J. S. Hill Gaston

Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility

Ankylosing spondylitis is a common form of inflammatory arthritis predominantly affecting the spine and pelvis that occurs in approximately 5 out of 1,000 adults of European descent. Here we report the identification of three variants in the RUNX3, LTBR-TNFRSF1A and IL12B regions convincingly associated with ankylosing spondylitis (P < 5 × 10−8 in the combined discovery and replication datasets) and a further four loci at PTGER4, TBKBP1, ANTXR2 and CARD9 that show strong association across all our datasets (P < 5 × 10−6 overall, with support in each of the three datasets studied). We also show that polymorphisms of ERAP1, which encodes an endoplasmic reticulum aminopeptidase involved in peptide trimming before HLA class I presentation, only affect ankylosing spondylitis risk in HLA-B27–positive individuals. These findings provide strong evidence that HLA-B27 operates in ankylosing spondylitis through a mechanism involving aberrant processing of antigenic peptides.

Frequency and phenotype of peripheral blood Th17 cells in ankylosing spondylitis and rheumatoid arthritis

OBJECTIVE To analyze the frequency, surface phenotype, and cytokine secretion of CD4+ T cells in peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis (AS) compared with both healthy control subjects and patients with rheumatoid arthritis (RA). METHODS Eight-color flow cytometry was used to analyze the surface phenotype and cytokine production of PBMCs from 20 patients with AS, 12 patients with RA, and 16 healthy control subjects, following stimulation ex vivo with phorbol myristate acetate and ionomycin for 5 hours. Secretion of interleukin-17 (IL-17) by PBMCs was measured by enzyme-linked immunosorbent assay, following stimulation with anti-CD3/CD28 for 4 days. RESULTS The percentages of IL-17-positive CD4+ T cells and IL-22-positive CD4+ T cells were increased in the PBMCs of both patients with AS and patients with RA compared with healthy control subjects, whereas there were no differences in the percentages of interferon-gamma (IFNgamma)-positive or IL-10-positive CD4+ T cells. Likewise, concentrations of IL-17 in supernatants from patients with AS were significantly higher compared with those from healthy control subjects. In patients with RA, the concentrations of IL-17 were increased but not significantly. There was a correlation between the percentages of IL-17-positive CD4+ T cells detected in PBMCs and the amounts of IL-17 in culture supernatants (r=0.414, P=0.0034). All IL-17-producing cells were CD4+CD45RO+; most expressed both CCR6 and CCR4, but only 50% expressed the IL-23 receptor (IL-23R). Nevertheless, there was a positive relationship between the percentage of IL-23R-positive CD4+ T cells and the frequency of IL-17-positive CD4+ T cells or IL-22-positive CD4+ T cells (r=0.57, P<0.0001 and r=0.46, P=0.001, respectively). A significant proportion of cells that produced IL-17 also produced IL-22 and IFNgamma, but none produced IL-10. CONCLUSION The frequencies of IL-17-positive and IL-22-positive CD4+ T cells were increased in PBMCs from patients with AS and patients with RA, resulting in secretion of higher quantities of IL-17 by PBMCs following stimulation. These data support the hypothesis that Th17 cells, particularly when present in excess of IL-10-producing cells, are involved in the pathogenesis of inflammatory arthritis.

Endoplasmic reticulum stress-induced transcription factor, CHOP, is crucial for dendritic cell IL-23 expression

The endoplasmic reticulum (ER) stress response detects malfunctions in cellular physiology, and microbial pattern recognition receptors recognize external threats posed by infectious agents. This study has investigated whether proinflammatory cytokine expression by monocyte-derived dendritic cells is affected by the induction of ER stress. Activation of ER stress, in combination with Toll-like receptor (TLR) agonists, markedly enhanced expression of mRNA of the unique p19 subunit of IL-23, and also significantly augmented secretion of IL-23 protein. These effects were not seen for IL-12 secretion. The IL-23 gene was found to be a target of the ER stress-induced transcription factor C/EBP homologous protein (CHOP), which exhibited enhanced binding in the context of both ER stress and TLR stimulation. Knockdown of CHOP in U937 cells significantly reduced the synergistic effects of TLR and ER stress on IL-23p19 expression, but did not affect expression of other LPS-responsive genes. The integration of ER stress signals and the requirement for CHOP in the induction of IL-23 responses was also investigated in a physiological setting: infection of myeloid cells with Chlamydia trachomatis resulted in the expression of CHOP mRNA and induced the binding of CHOP to the IL-23 promoter. Furthermore, knockdown of CHOP significantly reduced the expression of IL-23 in response to this intracellular bacterium. Therefore, the effects of pathogens and other environmental factors on ER stress can profoundly affect the nature of innate and adaptive immune responses.

Clinical phenotype is related to HLA genotype in the peripheral arthropathies of inflammatory bowel disease

BACKGROUND & AIMS The detection of phenotype-determining genes as opposed to disease susceptibility genes requires precise phenotypic characterization of patients. Peripheral arthropathies in inflammatory bowel disease (IBD) are well recognized and are classified with the HLA-B*27-related spondyloarthropathies by the European Spondyloarthropathy Study Group. However, previous HLA studies in IBD have only shown this association with axial disease rather than peripheral arthropathy. We recently reported a clinical classification that describes 2 types of peripheral arthropathy, distinguished by their natural history and articular distribution. We now report the results of immunogenetic studies in these patients and compare them with other spondyloarthropathies. METHODS IBD patients with type 1 (n = 57) and type 2 (n = 45) peripheral arthropathy were identified by case note review and questionnaire. Patients and 603 controls from Oxfordshire were assigned HLA-A, -B, -C, -DR, and -DQ genotypes by sequence-specific primer polymerase chain reaction. Patient results were compared with controls (corrected for multiple comparisons), then with each other in light of existing hypotheses. The results were compared with those of a cohort of 30 patients with postenteric reactive arthritis (ReA) and 16 patients with IBD-associated ankylosing spondylitis (IBD-AS). RESULTS Type 1 arthropathy was associated with HLA-DRB1*0103 (DR103; a rare subtype of DR1) in 33% (P < 0.0001; relative risk [RR], 12.1), B*35 in 30% (P = 0.01; RR, 2.2), and B*27 in 26% (P = 0. 001; RR, 4.0). In contrast, type 2 was associated with HLA-B*44 in 62% (P = 0.01; RR, 2.1). Similar significant associations to type 1 arthropathy were found in ReA, except that the HLA-B*27 association was significantly stronger and an association was found with DRB1*0101 (DR1) in 43% (P = 0.001; RR, 2.2). IBD-AS was associated only with HLA-B*27 and DRB1*0101. CONCLUSIONS These data suggest that the clinical classification into type 1 and type 2 arthropathies describes immunogenetically distinct entities and establish that in polygenic disorders, genes may determine clinical phenotype without conferring overall disease susceptibility (in this case, HLA genes). Type 1 arthropathy is clinically and immunogenetically similar to the spondyloarthropathies, but different HLA associations may define phenotypically distinct groups. Type 2 arthropathy has different HLA associations and may have a different etiology. Further studies are now required to confirm these associations and to elucidate the different pathogenetic mechanisms.

Arthritis associated with enteric infection

Reactive arthritis is classically seen following infection with enteric pathogens such as Yersinia, Salmonella, Campylobacter and Shigella. Inflammatory arthritis has also been described following other enteric infection with organisms such as Clostridium difficile, Brucella and Giardia. Furthermore, arthritis is seen in Whipples disease, caused by the actinomycete Tropheryma whippelii. This chapter reviews the current understanding of these conditions (with the exception of Brucella, which is discussed in a subsequent chapter). The epidemiology is reviewed, and the contribution of both host and organism to the aetiology and pathogenesis is discussed with particular discussion of the role of HLA-B27 in host susceptibility. Recent work exploring evidence for traffic of pathogenic organisms to the joint is highlighted. A practical approach to the diagnosis and management of the condition is then formulated based, where possible, on clinical trial evidence.

Autoreactive human peripheral blood CD8+ T cells with a regulatory phenotype and function

Despite substantial advances in our understanding of CD4+CD25+ regulatory T cells, a possible equivalent regulatory subset within the CD8+ T cell population has received less attention. We now describe novel human CD8+/TCRαβ+ T cells that have a regulatory phenotype and function. We expanded and cloned these cells using autologous LPS‐activated dendritic cells. The clones were not cytolytic, but responded in an autoreactive HLA class I‐restricted fashion, by proliferation and production of IL‐4, IL‐5, IL‐13 and TGFβ1, but not IFN‐γ. They constitutively expressed CD69 and CD25 as well as molecules associated with CD4+CD25+ regulatory T cells, including cytotoxic T lymphocyte‐associated antigen‐4 (CTLA‐4) and Foxp3. They suppressed IFN‐γ production and proliferation by CD4+ T cells in vitro in a cell contact‐dependent manner, which could be blocked using a CTLA‐4‐specific mAb. They were more readily isolated from patients with ankylosing spondylitis and may therefore be up‐regulated in response to inflammation. We suggest that they are the CD8+ counterparts of CD4+CD25+ regulatory T cells. They resemble recently described CD8+ regulatory cells in the rat that were able to abrogate graft‐versus‐host disease. Likewise, human HLA‐restricted CD8+ regulatory T cells that can be cloned and expanded in vitro may have therapeutic applications.

The Recognition of HLA-B27 by Human CD4+ T Lymphocytes

HLA-B27 transgenic animal models suggest a role for CD4+ T lymphocytes in the pathogenesis of the spondyloarthropathies, and murine studies have raised the possibility that unusual forms of B27 may be involved in disease. We demonstrate that CD4+ T cells capable of recognizing B27 can be isolated from humans by coculture with the MHC class II-negative cell line T2 transfected with B27. These CD4+ T cells recognize a panel of B27-transfected cell lines that are defective in Ag-processing pathways, but not the nontransfected parental cell lines, in a CD4-dependent fashion. Inhibition of responses by the MHC class I-specific mAb w6/32 and the B27 binding mAb ME1 implicates the recognition of a form of B27 recognized by both of these Abs. We suggest that B27-reactive CD4+ T cells may be pathogenic in spondyloarthropathies, particularly if factors such as infection influence expression of abnormal forms of B27.

CD27 expression discriminates between regulatory and non-regulatory cells after expansion of human peripheral blood CD4+ CD25+ cells

It is clear that regulatory T cells (Treg) have an important role in preventing autoimmunity and modulating responses to pathogens. Full characterization of Treg cell function in human patients would be greatly facilitated by practical methods for expanding Treg in vitro. Methods for expansion have been reported but whether expression of surface and intracellular markers associated with freshly isolated Treg following expansion correlates with the maintenance of function is unclear. Our aim was to investigate the various methods of expansion and to correlate regulatory activity with expression of these markers. We show that, of the markers associated with freshly isolated Treg, only CD27 expression correlated with regulatory activity and could be used to isolate cells with regulatory activity from lines expanded from CD4+ CD25+ cells. Also, cells expressing high levels of the transcription factor forkhead box P3 (Foxp3) were confined to the CD27+ population within these lines. Expression of CD27 by cells in lines expanded from CD4+ CD25– cells varied depending on the stimulus used for expansion, but these lines did not have significant regulatory activity even when the CD27+ cells were tested. Analysis of synovial CD4+ CD25+ cells from reactive arthritis patients revealed that they were predominantly CD27 positive. This also applied to CD25high and CD25intermediate CD4+ cells, despite their reported different abilities to regulate. We conclude that, whilst CD27 is useful for identifying Treg in the cell lines obtained after expansion of CD4+ CD25+ cells, its expression may not reliably identify the Treg cell population in other T‐cell populations such as those found in joints.

Uptake and processing of Chlamydia trachomatis by human dendritic cells

Chlamydia trachomatis (CT) causes several sexually transmitted diseases. In 2 – 5 % of cases, CT infection leads to the development of reactive arthritis. Dendritic cells (DC) are central in T cell priming and the induction of antigen specific immunity. Here we have studied the uptake and processing of CT serovar L2 by human DC, and their ability to present CT antigens to bothCD4+ and CD8+ T cells. We show that the entry of CT was mediated by the attachment of CT to heparan sulfates and could be inhibited by heparin. There was no inhibition of uptake by an agent which blocks micropinocytosis. Infecting DC with CT led to their activation and the production of IL‐12 and TNF‐α but not IL‐10. Following invasion, CT was confined to distinct vacuoles which were visualized with anti‐CT antibodies using confocal microscopy. Unlike with epithelial cells, these vacuoles did not develop into characteristic inclusion bodies. In the first 48 h, CT+ vacuoles were negative for Lamp‐1 and MHC class II. Despite no obvious co‐localization between CT vacuoles and MHC loading compartments, infected DC efficiently presented CT antigens to CD4+ T cells. Infected DC also expanded CT specific CD8+ T cells, allowing us to generate a number of CT‐reactive CD8+ T cell clones. There is still controversy about the importance of chlamydia‐specific CD8+ T cell responses in patients with arthritis. This is largely due to the difficulties in studying CTL responses at the clonal level. The use of DC as antigen‐presenting cells should enable more detailed characterization of these CTL responses.

Investigation of infectious agents associated with arthritis by reverse transcription PCR of bacterial rRNA

In reactive and postinfectious arthritis the joints are generally sterile but the presence of bacterial antigens and nucleic acids has been reported. To investigate whether organisms traffic to affected joints in these conditions, we performed reverse transcription PCR using universal primers to amplify any bacterial 16S rRNA sequences present in synovial fluid. Bacterial sequences were detected in most cases, even after treatment of the synovial fluid with DNase, implying the presence of bacterial RNA and therefore of transcriptionally active bacteria. Analysis of a large number of sequences revealed that, as reported in rheumatoid arthritis, most were derived from gut and skin commensals. Organisms known to have triggered arthritis in each case were not found by sequencing the products obtained using universal primers, but could in some cases be shown to be present by amplifying with species specific primers. This was the case for Yersinia pseudotuberculosis and Chlamydia trachomatis. However, in arthritis thought to be related to Campylobacter infection the sequences obtained were not from Campylobacter jejuni or C. coli, but from other Campylobacter spp. that are not known to be associated with reactive arthritis and are probably present as commensals in the gut. We conclude that although rRNA from reactive arthritis associated organisms can be detected in affected joints, bacterial RNA from many other bacteria is also present, as was previously noted in studies of other forms of inflammatory arthropathy.