Targeting ß2 adrenergic receptors regulate human T cell function directly and indirectly.

It is well-established that central nervous system activation affects peripheral blood mononuclear cell (PBMCs) function through the release of the catecholamines (Epi) and norepinephrine (NE), which act on ß2-adrenergic receptors (ß2AR). However, most studies have used non-specific stimulation of cells rather than antigen-specific responses. Likewise, few studies have parsed out the direct effects of ß2AR stimulation on T cells versus indirect effects via adrenergic stimulation of antigen presenting cells (APC). Here we report the effect of salmeterol (Sal), a selective ß2AR agonist, on IFN-γ(+) CD4 and IFN-γ(+) CD8 T cells following stimulation with Cytomegalovirus lysate (CMVL-strain AD169) or individual peptides spanning the entire region of the HCMV pp65 protein (pp65). Cells were also stimulated with Staphylococcal enterotoxin B. Additionally, we investigated the effect of Epi and Sal on cytotoxic cell killing of transfected target cells at the single cell level using the CD107a assay. The results show that Sal reduced the percentage of IFN-γ(+) CD4 and IFN-γ(+) CD8 T cells both when applied directly to isolated T cells, and indirectly via treatment of APC. These inhibitory effects were mediated via a ß2 adrenergic-dependent pathway and were stronger for CD8 as compared to CD4 T cells. Similarly, the results show that Sal suppressed cytotoxicity of both CD8 T and NK cells in vitro following stimulation with Chinese hamster ovary cell line transfected with MICA(*009) (T-CHO) and the human erythromyeloblastoid leukemic (K562) cell line. The inhibitory effect on cytotoxicity following stimulation with T-CHO was stronger in NK cells compared with CD8 T cells. Thus, targeting the ß2AR on lymphocytes and on APC leads to inhibition of inflammatory cytokine production and target cell killing. Moreover, there is a hierarchy of responses, with CD8 T cells and NK cells inhibited more effectively than CD4 T cells.

Glucocorticoid sensitivity in Behçet's disease.

OBJECTIVE: Glucocorticoid (GC) sensitivity is highly variable among individuals and has been associated with susceptibility to develop (auto-)inflammatory disorders. The purpose of the study was to assess GC sensitivity in Behçet's disease (BD) by studying the distribution of four GC receptor (GR) gene polymorphisms and by measuring in vitro cellular GC sensitivity. METHODS: Healthy controls and patients with BD in three independent cohorts were genotyped for four functional GR gene polymorphisms. To gain insight into functional differences in in vitro GC sensitivity, 19 patients with BD were studied using two bioassays and a whole-cell dexamethasone-binding assay. Finally, mRNA expression levels of GR splice variants (GR-α and GR-ß) were measured. RESULTS: Healthy controls and BD patients in the three separate cohorts had similar distributions of the four GR polymorphisms. The Bcll and 9ß minor alleles frequency differed significantly between Caucasians and Mideast and Turkish individuals. At the functional level, a decreased in vitro cellular GC sensitivity was observed. GR number in peripheral blood mononuclear cells was higher in BD compared with controls. The ratio of GR-α/GR-ß mRNA expression levels was significantly lower in BD. CONCLUSIONS: Polymorphisms in the GR gene are not associated with susceptibility to BD. However, in vitro cellular GC sensitivity is decreased in BD, possibly mediated by a relative higher expression of the dominant negative GR-ß splice variant. This decreased in vitro GC sensitivity might play an as yet unidentified role in the pathophysiology of BD.

Genetics in ocular inflammation--basic principles.

The ability to identify individuals at risk of ocular disease, or to determine the potential severity of disease or response to therapy is the current focus of much research. These studies are being led by genetic analysis of individuals to determine associations with alterations in gens that may explain manifestations of particular diseases. In this review we consider the basic principles behind genetic studies in general and of ocular disease in particular. We address the methodologies being utilised, and the results derived so far. The potential and pitfalls of such studies are relevant to the concept of personalised medicine and better defined clinical trials.

Serum cytokine profiles in Behçet's disease: is there a role for IL-15 in pathogenesis?

OBJECTIVES: Behçet's disease (BD) is a multisystem inflammatory disease characterised by recurrent orogenital ulceration, ocular inflammation and skin lesions whose aetiology is currently unknown. We hypothesized that levels of cytokines in the serum might provide either diagnostic or activity markers for the disease. METHODS: Levels of 10 cytokines were analysed in a multiplex bead analysis system as well as IL-15 by ELISA, in 79 serum samples from 52 patients with BD. The same cytokines were also measured in serum samples from 20 patients with recurrent aphthous stomatitis (RAS), as disease controls, and 15 healthy volunteers. The results were correlated with disease activity and current drug therapy. RESULTS: CXCL8 and TNF were the most abundant cytokines and were significantly raised compared to both patients with RAS and healthy controls. IL-15 was present in all samples and was significantly raised in both patients with BD and RAS compared to healthy controls. By comparison, cytokines associated with an adaptive immune response such as IFNgamma and IL-2 were found in few samples, while IL-4 and IL-10 were not detected in any sample. Levels of cytokines correlated with each other suggesting a response to the same stimulus, however, there was no association with either disease activity or treatment. CONCLUSION: Cytokines related to activity of the innate immune response were most prominent in this study and showed good correlation with each other. In particular, it was shown that IL-15 was raised in BD. However, there was no pattern of cytokine expression relating to disease activity or treatment.

Immunity and Toxoplasma retinochoroiditis.

Toxoplasma infection accounts for up to 50% of all cases of posterior uveitis worldwide. In this review the control of Toxoplasma infection generally, and specific in the eye, by the immune system is discussed.

CTLA-4 polymorphisms are not associated with ocular inflammatory disease.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is regarded with other molecules such as HLA, PTPN22 and CARD15 as genetic master switches of autoimmunity. Single nucleotide polymorphisms (SNPs) in the genes encoding these molecules have been associated with autoimmune conditions. We analysed the SNPs -318C/T and 49A/G in CTLA-4 in patients with Behcet's disease (BD), patients with intermediate uveitis and appropriate controls. Blood was collected from 236 patients with BD from the UK and the Middle East (ME), all fulfilling the International Study Group criteria for the diagnosis of BD, and 143 patients with idiopathic intermediate uveitis were recruited from the Medical Eye Unit at St Thomas' Hospital. Samples from healthy individuals from each geographical centre were used as controls. DNA was prepared by standard methods, and SNPs -318 and 49 in CTLA-4 were detected by a polymerase chain reaction-sequence specific primers (PCR-SSP) assay using primer mixes. The results showed that there was no association with either polymorphism in patients with BD from the UK or the ME. Similarly, there was no association in patients with intermediate uveitis. Moreover, there was no association with SNP in CTLA-4 and disease manifestations in BD or outcome in patients with intermediate uveitis. Both BD and intermediate uveitis have HLA associations, but there is no difference in distribution of CTLA-4 polymorphisms that are associated with other autoimmune diseases. The lack of association with polymorphisms in CTLA-4 and other master controlling genes of autoimmunity suggests that mechanisms that mediate such a description for BD and intermediate uveitis have still to be elucidated.

Expression analysis of immune response genes of Müller cells infected with Toxoplasma gondii.

PURPOSE: To investigate changes in immune response genes following Toxoplasma gondii infection of Müller cells. METHODS: Human Müller cells were infected or mock infected with two strains of T. gondii (RH and Prugniaud). RNA and supernatants were collected from infected and uninfected cells at 2 and 24 h. RNA from the two time points were compared using a custom made DNA microarray. Real time PCR or human cytokine antibody array was used to confirm up-regulation of immune molecules. RESULTS: Gene expression in infected cells showed up-regulation of CCL2, IL-6, CXCL8, and CXCL2. CCL2 and CXCL2 gene expression was confirmed by real time PCR. IL-6 and CXCL8 protein production was confirmed by a cytokine antibody array. IL-4 production was observed by cytokine antibody array but not by DNA microarray. In contrast, infection with T. gondii did not induce interferon-gamma (IFNgamma) and IL-12 expression, molecules conventionally associated with the inter-conversion of tachyzoite to bradyzoite. CONCLUSION: These results indicate that while in vitro infected Müller cells may be capable of inducing an immune response by attracting blood-borne leucocytes, they do not appear able to directly control the proliferation of T. gondii.

Associations of major histocompatibility complex class I chain-related molecule polymorphisms with Behcet's disease in Caucasian patients.

HLA-B*51 is known to be associated with Behcet's disease (BD) in many ethnic groups. The pathogenic gene, however, may lie close to the HLA-B locus and therefore be in linkage disequilibrium with HLA-B*51. On the basis of the proximity of MIC genes to HLA-B, their expression pattern and their affinity for the activating NKG2D receptor on natural killer (NK) cells and gammadelta T cells, these molecules have been postulated as susceptibility factors in BD. DNA from 56 western European Caucasians with BD and 90 Caucasian controls were analysed by polymerase chain reaction using allele-specific primers for MICA and MICB alleles. An increased allele frequency of MICA*009 was found in the BD patient group (25.0%) when compared with the controls (7.2%). This was associated with a corresponding decrease in MICA*008 in the BD patients (36.6%) compared with the controls (46.7%), which was not significant. MICA*009 was strongly associated with the presence of HLA-B*51 in patients and controls. No significant difference in frequency of MICB alleles was found between patients and controls. Both HLA-B*51 and MICA*009 are strongly associated with BD in a pure Caucasian BD patient group, and the two alleles are in linkage disequilibrium. No MICB allele was found to associate significantly with the disease, an unexpected finding considering the close proximity of the MICA and MICB loci. Our results suggest that while MICB does not influence the development of BD, polymorphisms in MICA may be pathogenic, perhaps through the interaction with NK and gammadelta T cells.

Are cytokine gene polymorphisms associated with outcome in patients with idiopathic intermediate uveitis in the United Kingdom?

BACKGROUND/AIM: Competing levels of cytokines, either locally within the eye or systemically, may influence the eventual outcome of ocular inflammation. Polymorphism in the promoter part of the genes controlling cytokine production may result in either higher or lower production of the relevant cytokine to a given stimulus. The authors hypothesised that such polymorphisms may relate to visual outcome in patients with idiopathic intermediate uveitis. METHODS: DNA was obtained from 125 patients with idiopathic intermediate uveitis and analysed for the interleukin 10 IL-10-1082G/Alpha and IL-10-819C/T, and interferon gamma IFNgamma 874T/A gene polymorphisms. Associations with disease were calculated by both allelic frequency and haplotype analysis, and associations between ocular disease outcomes and the presence of polymorphisms were identified. A bad outcome was defined as loss of vision <6/12 Snellen in both eyes at 5 years from presentation when the eyes were quiet. RESULTS: An initial screen showed that the 874T allele of the IFNgamma gene was more prevalent in patients than controls (chi2= 7.9; p = 0.004 OR 1.7; 95% CI 1.2 to 2.6 (Pc = 0.02), whereas the IL-10-1082/-819 AT haplotype of the interleukin 10 (IL-10) gene was not. Analysis of disease outcome showed an association between IL-10-1082 AA homozygosity and bad outcome (chi2= 13; p = 0.0003). Moreover, the two cytokine polymorphisms taken together showed that up to 75% of patients with a poor visual outcome had the combined IFNgamma 874TA or TT genotype together with the IL-10-1082AA genotype (chi2= 13.2 p = 0.0008 OR 6.4; 95% CI 1.85 to 23.6 Pc = 0.1). CONCLUSION: These results show that disease outcome in intermediate uveitis may be partly determined by a complex interplay between cytokine genes and these results may have implications for future treatment with biological agents that target these cytokines.

The effect of Toxoplasma gondii infection on expression of chemokines by rat retinal vascular endothelial cells.

Cells infected by Toxoplasma gondii undergo up-regulation of proinflammatory cytokines, organelle redistribution, and protection from apoptosis. During infection in man, the parasite encysts within the retina, a process that results in retinochoroiditis which can lead to permanent loss of sight. The reasons for the parasite to infect retinal tissue and the mechanisms by which it encysts are not clearly understood. We studied the effect of infection with T. gondii of retinal vascular endothelial cells using the Clontech Atlastrade mark array system in order to elucidate changes in gene expression. We compared hybridization of RNA to the array from infected and uninfected cells at two time points; 2 and 24 h. Exposure to T. gondii after 2 h resulted in change of expression of approximately 6% of genes on the array, including those involved in cell structure, protein and vesicle trafficking, cell-cycle regulation, transcriptional and translational machinery, and apoptosis. Among the genes involved in the inflammatory response, chemokine genes such as GRO1 (Growth Regulated Oncogene 1), MCP-1 (Monocyte Chemotactic Protein-1), FKN (Fractalkine) and RANTES (Regulated upon Activation, Normal T Cell Expressed and Secreted) were found to be up-regulated and protein production was confirmed by ELISA. However after 24 h of infection, GRO1, MCP-1 and FKN were down-regulated, confirmed by RT-PCR. Thus, invasion of retinal vascular endothelium (RVE) cells by T. gondii leads to the production of chemokines important in directing the traffic of inflammatory cells to the infected area.

Analysis of the CC chemokine receptor 5 (CCR5) Delta32 polymorphism in Behçet's disease.

Chemokines are important determinants of the early inflammatory response. The CC chemokine receptor 5 (CCR5) Delta32 variant results in a non-functional form of the chemokine receptor, and has been implicated in a variety of immune-mediated diseases. To investigate its role in the pathogenesis of Behçet's disease, we studied 350 patients and 519 healthy controls from three ethnic groups. While significant inter-ethnic variation in allele frequency was observed, no association was identified with disease, even when data were stratified by the known susceptibility gene HLA-B*51.

Chemokine gene polymorphisms associate with gender in patients with uveitis.

Uveitis is an inflammatory condition of ocular tissue characterized by leukocyte infiltration, tissue damage, and decreased visual acuity. Chemokines have been implicated in the pathogenesis of uveitis. Polymorphisms in the genes encoding chemokines have been described as affecting chemokine production or function. We analyzed the frequency of single-nucleotide polymorphisms (SNPs) in genes encoding CCL2 (-2518 and -2076) and CCL5 (-403 and -28) in patients with Behçet's disease (BD), a systemic form of uveitis, and patients with retinal vasculitis (RV), an organ-specific form of disease. We report that there was no association between any SNP and disease. However, when segregated on the basis of gender the CCR5 -403 AA genotype was only found in male patients with BD. Similarly, CCL2 genotypes 1/2 were predominant in males, while genotype 4 was significantly associated with disease in female patients with BD. Differences in disease symptoms and severity between males and females have been described in BD and gender-specific genetic differences in chemokine gene function may be involved.

Behçet's disease: from Hippocrates to the third millennium.

Behçet's disease (BD) is characterised by recurrent episodes of orogenital aphthae, systemic vasculitis, and systemic and retinal venous thrombosis. An association between HLA-B51 and BD was first identified over 20 years ago, but recently identified gene associations implicate regions both within and without the MHC in the immunological events underlying the lesions in BD. These include allelic variants within the tumour necrosis factor gene region and within the MHC class I chain related gene region, the factor V Leiden mutation, which is associated with retinal vascular occlusion, and alleles of the intercellular adhesion molecule gene. No single causative gene for BD has emerged; the evidence indicates that the underlying immune events in BD are triggered by a microbial antigen and subsequently driven by genetic influences which control leucocyte behaviour and the coagulation pathways. Knowledge of these risk factors may permit a more accurate prognosis for a given patient, and identify new pathways for more targeted intervention than is currently available.

Urinary neopterin in idiopathic retinal vasculitis.

AIMS: To determine whether urinary neopterin:creatinine (UNC) ratios relate to disease activity in idiopathic retinal vasculitis (RV). METHODS: 18 patients with RV were prospectively recruited into a year long longitudinal study. Patients collected first morning urine samples on a weekly basis and on the same day completed a diary which documented their subjective view of RV activity and any concurrent infection. They were examined in clinic on a 6-8 weekly basis and an objective assessment was made of RV disease activity. 14 healthy controls collected urine samples in the same way. RESULTS: UNC ratios were significantly higher in patients than in controls (p=0.004, Mann-Whitney U test). UNC ratios were significantly higher when, according to their diaries, the patients had a subjective flare up of RV (p=0.001, Mann-Whitney U test). Subjective increased RV activity occurred more often when the patients had a concurrent infection (p<0.0001, chi(2) test). There was no significant difference in the UNC ratio between objective clinical relapse and non-relapse of RV. There was moderate agreement between the clinical assessment and patients' subjective impression of RV activity (kappa=0.48). CONCLUSIONS: Higher neopterin levels reflect cell mediated disease that occurs in RV, but UNC ratios are not recommended as a means of monitoring clinical disease activity in RV.

Intercellular adhesion molecule-1 gene polymorphisms in Behçet's disease.

Intercellular adhesion molecule-1 (ICAM-1) gene polymorphisms have been implicated in the susceptibility to inflammatory diseases, including multiple sclerosis and inflammatory bowel disease. The expression of both soluble and tissue ICAM-1 is increased in Behçet's disease (BD) but the contribution of ICAM-1 gene polymorphisms to this disease remains unknown. Associations with BD have been reported for genes within the MHC, including HLA-B51, TNF and MICA, but the role of non-MHC genes in BD remains largely unexplored. We have investigated the frequency of the R/G 241 and K/E 469 ICAM-1 gene polymorphisms in 83 patients with BD disease and 103 healthy controls, all of Palestinian and Jordanian descent, and demonstrated an association between BD and the ICAM-1 E469 allele (Pc = 0.046, OR = 2.1). Among patients, no association was found between the presence of ocular disease and ICAM-1 polymorphisms. While the functional correlate of this polymorphism remains unclear, this finding indicates that a genetic polymorphism in the ICAM-1 gene domain, which is independent of the MHC, may contribute to disease.

The effect of cytokines on the replication of T. gondii within rat retinal vascular endothelial cells.

Toxoplasma gondii infection of the eye can result in a recurrent necrotising retinochoroiditis (TR) which may lead to a permanent loss of visual acuity. The mechanisms responsible for the control of TR within the retina are unknown. The aim of this study was to examine the effects of cytokines on the replication of T. gondii RH strain tachyzoites within rat retinal vascular endothelial (rRVE) cells. Pretreatment of rRVE with IFNgamma, TNF or IL-1beta resulted in a significant decrease in T. gondii replication from day 2 onwards. There was no significant difference in nitric oxide (NO) production by IFNgamma, TNF or IL-1beta treated rRVE as compared to controls at any time point. By comparison, the addition of L-tryptophan to IFNgamma treated cultures significantly restored T. gondii replication from 48 h post inoculation. Thus, IFNgamma, TNF and IL-1beta can significantly inhibit the replication of T. gondii within rRVE. However, this inhibition appears to be independent of NO production. L-tryptophan catabolism may have a role in IFNgamma mediated inhibition of T. gondii replication in rRVE cells.