclonal antibody (GB3) specific for the FcgammaRIIb isoform. FcgammaRIIb was abundantly expressed in synovia of RA patients, in sharp contrast to the absence or weak staining of FcgammaRIIb in synovial biopsies from healthy volunteers. In addition, the expression of FcgammaRI, FcgammaRII and FcgammaRIII was analyzed in synovia obtained from early and late stages of RA. Compared with healthy synovia, which expressed FcgammaRII, FcgammaRIII but not FcgammaRI, all activating FcgammaRs were expressed and significantly up-regulated in RA, regardless of disease duration. Macrophages were one of the major cell types in the RA synovium expressing FcgammaRIIb and the activating FcgammaRs. Anti-inflammatory treatment with glucocorticoids reduced FcgammaR expression in arthritic joints, particularly that of FcgammaRI. This study demonstrates for the first time that RA patients do not fail to up-regulate FcgammaRIIb upon synovial inflammation, but suggests that the balance between expression of the inhibitory FcgammaRIIb and activating FcgammaRs may be in favour of the latter throughout the disease course. Anti-inflammatory drugs that target activating FcgammaRs may represent valuable therapeutics in this disease.     

Characterization and functional consequences of underexpression of clusterin in rheumatoid arthritis

We previously compared by microarray analysis gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) tissues. Among the set of genes identified as a molecular signature of RA, clusterin (clu) was one of the most differentially expressed. In the present study we sought to assess the expression and the role of CLU (mRNA and protein) in the affected joints and in cultured fibroblast-like synoviocytes (FLS) and to determine its functional role. Quantitative RT-PCR, Northern blot, in situ hybridization, immunohistochemistry, and Western blot were used to specify and quantify the expression of CLU in ex vivo synovial tissue. In synovial tissue, the protein was predominantly expressed by synoviocytes and it was detected in synovial fluids. Both full-length and spliced isoform CLU mRNA levels of expression were lower in RA tissues compared with OA and healthy synovium. In synovium and in cultured FLS, the overexpression of CLU concerned all protein isoforms in OA whereas in RA, the intracellular forms of the protein were barely detectable. Transgenic overexpression of CLU in RA FLS promoted apoptosis within 24 h. We observed that CLU knockdown with small interfering RNA promoted IL-6 and IL-8 production. CLU interacted with phosphorylated IkappaBalpha. Differential expression of CLU by OA and RA FLS appeared to be an intrinsic property of the cells. Expression of intracellular isoforms of CLU is differentially regulated between OA and RA. We propose that in RA joints, high levels of extracellular CLU and low expression of intracellular CLU may enhance NF-kappaB activation and survival of the synoviocytes.     

Molecular profile of synovial fibroblasts in rheumatoid arthritis depends on the stage of proliferation

The aim of this study was to explore the molecular profile of proliferating rheumatoid arthritis synovial fibroblasts (RA-SF). Total RNA was extracted from two cultures of RA-SF (low-density [LD] proliferating cells and high-density [HD] nonproliferating cells) and suppression subtractive hybridization was performed to compare differential gene expression of these two cultures. Subtracted cDNA was subcloned, and nucleotide sequences were analyzed to identify each clone. Differential expression of distinct clones was confirmed by semiquantitative RT-PCR. The expression of certain genes in synovial tissues was examined by in situ hybridization. In both LD and HD cells, 44 clones were upregulated. Of the 88 total clones, 46 were identical to sequences that have previously been characterized. Twenty-nine clones were identical to cDNAs that have been identified, but with unknown functions so far, and 13 clones did not show any significant homology to sequences in GenBank (NCBI). Differential expression of distinct clones was confirmed by RT-PCR. In situ hybridization showed that certain genes, such as S100A4, NFAT5, unr and Fbx3, were also expressed predominantly in synovial tissues from patients with RA but not from normal individuals. The expression of distinct genes in proliferating RA-SF could also be found in RA synovium, suggesting that these molecules are involved in synovial activation in RA. Most importantly, the data indicate that the expression of certain genes in RA-SF depends on the stage of proliferation; therefore, the stage needs to be considered in any analysis of differential gene expression in SF.     

Clonal expansions in acute EBV infection are detectable in the CD8 and not the CD4 subset and persist with a variable CD45 phenotype

We have applied a sensitive global analysis of TCR heterogeneity to compare clonal dynamics of CD4(+) and CD8(+) T cells in acute infectious mononucleosis. Using this approach, we are able to identify a broad representation of the total virus-specific population without the bias of in vitro culture and then to track their phenotype and fate by their unique molecular footprint. We demonstrate a large number of Ag-driven clones using different TCRs in the acute phase, all CD8(+). The diverse large clones generated in the CD8 subset in response to this virus contrast with the complete lack of detectable clonal expansion in the CD4 compartment. Many of the same clones remain detectable in directly ex vivo CD8(+) T cells for at least a year after resolution of infectious mononucleosis, although the clone size is reduced. Thus, memory CD8 cells following EBV infection persist at relatively high circulating frequency and represent a subset of the large range of clonotypes comprising the acute effectors. Separation of samples into CD45RA (naive) and CD45RO (memory) fractions shows the accumulation of identical CDR3 region defined clonotypes in both CD45RO and CD45RA fractions and sequencing confirms that dominant long-lived monoclonal expansions can reside in the CD45RA pool.     

High synovial expression of the inhibitory FcγRIIb in rheumatoid arthritis

Activating Fc gamma receptors (FcγRs) have been identified as having important roles in the inflammatory joint reaction in rheumatoid arthritis (RA) and murine models of arthritis. However, the role of the inhibitory FcγRIIb in the regulation of the synovial inflammation in RA is less known. Here we have investigated synovial tissue from RA patients using a novel monoclonal antibody (GB3) specific for the FcγRIIb isoform. FcγRIIb was abundantly expressed in synovia of RA patients, in sharp contrast to the absence or weak staining of FcγRIIb in synovial biopsies from healthy volunteers. In addition, the expression of FcγRI, FcγRII and FcγRIII was analyzed in synovia obtained from early and late stages of RA. Compared with healthy synovia, which expressed FcγRII, FcγRIII but not FcγRI, all activating FcγRs were expressed and significantly up-regulated in RA, regardless of disease duration. Macrophages were one of the major cell types in the RA synovium expressing FcγRIIb and the activating FcγRs. Anti-inflammatory treatment with glucocorticoids reduced FcγR expression in arthritic joints, particularly that of FcγRI. This study demonstrates for the first time that RA patients do not fail to up-regulate FcγRIIb upon synovial inflammation, but suggests that the balance between expression of the inhibitory FcγRIIb and activating FcγRs may be in favour of the latter throughout the disease course. Anti-inflammatory drugs that target activating FcγRs may represent valuable therapeutics in this disease.     

Phenotypic and functional heterogeneity of EBV epitope-specific CD8+ T cells

High frequencies of EBV-specific CD8(+) T cells have been detected during acute EBV infection, yet persistent infection inevitably results. To address this issue, we characterized the phenotype and function of epitope-specific CD8(+) T cell populations from presentation with acute through latent infection. Considerable phenotypic and functional heterogeneity within, as well as between, two different epitope-specific populations was observed over time following acute infection. B7 EBV-encoded nuclear Ag (EBNA)-3A-specific CD8(+) T cells expressed only CD45RO from acute through latent EBV infection. A2 BMLF-1-specific CD8(+) T cells expressed CD45RO during acute infection and either CD45RA or CD45RO during latent EBV infection. This difference in CD45 isoform expression between the two epitope-specific populations did not translate into differences in perforin content, the ability to produce IFN-gamma, or the ability to proliferate in response to Ag in vitro. In individuals with latent EBV infection, the frequencies of A2 BMLF-1- or B7 EBNA-3A-specific CD8(+) T cells that expressed CD45RA, CD45RO, CD62 ligand, CCR7, and perforin were stable over time. However, the expression of CD62 ligand and CCR7 was significantly higher among EBNA-3A-specific CD8(+) T cells than among BMLF-1-specific CD8(+) T cells. Further work is necessary to understand how phenotypic and functional differences between EBV epitope-specific CD8(+) T cells are related to the biology of the virus and to the equilibrium between the virus and the host during persistent infection.     

Impaired transporter associated with antigen processing-dependent peptide transport during productive EBV infection

Human herpesviruses, including EBV, persist for life in infected individuals. During the lytic replicative cycle that is required for the production of infectious virus and transmission to another host, many viral Ags are expressed. Especially at this stage, immune evasion strategies are likely to be advantageous to avoid elimination of virus-producing cells. However, little is known about immune escape during productive EBV infection because no fully permissive infection model is available. In this study, we have developed a novel strategy to isolate populations of cells in an EBV lytic cycle based on the expression of a reporter gene under the control of an EBV early lytic cycle promoter. Thus, induction of the viral lytic cycle in transfected EBV(+) B lymphoma cells resulted in concomitant reporter expression, allowing us, for the first time, to isolate highly purified cell populations in lytic cycle for biochemical and functional studies. Compared with latently infected B cells, cells supporting EBV lytic cycle displayed down-regulation of surface HLA class I, class II, and CD20, whereas expression levels of other surface markers remained unaffected. Moreover, during lytic cycle peptide transport into the endoplasmic reticulum, was reduced to <30% of levels found in latent infection. Because steady-state levels of TAP proteins were unaffected, these results point toward EBV-induced interference with TAP function as a specific mechanism contributing to the reduced levels of cell surface HLA class I. Our data implicate that EBV lytic cycle genes encode functions to evade T cell recognition, thereby creating a window for the generation of viral progeny.     

Involvement of PDCD5 in the regulation of apoptosis in fibroblast-like synoviocytes of rheumatoid arthritis

Apoptosis is reduced in the synovial tissue of patients with rheumatoid arthritis (RA), possibly due to decreased expression of pro-apoptotic genes. Programmed Cell Death 5 (PDCD5) has been recently identified as a protein that mediates apoptosis. Although PDCD5 is down-regulated in many human tumors, the role of PDCD5 in RA has not been investigated. Here we report that reduced levels of PDCD5 mRNA and protein are detected in RA synovial tissue (ST) and fibroblast-like synoviocytes (FLS) than in tissue and cells from patients with osteoarthritis (OA). We also report differences in the PDCD5 expression pattern in tissues from patients with these two types of arthritis. PDCD5 showed a scattered pattern in rheumatoid synovium compared with OA, in which the protein labeling was stronger in the synovial lining layer than in the sublining. We also observed increased expression and nuclear translocation of PDCD5 in RA patient-derived FLS undergoing apoptosis. Finally, overexpression of PDCD5 led to enhanced apoptosis and activation of caspase-3 in triptolide-treated FLS. We propose that PDCD5 may be involved in the pathogenesis of RA. These data also suggest that PDCD5 may serve as a therapeutic target to enhance sensitivity to antirheumatic drug-induced apoptosis in RA.     

Dendritic cells provide a potential link between smoking and inflammation in rheumatoid arthritis

Introduction

Smoking increases the risk of developing rheumatoid arthritis (RA) and affects the severity of established RA. Smoking can impact on Th17 lymphocyte differentiation and function through activation of the aryl hydrocarbon receptor (AHR), a process with implications for the pathogenic mechanisms in RA that involve the cytokine, interleukin (IL)-17A. The objective of this study was to establish any effect of smoking on the inflammatory tissue lesions of rheumatoid arthritis via the AHR and IL-17A.

Methods

Twenty synovial and eighteen subcutaneous nodule tissue samples from 31 patients with RA were studied. Patient smoking status at the time of tissue collection was established. Expression of AHR, CYP1A1, AHRR, IL6, IL17A, IL17F, IL22, IL23, IL23R, IFNG, TBX21, IDO1 and FOXP3 genes were assessed in tissues and cultured cells using real-time PCR. Two-colour immunofluorescence was used to co-localise AHR and CYP1A1 protein in synovial tissues. The response of monocytes and monocyte-derived dendritic cells (mo-DCs) to the AHR agonist, benzo(a)pyrene (BaP) was compared in vitro.

Results

AHR gene expression was demonstrated in rheumatoid synovial tissues and nodules with significantly greater expression in synovia. Expression was not influenced by smoking in either tissue. Evidence of AHR activation, indicated by CYP1A1 and AHRR gene expression, was found only in synovia from patients who smoked. However, IL17A gene expression was lower in synovia from smokers. TBX21 and FOXP3 expression was not affected by smoking. Within the synovial tissues of smokers the principal cell type with evidence of AHR activation was a subset of synovial DCs. This observation was consistent with the sensitivity of human mo-DCs to BaP stimulation demonstrated in vitro. Exposure to BaP affected mo-DC function as demonstrated by decreased IL6 expression induced by PolyI:C, without affecting indoleamine 2,3 dioxygenase (IDO)1 expression.

Conclusion

Our findings show that one effect of smoking on inflamed rheumatoid synovial tissue involves activation of the AHR pathway. A subset of synovial DCs is important in the response to cigarette smoke. The potential for smoking to affect DC behaviour in joint tissues has relevance to both early and late phases of RA pathogenesis and warrants further investigation.     

IL-1 receptor antagonist protein production and gene expression in rheumatoid arthritis and osteoarthritis synovium.

IL-1 can participate in the perpetuation of arthritis through direct stimulation of synoviocytes and augmentation of matrix degradation. Hence, local production of the IL-1R antagonist protein (IRAP) might be an important negative feedback signal that regulates synovitis. We assessed synovial IRAP production in synovia from 30 individuals, by using a specific mAb and the immunoperoxidase staining method. IRAP was detected in 11 of 12 rheumatoid arthritis (RA) synovial tissues (ST) and was located primarily in the sublining, particularly in perivascular regions enriched for macrophages. Some staining was observed in the intimal lining of the synovium, although this was significantly less than in the sublining (p less than 0.05). Nine of 12 osteoarthritis (OA) tissues were positive for IRAP. In contrast to RA, the staining was observed primarily in the synovial lining in OA, with only minimal sublining IRAP being detected. Synovia from four patients without arthritis were negative (three autopsy specimens and one post-traumatic sample). Of the other two patients with miscellaneous diagnoses, one sample was negative (tenosynovitis) and one was positive (seronegative inflammatory arthritis) (sublining). Studies of serial sections and double-immunostaining experiments indicated that macrophages are the major cells containing immunoreactive IRAP. IRAP gene expression in vivo was determined by performing in situ hybridization on ST from 17 arthritis patients. RNA sense IRAP probes did not hybridize to any tissues. Anti-sense IRAP probes bound to two of nine RA tissues, two of six OA tissues, one of one seronegative inflammatory arthropathy tissue, and none of one flexor tenosynovitis tissue. As with immunoreactive protein, IRAP mRNA was primarily localized to cells in the synovial lining in OA but was more prominent in perivascular lymphoid aggregates in RA and seronegative inflammatory arthropathy. Northern blot analysis was performed on RNA isolated from nine ST. The appropriately sized IRAP band was identified in six of nine samples (five of six RA and one of three OA). Supernatants from cultured RA and OA ST cells contained immunoreactive and biologically active IRAP. Hence, IRAP gene expression and protein production occur in RA and OA synovium, albeit in different distributions.     

Long-term MHC class II presentation of the EBV lytic protein BHRF1 by EBV latently infected b cells following capture of BHRF1 antigen

Although T lymphocytes are considered essential for the control of EBV infection, it remains uncertain how this control occurs. We previously reported unexpected killing of EBV-transformed B-lymphoblastoid cells (LCLs) that did not express BHRF1 by CD4+ T cells specific for BHRF1, an EBV lytic cycle protein. Using LCLs transformed with an EBV mutant, in which the BHRF1 gene was deleted, we showed that killing of latently infected cells through the recognition of a protein produced during the lytic cycle is due to transfer of BHRF1 from lytically infected to latently infected cells, which occurs in culture. Accordingly, LCLs efficiently presented exogenous BHRF1 protein. Furthermore, we present evidence for persistence of captured BHRF1 Ag for several days. Due to this long-term persistence, repeated loading of suboptimal amounts of BHRF1 led to accumulation of BHRF1 Ags in LCLs and, ultimately, to their optimal recognition by BHRF1-specific CD4+ T cells. These results unveil an MHC class II-dependent pathway that could be important for the control of EBV latent infection through recognition of lytic cycle Ags.     

EBV阳性胃癌细胞系与阴性胃癌细胞系全基因组表达谱差异比较

目的：探讨EB病毒阳性胃癌细胞系与EB 病毒阴性胃癌细胞系全基因组表达谱的差异。方法：利用Agilent 人类全基因组 表达谱芯片技术在3 种EB病毒阳性胃癌细胞系和3 种EB 病毒阴性胃癌细胞系中筛选EB病毒相关胃癌肿瘤相关基因。选取6 条差异表达基因,应用实时荧光定量PCR验证芯片结果的可靠性。结果：聚类热图及矩阵图分析显示,EB病毒阳性胃癌细胞系与 阴性胃癌细胞系之间表达谱存在明显差异。差异表达基因涉及多种生物学过程。选取的6 条差异基因进行验证,其表达趋势与芯 片结果一致。结论：感染EB病毒可能会改变肿瘤相关基因的表达,进而在EB病毒相关胃癌的发生、发展及预后中发挥作用。筛 选出的肿瘤相关基因涉及多种生物学过程,提示多基因及相关基因的变异参与了EB病毒相关胃癌的形成。     

Peptides presented by HLA-DR molecules in synovia of patients with rheumatoid arthritis or antibiotic-refractory Lyme arthritis

Disease-associated HLA-DR molecules, which may present autoantigens, constitute the greatest genetic risk factor for rheumatoid arthritis (RA) and antibiotic-refractory Lyme arthritis (LA). The peptides presented by HLA-DR molecules in synovia have not previously been defined. Using tandem mass spectrometry, rigorous database searches, and manual spectral interpretation, we identified 1,427 HLA-DR-presented peptides (220-464 per patient) from the synovia of four patients, two diagnosed with RA and two diagnosed with LA. The peptides were derived from 166 source proteins, including a wide range of intracellular and plasma proteins. A few epitopes were found only in RA or LA patients. However, two patients with different diseases who had the same HLA allele had the largest number of epitopes in common. In one RA patient, peptides were identified as originating from source proteins that have been reported to undergo citrullination under other circumstances, yet neither this post-translational modification nor anti-cyclic citrullinated peptide antibodies were detected. Instead, peptides with the post-translational modification of S-cysteinylation were identified. We conclude that a wide range of proteins enter the HLA-DR pathway of antigen-presenting cells in the patients' synovial tissue, and their HLA-DR genotype, not the disease type, appears to be the primary determinant of their HLA-DR-peptide repertoire. New insights into the naturally presented HLA-DR epitope repertoire in target tissues may allow the identification of pathogenic T cell epitopes, and this could lead to innovative therapeutic interventions.