The interplay between HLA-B27 and ERAP1/ERAP2 aminopeptidases: from anti-viral protection to spondyloarthritis.

The human leukocyte antigen class I gene HLA-B27 is the strongest risk factor for ankylosing spondylitis (AS), a chronic inflammatory arthritic disorder. More recently, the Endoplasmic Reticulum Aminopeptidase (ERAP) 1 and 2 genes have been identified by genome wide association studies (GWAS) as additional susceptibility factors. In the ER, these aminopeptidases trim the peptides to a length suitable to fit into the groove of the major histocompatibility complex (MHC) class I molecules. It is noteworthy that an epistatic interaction between HLA-B27 and ERAP1, but not between HLA-B27 and ERAP2, has been highlighted. However, these observations suggest a paramount centrality for the HLA-B27 peptide repertoire that determines the natural B27 immunological function, i.e. the T cell antigen presentation and, as a by-product, elicits HLA-B27 aberrant behaviours: (i) the misfolding leading to ER stress responses and autophagy and (ii) the surface expression of homodimers acting as ligands for innate immune receptors. In this context, it has been observed that the HLA-B27 carriers, besides being prone to autoimmunity, display a far better surveillance to some viral infections. This review focuses on the ambivalent role of HLA-B27 in autoimmunity and viral protection correlating its functions to the quantitative and qualitative effects of ERAP1 and ERAP2 polymorphisms on their enzymatic activity.

Susceptibility to ankylosing spondylitis but not disease outcome is influenced by the level of HLA-B27 expression, which shows moderate variability over time.

OBJECTIVE: Previous reports have highlighted the relevance of HLA-B27 expression in the pathogenesis of ankylosing spondylitis (AS). The aim of the current study was to estimate the level of HLA-B27 expression on the cell surface of ex vivo monocytes and lymphocytes by a quantitative method and to correlate this with AS disease susceptibility, disease clinical indexes, and the occurrence of acute anterior uveitis (AAU). METHOD: We recruited 32 B27-positive patients with AS and 32 B27-positive healthy normal controls (NCs) for evaluation at different time points. The expression of HLA-B27 molecules was quantified by flow cytometry on ex vivo peripheral blood mononuclear cells (PBMCs). Patients were also evaluated by scores on the Bath AS disease activity (BASDAI), functional (BASFI), and metrology (BASMI) indexes. RESULTS: The expression of HLA-B27 molecules was significantly higher in patients with AS than in B27-matched controls in the case of both monocytes [219K (IQR 174K-308K) vs. 137K (IQR 96K-170K), p < 0.0001] and lymphocytes [82K (IQR 58K-118K) vs. 54K (IQR 44K-61K), p < 0.0001]; AS only vs. AS with AAU: p = 0.744 in monocytes and p = 0.701 in lymphocytes. Comparisons with metrology and functional indexes were also not significant (BASMI: r = 0.05, p = 0.77; BASFI: r = -0.09, p = 0.67). The overexpression of HLA-B27 molecules was stable after 1 week of follow-up. At 3 years follow-up, the variability was moderate and did not correlate with variations in disease activity (BASDAI: r = -0.01, p = 0.92 ns). CONCLUSIONS: The level of HLA-B27 expression in PBMCs correlates with the susceptibility to AS but not with the disease outcome, nor with the occurrence of extra-articular manifestations such as AAU.

Age-dependent association of idiopathic achalasia with vasoactive intestinal peptide receptor 1 gene.

Idiopathic achalasia is a rare disorder of the oesophagus of unknown aetio-pathogenesis characterized by a myenteric inflammation, aperistalsis and insufficient lower oesophageal sphincter relaxation. Vasoactive intestinal peptide (VIP), present in the myenteric plexus, is involved in smooth muscle relaxation and acts as an anti-inflammatory cytokine. The human VIP receptor 1 gene (VIPR1) is highly polymorphic and may play a role in idiopathic achalasia. One hundred and four consecutive patients and 300 random controls from the same geographic area were typed for five SNPs mapping in the VIPR1 gene. Patients with idiopathic achalasia show a significant difference in allele, genotype and phenotype distribution of SNP rs437876 mapping in intron 4. This association, however, was almost entirely due to the group of patients with late disease onset (P = 0.0005). These results strongly suggest that idiopathic achalasia is a heterogeneous disease with a different aetiology in cases with early or late disease onset.

Antiviral treatment for hepatitis C virus infection: effectiveness at general population level in a highly endemic area.

BACKGROUND: Peginterferon plus ribavirin treatment induced a sustained virological response in >50% of HCV-RNA-positive individuals enrolled in published clinical trials. AIM: To determine anti-HCV treatment effectiveness at a general population level. PATIENTS AND METHODS: In 2002, a 1:5 random sample of >11 years old inhabitants of a small Italian town (Cittanova) was invited for HCV screening. HCV-RNA-positive individuals were evaluated for antiviral treatment. RESULTS: 1645 of 1924 invited individuals (85.5%) participated in the screening. 84 HCV-RNA-positive individuals were detected: median age was 65 years (range: 32-87); 67% was infected with genotype 1 or 4. Antiviral treatment was judged unnecessary for 43 (51.2%), due to persistently normal alanine aminotransferases, mild disease at liver biopsy or age >70 years without cirrhosis. Twenty-eight of the remaining 41 patients (68.3%) were ineligible for treatment, because of medical/psychiatric contraindications (42.9%), alcohol/drug abuse (17.9%), decompensated cirrhosis/hepatocellular carcinoma (17.9%), not attending official appointments (10.7%), previous intolerance/non-response to interferon plus ribavirin (10.7%). 5 of 13 eligible patients (38.5%) did not receive treatment (4 refused and 1 accidental death). 3 of 8 treated patients (37.5%) reached a sustained virological response. CONCLUSIONS: Although efficacy of anti-HCV therapy improved in recent years, we found that low eligibility to treatment still limited its effectiveness at general population level in a highly endemic town.

Association of MICA alleles with psoriatic arthritis and its clinical forms. A multicenter Italian study.

OBJECTIVE: Analysis of the association between psoriatic arthritis (PsA) clinical forms and MICA gene transmembrane polymorphisms. METHODS: Patients were classified as having peripheral asymmetric oligoarthritis (AO), peripheral symmetric poly-arthritis (PA) and spondylitis (SP), or disease combinations (PA/SP, OA/SP). Two hundred and twenty-six patients with PsA were typed for MICA exon 5 microsatellite (TM) by heteroduplex analysis and compared with 225 normal controls. RESULTS: MICA-TM microsatellite typing revealed that, among the different clinical forms of PsA, only the combined PA/SP subset shows a significant positive association with MICA-A9 and a lower frequency of MICA-A4, A5 genotype in PsA patients with a decrease, only in the PA/SP cohort, of all MICA-A5 combinations except MICA-A5, -A9. CONCLUSION: These results suggest a role for genes within the HLA region in the pathogenesis of PsA, and reinforce the idea that the different forms of PsA may have heterogeneous genetic basis.

A functional polymorphism of the vasoactive intestinal peptide receptor 1 gene correlates with the presence of HLA-B*2705 in Sardinia.

The association of HLA-B27 with ankylosing spondylitis (AS) is the strongest among all inflammatory diseases. However, the exact role of these molecules in disease pathogenesis is still unknown. The existence of HLA-B27 variants rarely found in patients introduces a further level of complexity. It is now accepted that other genes of minor impact contribute to modify disease susceptibility and these genes might be diverse in different populations depending on the genetic background. We report here a study performed in Sardinia, an outlier population in which two major HLA-B27 subtypes are present, B (*)2705 strongly associated with AS and B (*)2709 which is not, and show the co-occurrence of the B (*)2705 allele with a single nucleotide polymorphism (SNP) mapping at 3'-UTR of the receptor 1 (VIPR1) for the vasoactive intestinal peptide (VIP), a neuropeptide with anti-inflammatory properties. This same SNP is associated with a different kinetics of down-modulation of the VIPR1 mRNA in monocytes after exposure to lipopolysaccharide (P=0.004). This particular setting, HLA-B (*)2705 and a functional polymorphism in VIPR1 gene, might be due to a founder effect or might be the result of a selective pressure. Irrespectively, the consequent downregulation of this receptor in the presence of a 'danger' signal might influence susceptibility to AS.

HLA-B*2709 and lack of susceptibility to sacroiliitis: further support from the clinic.

Interferons (IFN) are well known triggers of immunomediated diseases in genetically predisposed subjects. We describe the unique case of a HLA-B*2709 positive subject who underwent IFN-alpha treatment for essential thrombocythemia and developed arthritis of the proximal interphalangeal joints of the hands but not sacroiliitis. The possible mechanisms of IFN-induced arthritis are discussed.

Evaluation of chromogranin A expression in patients with non-neuroendocrine tumours.

BACKGROUND: Chromogranin A (CgA) is well established as a serum marker for neuroendocrine tumours and has also been associated with some non-neuroendocrine tumours, suggesting a possible role for somatostatin analogues such as octreotide in the treatment of these tumours. OBJECTIVE: The aim of this study was to measure plasma CgA levels in patients with various non-neuroendocrine tumours in order to identify those patients who might benefit from octreotide therapy. METHODS: Plasma CgA levels were tested in 151 patients with metastatic non-neuroendocrine tumours. Patients with highly elevated levels were assessed by OctreoScan scintigraphy to determine their somatostatin receptor status, and those with positive results were offered treatment with the somatostatin analogue octreotide, 20 mg every 4 weeks, and followed up every 3 months. RESULTS: CgA levels were elevated (>18 U/L) in 34/72 patients with breast cancer, 11/21 with lung cancer, 10/28 with gastrointestinal cancer, 7/12 with gynaecological cancer, 6/9 with genitourinary cancer, 5/5 with haematological cancer, and 3/4 with head and neck cancer. Eight patients with CgA levels >150 U/L underwent scintigraphy, five of whom (two colorectal, two prostate, one non-small cell lung cancer [NSCLC]) showed positive results and received treatment with octreotide. Follow-up for a mean 12-16 months showed improvements in biochemical parameters, cenesthesis and quality of life. CONCLUSION: CgA levels were found to be elevated in approximately 50% of patients with non-neuroendocrine tumours. Further studies are required to determine the value of CgA as a marker for non-neuroendocrine tumours and the role of somatostatin analogues as a treatment for these tumour types.

Increased level of HLA-B27 expression in ankylosing spondylitis patients compared with healthy HLA-B27-positive subjects: a possible further susceptibility factor for the development of disease.

OBJECTIVE: In B27 transgenic rats, susceptibility to the development of a spondyloarthropathy-like disease has been shown to correlate with the level of B27 transgene expression on lymphoid cells. The aim of this work was to study HLA-B27 molecule expression in peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis (AS) and from normal controls (NC). METHODS: Twenty B27(+) AS patients and 16 B27(+) NC were studied. HLA-B27 whole molecules and free heavy chains (HCs) and total HLA class I molecules were evaluated at the surface of PBMCs by immunofluorescence and flow cytometry. B27 subtypes were defined with the PCR-SSP (polymerase chain reaction-sequence-specific primer) technique. Cellular activation was evaluated by the expression of CD69, CD25 molecules and interferon gamma (IFN-gamma) production. RESULTS: B27 expression was 55,536.3+/-18,961.0 MESF (molecules of equivalent soluble fluorochromes) units in AS and 25,936.0+/-12,117.5 MESF in NC (P=0.00009), total HLA class I expression was 448,840.2+/-136,293.8 MESF in AS and 533,494.4+/-232,931.1 MESF in NC (not significant), HC expression was 10,593.4+/-6,396.1 MESF in AS and 14,843.0+/-7,544.2 MESF in NC (not significant). The higher B27 expression in the SA group was not due to higher cell activation as it was not correlated with CD69 and CD25 expression in PBMCs or with the level of IFN-gamma. HLA-B27 expression did not correlate with indexes of disease status [Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI) and Bath Ankylosing Spondylitis Metrology Index (BASMI)]. CONCLUSIONS: We found greater expression of HLA-B27 molecules in patients with AS than in healthy subjects. This phenomenon was not accompanied by general up-regulation of HLA class I molecules or by greater expression of classical T-cell activation markers. On this basis we propose that the higher expression of the HLA-B27 molecules is a further predisposing factor for the development of AS.

T cell response to N-formylated peptides in humans.

We present the first evidence of a T lymphocyte response to N-formylated peptides in humans. N-formylated peptide sequences from self (mitochondrial) and foreign (microbial) antigens were used to isolate antigen-specific T cell clones from healthy individuals, including a set of monozygotic twins. The observed response differed from that previously described in mouse (CD4(+) phenotype and MHC class II restriction in humans vs. CD8(+) phenotype and class I restriction in mice). These lymphocytes produce substantial amounts of IFN-gamma. They were isolated in only one of the monozygotic twins, which suggests that their expansion in the healthy immune repertoire is independent of the genetic background. Our result will help in assessing the relevance of N-formylated peptide-specific T cells in protection against infections within the human immune system.

HLA-DP allele-specific T cell responses to beryllium account for DP-associated susceptibility to chronic beryllium disease.

Occupational exposure to small molecules, such as metals, is frequently associated with hypersensitivity reactions. Chronic beryllium (Be) disease (CBD) is a multisystem granulomatous disease that primarily affects the lung, and occurs in approximately 3% of individuals exposed to this element. Immunogenetic studies have demonstrated a strong association between CBD and possession of alleles of HLA-DP containing glutamic acid (Glu) at position 69 in the HLA-DP beta-chain. T cell clones were raised from three patients with CBD in whom exposure occurred 10 and 30 years previously. Of 25 Be-specific clones that were obtained, all were restricted by HLA-DP alleles with Glu at DP beta69. Furthermore, the proliferative responses of the clones were absolutely dependent upon DP beta Glu(69) in that a single amino acid substitution at this position abolished the response. As befits a disease whose pathogenesis involves a delayed type hypersensitivity response, the large majority of Be-specific clones secreted IFN-gamma (Th1) and little or no IL-4 (Th2) cytokines. This study provides insights into the molecular basis of DP2-associated susceptibility to CBD.

CD8(+) T-cell autoreactivity to an HLA-B27-restricted self-epitope correlates with ankylosing spondylitis.

HLA-B27 is highly associated with ankylosing spondylitis (AS), but the mechanism is unknown. Among the HLA-B27 alleles, B*2709, which differs by one amino acid from the susceptible B*2705, is not associated with the disease. Here, we analyze the reactivity, in patients with AS and in healthy controls carrying the B*2709 or B*2705 alleles, to an EBV epitope derived from LMP2 (236-244) and to a sequence-related self-peptide from vasoactive intestinal peptide receptor 1 (VIP1R 400-408). We found that both B*2705(+) and B*2709(+) subjects possess LMP2 236-244-specific, HLA-B27-restricted T cells, whereas only the B*2705(+) individuals respond significantly to VIP1R 400-408. These results prompted us to compare, by IFN-gamma ELISPOT analysis, the T-cell response to VIP1R 400-408 in patients with AS versus B*2705 healthy controls. The data show that VIP1R 400-408-specific reactivity is a major feature of the patients with AS. These findings show, for the first time to our knowledge, a widespread reactivity in patients with AS against a self-epitope that exhibits some features of a putative "arthritogenic" peptide.

The naturally occurring polymorphism Asp116-->His116, differentiating the ankylosing spondylitis-associated HLA-B*2705 from the non-associated HLA-B*2709 subtype, influences peptide-specific CD8 T cell recognition.

HLA-B27 molecules are interesting because of their strong association with ankylosing spondylitis (AS) and reactive arthritis (ReA). A pathogenetic role for these molecules has been postulated in presenting a putative "arthritogenic" peptide to CD8 T cells. The HLA-B*2709 subtype, although differing by a single amino acid (His116-->Asp116) from the widespread and strongly AS-associated subtype HLA-B*2705, is not found in patients. Since residue 116 interacts with the C terminus of the peptide, it is possible that the two subtypes differ in their antigen-presenting features. We show here that CD8 T cells can distinguish the two HLA-B27 subtypes when presenting a same epitope derived from Epstein-Barr virus-latent membrane protein 2. Moreover, alanine scanning mutagenesis analysis revealed that the peptide residues relevant for such recognition are different depending on whether HLA-B*2705 or -B*2709 molecules present the epitope. These results give support to the belief that functional differences determined by subtype-specific polymorphisms can have a pathogenetic relevance and open up a new scenario where subtle modifications within the peptide/HLA ligand might be responsible for the differential association between HLA-B27 subtypes and spondyloarthropathies.

Susceptibility to ankylosing spondylitis correlates with the C-terminal residue of peptides presented by various HLA-B27 subtypes.

Susceptibility to spondyloarthropaties is strongly associated with some HLA-B27 alleles. Evidence suggests a direct pathogenic role for the B27 molecules which possibly present an arthritogenic peptide to the T cells. If this hypothesis is true, B27 subtypes that differ structurally but are disease-associated ought to be capable of presenting such peptide(s), while non-disease-associated ones would not. We have recently described a B27 subtype, B*2709, and shown its absence in ankylosing spondylitis (AS) patients. Here, we show the elution and sequence of peptides from HLA-B*2709 molecules. Similar to other B27 subtypes, these peptides are mainly nonamers with an Arg at position P2. Comparison of the C-terminal anchors of peptides eluted from B*2702 and B*2705 with those eluted from B*2709 reveals that, while B*2702 and B*2705 have a broader specificity, B*2709 molecules appear to only accept C-terminal hydrophobic residues. A common feature shared by the two caucasoid AS-associated subtypes (B*2702 and B*2705) but different from B*2709, is the presence of a Tyr as peptide C-terminal anchor. The substitution of Val for Tyr at the C terminus in one of the eluted peptides greatly reduces the binding to B*2709 molecules. This finding suggests Tyr as a discriminative amino acid allowed at the C terminus of peptides bound to the AS-associated B27 subtypes, but not to those which are not associated with AS.

Relevance of residue 116 of HLA-B27 in determining susceptibility to ankylosing spondylitis.

Ankylosing spondylitis (AS) is an autoimmune disorder strongly associated with HLA-B27. A direct role of B27 molecules in the disease pathogenesis has been postulated, possibly by presenting to T cells an as-yet unidentified arthritogenic peptide that triggers the autoimmune response. There are nine HLA-B27 alleles differing from each other at one or more amino acid positions. It is important, for the identification of the arthritogenic peptide, to define which alleles, and therefore which polymorphic positions, predispose to the disease. Here, we report that HLA-B*2709 is not associated with AS, as it was not found in patients. HLA-B*2709 differs from the most frequent and disease-associated HLA-B*2705 allele for a single substitution (His vs. Asp) at position 116. Amino acid 116 is located at the bottom of the groove where the antigenic peptide sits, and it has been proven to influence the peptide-binding specificity of HLA class I molecules. The most likely interpretation of these data is that the differences in charge and size that accompany the His-to-Asp substitution exclude the acceptance of the arthritogenic peptide.

Identification of a novel HLA-B27 subtype by restriction analysis of a cytotoxic gamma delta T cell clone.

Seven HLA-B27 alleles are known, which share the same allospecificity, but differ by one to six amino acid substitutions. Herein, we describe a novel HLA-B27 allele, provisionally named B27-ci, which is expressed by an individual from whom a B27-restricted gamma delta T cell clone has been derived. This clone recognizes B cell lines from the proband and all of the other B27-positive members of the family, but does not lyse B cell lines that express other HLA-B27 alleles. The amino acid sequence deduced from three B27-ci cDNA clones was found to differ from the B*2705 sequence by one amino acid substitution (Asp to His) in position 116 of the alpha 2 domain. This position has been shown to lie in the floor of the F pocket, where it plays a key role in determining the nature of the amino acid side chain that will fit into this pocket. Moreover, the fact that the clone described here possesses a TCR-gamma delta indicates that this subset of cells not only can be HLA-restricted, but also can finely discriminate among classical class I molecules.

Analysis of human/mouse interleukin-6 hybrid proteins: both amino and carboxy termini of human interleukin-6 are required for in vitro receptor binding.

The multifunctional cytokine interleukin-6 (IL-6) is a single polypeptide chain consisting of 184 amino acids in man and 187 amino acids in mouse. Despite the relatively high degree of sequence similarity of these two molecules (about 57%), the biological activity in mouse and human IL-6 shows species specificity. Starting with this observation, we constructed interspecies hybrids with the goal of defining which segments of the human IL-6 molecule are involved in human receptor binding. In this manner we generated multiple amino acid substitution mutants which do not contain insertions or deletions as compared with the parental proteins, and which, therefore, should not show dramatic changes in folding. Using two biological assays on cells of human and mouse origin and a recently developed in vitro binding assay to recombinant soluble human IL-6 receptor, we obtained results which indicate that both the amino and carboxy termini are necessary and sufficient for efficient binding, but that the carboxy terminus plays the dominant role in receptor recognition.