ß-Glucan Size Controls Dectin-1-Mediated Immune Responses in Human Dendritic Cells by Regulating IL-1ß Production.

Dectin-1/CLEC7A is a pattern recognition receptor that recognizes ß-1,3 glucans, and its stimulation initiates signaling events characterized by the production of inflammatory cytokines from human dendritic cells (DCs) required for antifungal immunity. ß-glucans differ greatly in size, structure, and ability to activate effector immune responses from DC; as such, small particulate ß-glucans are thought to be poor activators of innate immunity. We show that ß-glucan particle size is a critical factor contributing to the secretion of cytokines from human DC; large ß-glucan-stimulated DC generate significantly more IL-1ß, IL-6, and IL-23 compared to those stimulated with the smaller ß-glucans. In marked contrast, the secretion of TSLP and CCL22 were found to be insensitive to ß-glucan particle size. Furthermore, we show that the capacity to induce phagocytosis, and the relative IL-1ß production determined by ß-glucan size, regulates the composition of the cytokine milieu generated from DC. This suggests that ß-glucan particle size is critically important in orchestrating the nature of the immune response to fungi.

Cytokine secretion by pathogen recognition receptor-stimulated dendritic cells in rheumatoid arthritis and ankylosing spondylitis.

OBJECTIVE: Interleukin 23 (IL-23) plays a major role in differentiation and survival of IL-17-secreting CD4+ Th17 cells. Having noted a higher frequency of Th17 cells in ankylosing spondylitis (AS) and rheumatoid arthritis (RA) than in healthy donors (HD), we investigated whether IL-23 secretion is increased in these conditions. METHODS: Monocyte-derived dendritic cells (moDC) were obtained from peripheral blood of 17 HD, 16 patients with RA, and 30 patients with AS, and stimulated with ligands for several pathogen recognition receptors. Messenger RNA (mRNA) expression and cytokine secretion were analyzed by real-time polymerase chain reaction and ELISA, respectively. RESULTS: The combination of ligands for Toll-like receptors (TLR) 7/8 and TLR3 led to synergistic secretion of both IL-23 and IL-12p70 from all subjects; similar synergy was seen with TLR2 ligands and curdlan. However, for both combinations, moDC from patients with RA produced significantly lower amounts of IL-23 than moDC from patients with AS; in contrast, IL-12p70 secretion did not differ. Similarly, tumor necrosis factor-α, IL-6, and IL-10 were secreted at comparable levels in all subjects, whereas CXCL8 and CCL3 production was actually enhanced in moDC of patients with RA. Equivalent levels of mRNA for both IL-23p19 and IL-12p35 subunits were found in moDC from all donors, suggesting posttranscriptional regulation of IL-23 production in RA. CONCLUSION: Our observations show that IL-23 production is decreased in RA and maintained in AS. Because increased numbers of CD4+IL-17+ T cells are seen in both diseases, these observations imply that there are different mechanisms underlying chronic inflammation in these 2 forms of inflammatory arthritis.

Characterisation of Foxp3 splice variants in human CD4+ and CD8+ T cells--identification of Foxp3Δ7 in human regulatory T cells.

Foxp3 is proposed to play a critical role in the development and function of regulatory T cells. Functional and transgenic studies in mice propose Foxp3 as a "regulatory T cell lineage specification factor" but conflicting data exist in humans. Expression of multiple Foxp3 splice variants in humans represents an additional layer of complexity for this transcription factor and acts as a possible mechanism of regulating protein diversity. We report the identification of a novel splice variant of Foxp3, called Foxp3Δ7, in ex vivo CD4+CD25+ T cells and CD8+ regulatory T cell clones. Foxp3Δ7 lacks the 81bp region that encodes exon 7 of Foxp3, which is a part of the leucine zipper domain of the protein. The three splice variants of Foxp3 namely Foxp3FL, Foxp3Δ2 and Foxp3Δ7 are co-expressed in ex vivo human CD4+CD25+ T cells and CD8+ Treg clones. Stimulation of freshly isolated CD4+CD25+ T cells with anti-CD3 and anti-CD28 antibodies leads to a 140-fold upregulation of Foxp3Δ7 within 24h of stimulation, which is ∼10-fold greater than that observed in stimulated CD4+CD25- T cells. In addition, resting CD8+ Treg cells have decreased expression of Foxp3FL and Foxp3Δ2; however they have a 10-fold higher expression of Foxp3Δ7, in comparison to ex vivo CD4+CD25+ T cells. In order to assess the functional effects of these Foxp3 isoforms, we carried out lentivirus expression studies. All three isoforms were capable of inducing increased levels of CD25 expression in primary human CD4+ T cells, along with a tendency to decreased levels of CD127. Further investigation into pathways that alter the relative proportions of Foxp3 isoforms, and hence their interaction with other transcriptional co-regulators, will help to define the role of Foxp3 isoforms in immune regulation.

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.

Consistent patterns of expression of HLA class I free heavy chains in healthy individuals and raised expression in spondyloarthropathy patients point to physiological and pathological roles.

OBJECTIVES: Major histocompatibility complex class I (MHC-I) proteins exist at the cell surface in antigen presenting forms and as beta2m-independent free heavy chains (FHCs). FHCs have been implicated in spondyloarthritis, but little is known about their expression in healthy individuals. We studied FHC expression on various human cell types, comparing spondyloarthropathy patients with healthy and rheumatoid arthritis (RA) patient controls. METHODS: MHC-I expression was analysed by flow cytometry. FHC levels were normalized for overall MHC-I to generate a relative expression level. Relative FHC levels were analysed for peripheral blood and trophoblast samples from healthy volunteers, RA and spondyloarthropathy patients. Macrophages and dendritic cells were cultured in vitro to analyse changes following activation. Peripheral blood leucocytes from patients with ankylosing spondylitis (AS) and RA were treated with inflammatory stimuli and subsequent alterations in their relative FHC levels were analysed. RESULTS: We found consistent patterns of differential relative FHC expression across lymphocyte subpopulations and particularly high expression on extravillous trophoblast. FHCs were present at higher levels in a reactive arthritis (ReA) population than in healthy controls and RA patients; differences not merely due to the presence of Human Leucocyte Antigen (HLA) B27. Treatment of leucocytes from arthritic patients with bacterial lipopolysaccharide resulted in significant up-regulation of FHC compared with an HLA B27+ control population. CONCLUSIONS: Our findings define normal levels and tissue expression of FHCs, and support the hypothesis that disregulation of heavy chain expression may play a pathogenic role in spondyloarthropathy.

Spondylarthritis-associated and non-spondylarthritis-associated B27 subtypes differ in their dependence upon tapasin for surface expression and their incorporation into the peptide loading complex.

OBJECTIVE: B27 subtypes associated with susceptibility to ankylosing spondylitis (AS), and those reported not to be associated with AS, are found to differ in the amino acids that are known in other HLA class I molecules to alter the requirements for tapasin and incorporation into the peptide loading complex. The purpose of this study was to examine the behavior of B*2704 and B*2705 in comparison with B*2706 and B*2709 during early events in HLA class I antigen expression, and determine if their behavior correlates with disease association. METHODS: Cell lines with nonfunctional tapasin were transiently transfected with different B27 subtypes and their site-directed mutants, and surface expression analyzed by flow cytometry. The association with the peptide loading complex was determined by immunoprecipitation of heterodimeric transporter-associated peptide and analysis of coprecipitated B27. RESULTS: Amino acids at positions 114, 116, and 152 in the different B27 subtypes were shown to perform key roles in defining a requirement for interaction with tapasin. Not all disease-associated alleles were expressed optimally in the absence of tapasin; furthermore, dependence on tapasin for cell surface expression did not correlate with disease association. Although B*2706, which is not associated with disease, exhibited a number of properties different from those of the disease-associated subtypes, these properties were not displayed by the non-disease-associated allele B*2709. CONCLUSION: These results indicate that the ability to exhibit optimal cell surface expression in the absence of tapasin is not a prerequisite for susceptibility to AS.

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 Whipple's 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.

Breaking the rules: the unconventional recognition of HLA-B27 by CD4+ T lymphocytes as an insight into the pathogenesis of the spondyloarthropathies.

Despite extensive research, it remains unclear why a small proportion of HLA- B27(+) individuals develop spondyloarthropathies (SpA). Because the function of HLA-B27, as a major histocompatibility complex (MHC) class I molecule, is peptide presentation to CD8(+) T cells, research has concentrated on the role of HLA-B27 as a restriction element for CD8(+) cytotoxic T lymphocytes in pathogenesis. However, findings in the B27-transgenic animal models, together with the identification of unusual processing and presentation features of HLA-B27, have raised alternative hypotheses for the pathogenic role of HLA-B27. One such hypothesis is that HLA-B27 can be recognized by CD4(+) T lymphocytes. Here we report the identification of such unusual cells, which break the conventional rules of MHC restriction, and propose a model for the role of such CD4(+) T cells in SpA.

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.

Conserved TcR beta chain usage for a single MHC class II-restricted heat shock protein peptide.

Previously we have shown that the T-cell response against the HLA-DR3 (17)-restricted heat shock protein (Mr 65 000)-derived peptide amino acids (aa) 3-13 (hsp65 aa 3-13) is recognized by the exclusive usage of the TRBV5 gene as well as a conserved CDR3 region in a tuberculoid leprosy patient. In the present study we analyzed the TcR of T-cell clones specific for hsp65 aa 3-13 derived from three healthy individuals with a response level similar to that of the leprosy patient. We show that unlike the tuberculoid leprosy patient, healthy high responders have a diverse T-cell response to hsp65 aa 3-13. However, a striking observation was made: even though high responders have a diverse specific TcR repertoire, TRBV5-expressing clones from two healthy individuals could be isolated that were nearly identical to a dominant clone in the tuberculoid leprosy patient. In conclusion, the data show that restriction of TcR specific for an antigen correlates with the presence of that antigen in disease. However, the preferred TcR can also be detected in healthy high responders. A natural infection in vivo, as with the tuberculoid leprosy patient, may be responsible for the observed trimming and preferential outgrowth of a certain TcR.