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.

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.

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.

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.

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.

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.

Leukocyte trafficking in experimental autoimmune uveitis in vivo.

Leukocyte trafficking from blood into tissue is a fundamental process in immune surveillance and the immune response to stimuli. Experimental autoimmune uveitis (EAU) is an animal model for posterior uveitis and is mediated by T lymphocytes and macrophages that infiltrate the posterior segment of the eye. To analyze leukocyte migration into retinal tissue during the course of EAU, labeled cells were identified in vivo by scanning laser ophthalmoscopy and in retinal flatmounts by confocal microscopy. Adhesion of blood leukocytes to retinal endothelial cells in vivo was significantly raised 48 h before the appearance of clinical disease, and this correlated with the increased expression of CD54 on retinal vessels. Mitogen-activated spleen cells and CD4+ T cells only entered into retinal tissue in animals with clinical disease and not naive recipients. The disease status of the donor animal had no effect on leukocyte trafficking. These results, which identify leukocyte-endothelial cell interactions in vivo, suggest that the activation of the retinal endothelium is a prerequisite to leukocyte adhesion and extravasation into ocular tissue during EAU.

Characterisation of an epitope recognised by a monoclonal antibody against horse alcohol dehydrogenase using peptides synthesised on solid support.

Immunological analysis, using the Pepscan technique, of the tetradecapeptide, Pro344-Glu357 (PLITHVLPFEKINE), from horse liver alcohol dehydrogenase has identified a five amino acid sequence, HVLPF, which binds a monoclonal antibody. The epitope seems to be rather flexible with only two of the amino acids, Pro and Phe, having the characteristics of contact residues. However, the presence of the adjacent glutamic acid residue as part of the Pepscan peptide has a dramatic negative neighbourhood effect and inhibits binding. This highlights the potential risk of missing an epitope altogether when using the Pepscan procedure for epitope mapping.

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.

Toxoplasma gondii: its effect on the ovine popliteal lymph node.

Four non-immune sheep and two with naturally acquired antibody were inoculated subcutaneously in the lower part of the leg with 100 cysts of Toxoplasma gondii. Two other non-immune sheep were given a control inoculum. Efferent lymph from the popliteal nodes on the side of the injection was collected via a cannula and injected into mice. Live toxoplasms were present in the lymph of non-immune sheep from day 2 until day 15, at which time the experiment was terminated. Corresponding samples of lymph from the one immune animal tested were almost always negative. Severe pathological changes were present in lymph nodes from non-immune sheep. Gross enlargement, loss of architecture, haemorrhages, and some necrosis occurred, and the sinuses were packed with plasma cells and plasmablasts. Changes in the nodes of immune sheep were similar but less striking, with retention of architecture, no haemorrhages and no necrosis. It was concluded that the lymphadenopathy in sheep is similar to that in rabbits, mice and man with toxoplasmosis.

Factor V Leiden mutation is associated with ocular involvement in Behçet disease.

PURPOSE: Behçet disease is a systemic disease of young adults characterized by venous occlusion in both the deep venous and retinal circulations. In severe ocular disease, blindness may occur despite immunosuppressive treatment. The most common inherited risk factor for the development of idiopathic venous thrombosis is the presence of the Factor V (FV Leiden) mutation, which confers resistance to activated protein C. The association of FV Leiden with Behçet disease has been reported, but its influence on ocular disease is not known. We therefore investigated the prevalence of this mutation in patients with Behçet disease to determine its contribution to the presence and severity of ocular disease. METHODS: One hundred and six Middle Eastern patients satisfying international criteria, and 120 healthy control subjects without a history of venous thrombosis were included in the study, and patients underwent standard examination by two ophthalmologists with an interest in Behçet disease. Genomic DNA was extracted from peripheral blood leukocytes and screened for the FV Leiden mutation with the polymerase chain reaction method with sequence-specific primers (PCR-SSP). RESULTS: FV Leiden was detected in 19% (23/120) of the control population compared with 27% (29/106) of all patients with Behçet disease (P = .13). However, among patients with Behçet disease who had ocular disease (75/106), the prevalence of FV Leiden was significantly higher (32%) than it was in control subjects (P = .04). Furthermore, ocular patients with Behçet disease in whom retinal occlusive disease was observed (25/75) had the highest expression of FV Leiden (44%). CONCLUSIONS: These data suggest that FV Leiden may be an additional risk factor for the development of ocular disease and, in particular, retinal vaso-occlusion, and it may contribute to the poor visual outcome in these patients.

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.

Human antiretinal antibodies in toxoplasma retinochoroiditis.

BACKGROUND/AIMS: Toxoplasma retinochoroiditis (TR) is an important cause of blindness and visual morbidity, affecting young adults. It has been postulated that some of the retinal damage observed in TR is due to antiretinal autoimmune mechanisms. METHODS: Humoral antiretinal autoimmunity in TR was investigated by indirect immunofluorescence (IIF) on normal human cadaveric retina and by a human retinal S-antigen ELISA. 36 patients with TR were separated on clinical grounds into those with first recurrence of disease (n = 18) or those with multiple recurrences (n = 18). Patients were also segregated into those with active (n = 28) or quiescent disease (n = 8). Serum from 16 normal controls (six with positive toxoplasma serology and 10 without) with no evidence of eye disease and 12 patients with idiopathic retinal vasculitis (IRV) were also tested. RESULTS: Sera from 34 of the 36 patients (94%) with TR demonstrated photoreceptor layer reactivity by IIF contrasting with six of 16 normal controls (p = < 0.001) and three of 12 IRV patients (p = < 0.001). Titres of antiphotoreceptor antibody were also higher among TR patients than controls. Sera from 27 of the 36 TR patients, 10 of 16 normals, and nine of 12 retinal vasculitis patients possessed anti-human retinal S-antigen antibodies at a titre of 1:400 or more as assessed by ELISA (p = > 0.05). Antiretinal autoantibody as detected by IIF did not run in parallel with S-antigen reactivity. CONCLUSIONS: The data indicate that the extent of antiretinal reactivity within TR is not accounted for by anti-S-antigen antibodies alone. This remarkably high prevalence of antiphotoreceptor antibody in TR as opposed to that found in either healthy or disease controls suggest that these antibodies may be co-pathogenic in toxoplasma retinochoroiditis.

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.

The pituitary-adrenal axis in idiopathic retinal vasculitis.

AIMS: To determine whether patients with idiopathic retinal vasculitis have altered production of cortisol and dehydroepiandrosterone sulphate (DHEA-S), and whether differences in these variables occur between those who are sensitive (SS) and resistant (SR) to steroids. METHODS: 20 patients with retinal vasculitis (off treatment) and 10 control subjects were prospectively recruited. Morning cortisol and DHEA-S levels were measured, and cortisol secretion rates and short synacthen tests (SST) carried out in patients before treatment, when on prednisolone 20 mg/day, and in controls. RESULTS: There were no differences in any variables between patients and controls. For retinal vasculitis patients pretreatment, the SST was lower in SR patients (p=0.02). More of the SR patients had ischaemic retinal vasculitis ( p<0.001). CONCLUSIONS: Cortisol and DHEA-S are not involved in the pathogenesis of retinal vasculitis. SR in retinal vasculitis may be associated with a defective hypothalamic-pituitary-adrenal axis.

Limitations of SCID-Hu mouse model for antiretinal autoantibody responses.

The transfer of human peripheral blood mononuclear cells (PBMC) to mice with severe combined immune deficiency (SCID) allows the investigation of immune function. The authors investigated the potential of SCID mice to produce anti-retinal antibodies from PBMC derived from retinal vasculitis patients, and in vivo primed with retinal antigen on days 2 and 22 following transfer. Using Western blotting they could not detect any anti-retinal humoral response in sera from reconstituted animals. Human CD(4)(+) or CD8(+) T cells in spleen or lymph nodes from reconstituted animals were not detected by histological examination. Similarly, no ocular pathology was apparent. The possible mechanisms involved in the lack of an anti-retinal specific antibody response in reconstituted SCID mice are discussed.

Adhesion molecule expression on circulating lymphocytes and monocytes in patients with isolated idiopathic retinal vasculitis.

Idiopathic retinal vasculitis (RV) is a disease of unknown aetiology in which immune responses are involved in the pathogenesis of disease. T cells are thought to be important in this disease and there is evidence of peripheral T cell activation in a significant proportion of patients. The authors examined the expression of the leukocyte adhesion molecules (LeuCAMs) CD11a and CD18 on the peripheral T cells and monocytes of 11 patients with active idiopathic retinal vasculitis compared with 11 age, sex and race matched controls. Although the percentage of T cells expressing HLA DR was increased in the patient group the percentage of cells expressing CD11a and CD18 and the density, expressed as mean fluorescence intensity (MFI) were no different in the two groups. The expression of CD11a and CD18 on peripheral blood monocytes was also not found to be different between patients and controls. Adhesion between leukocytes and endothelial cells is essential for emigration of leukocytes and their accumulation in disease. Our findings suggest that any upregulation of leukocyte adhesion molecules occurring as part of this process is taking place in response to locally produced cytokines.

Soluble adhesion molecules in Behçet's disease.

BACKGROUND: Behçet's disease (BD) may lead to blindness in up to 25% of eyes. Soluble (s)ICAM-1 but not sVCAM-1 is associated with relapse in idiopathic uveoretinitis and is reported to be raised in BD patients. We have investigated the levels of sICAM-1 and sVCAM-1 in Palestinian patients with BD and related them to both ocular and systemic disease activity and to immunosuppressive treatment. METHODS: A total of 51 patients (43 male, 8 female; mean ages 29.8 & 31.9 yr) were examined at the St John Ophthalmic Hospital during a one year period (135 consultations). Disease activity was determined from history and standard ocular examination. Anterior uveitis, vitritis and retinal vasculitis acted as markers of ocular inflammation. Peripheral venous sICAM-1 and sVCAM-1 levels were determined by standard ELISA. A total of 53 healthy age- and sex-matched clinic staff members acted as controls. RESULTS: sICAM-1 and sVCAM-1 were both significantly lower in patients on systemic immunosuppression than in those off treatment (p < 0.001). Among patients off systemic treatment, sICAM was higher in the group with active systemic disease but quiet eyes (p = 0.003), but not in those with active ocular disease (p = 0.09), compared to controls. sVCAM was not raised in either group. CONCLUSION: Systemic immunosuppression was associated with reduced sICAM-1 and sVCAM-1, supporting a role for adhesion molecules in the pathogenesis of BD. sICAM-1 levels were raised in association with inflammatory features implicating endothelial activation in active BD. The mean sICAM-1 was higher in active ocular patients than controls.

Quantitative recovery of isolated mucosal mast cells and globule leucocytes from parasitised sheep.

A method of the quantitative recovery of mast cells and globule leucocytes from the gastric mucosa of parasitised sheep is described. The optimal yields of viable cells from small samples of tissue digested with collagenase and hyaluronidase were measured. Mast cell/globule leucocyte yields were significantly increased in sheep orally challenged with 3000 to 5000 Ostertagia circumcincta per day. In these groups as many as 23 per cent of the cells were mast cells/globule leucocytes. Enrichment of these cells was achieved by Percoll density gradient centrifugation and fractions containing 34 to 84 per cent mast cells/globule leucocytes were isolated.