Pegylated interferon-α-2b reduces corticosteroid requirement in patients with Behçet's disease with upregulation of circulating regulatory T cells and reduction of Th17.

OBJECTIVE: To determine whether the addition of 26 weeks of subcutaneous peginterferon-α-2b could reduce the requirement for systemic corticosteroids and conventional immunosuppressive medication in patients with Behçet's disease (BD). METHODS: We conducted a multicentre randomised trial in patients with BD requiring systemic therapy. Patients were randomised to 26 weeks of peginterferon-α-2b in addition to their standard care or to standard care only and followed 6-monthly for 3 years with BD activity scores and quality of life questionnaires. Patients at one centre had blood taken to measure regulatory T cells (Tregs) and Th17 cells. RESULTS: 72 patients were included. At months 10-12, while among the entire patient population there was no difference in the corticosteroid dose or immunosuppression use between the treatment groups (adjusted OR 1.04, 95% CI 0.34 to 3.19), post hoc analysis revealed that in patients who were on corticosteroids at baseline the corticosteroid requirement was significantly lower in the peginterferon-α-2b (6.5 (5-15) mg/day) compared with the non-interferon group (10 (8.25-16.5) mg/day, p=0.039). Furthermore, there was a trend towards an improved quality of life that became significant by 36 months (p=0.008). This was associated with a significant rise in Tregs and a decrease in Th17 cells which was still present at 1 year and 6 months after the interferon was stopped. The safety profile was similar with adverse events in 10% in both groups. CONCLUSIONS: The addition of peginterferon-α-2b to the drug regime of BD patients did not significantly reduce their corticosteroid dose required at 1 year. However, in those on corticosteroids at baseline post hoc analysis demonstrated that the addition of peginterferon-α-2b did result in a significant reduction in corticosteroid dose with a significantly improved quality of life and trend to reduce other required immunosuppressive agents. This effect was seen at 1 year and associated with a rise in Tregs suggesting a possible mode for interferon action. TRIAL REGISTRATION NUMBER: ISRCTN 36354474; EudraCT 2004-004301-18.

Multiple cytokines in human tear specimens in seasonal and chronic allergic eye disease and in conjunctival fibroblast cultures.

BACKGROUND: Several cytokines are involved in the recruitment and activation of inflammatory cells in ocular allergic diseases. The purpose of the study was to assay multiple cytokines and chemokines in tears, to compare subgroups of allergic conjunctivitis (AC) with controls, and in culture supernatants to determine whether conjunctival fibroblasts produce some of these cytokines. METHODS: Fifty to one hundred microlitre tears were obtained from patients with active seasonal allergic conjunctivitis (SAC; n=12), vernal keratoconjunctivitis (VKC; n=18), atopic keratoconjunctivitis (AKC; n=6) and non-atopic controls (n=14). Primary conjunctival fibroblasts grown in vitro were stimulated with IL-4, IL-13 or TNF-alpha for 24 h. Cell-free tear and culture supernatants were assayed for IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IFN-gamma, TNF-alpha, eotaxin, MCP-1 and RANTES using multiplex bead analysis. Induction of chemokine gene expression was determined by PCR. RESULTS: IL-1beta, IL-2, IL-5, IL-6, IL-12, IL-13, MCP-1 were increased in all tears groups compared with controls, with highly significant correlations between many of these molecules. In addition IL-4, IFN-gamma, and IL-10 were elevated in SAC and VKC, while eotaxin and TNF-alpha were only increased in VKC. IL-6, IL-8, MCP-1, RANTES and eotaxin were detected from fibroblasts cultures, and were all up-regulated by TNF-alpha. By PCR, fibroblasts expressed MCP-1 transcripts constitutively, whereas IP-10 and Mig were up-regulated by TNF-alpha. CONCLUSIONS: Differential cytokine levels support tears as a useful indicator of immune mechanisms occurring during AC. The striking similarities in chemokine profiles between tears and fibroblasts suggest these cells as likely sources of chemokines in tears.

Antigen-specific T-cell downregulation by human dendritic cells following blockade of NF-kappaB.

Dendritic cells (DCs) are important for presenting antigen to T cells, especially naïve T cells. It has recently been shown that blocking the transcription factor, nuclear factor kappa B (NF-kappaB) in human DCs inhibited the mixed leukocyte reaction. The aim of this study was to investigate the effect of blocking NF-kappaB in DCs during presentation of antigen to memory T cells in vitro. Peripheral blood monocytes were differentiated into immature DCs with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor, and pulsed with an immunogenic tetanus toxoid peptide. Upon maturation, the antigen-pulsed DCs were highly effective in presenting antigen to autologous T cells. However, stimulation with antigen-pulsed DCs overexpressing IotakappaBetaalpha, the endogenous inhibitor of NF-kappaB, led to a significant reduction in T-cell proliferation, and decreased production of interferon-gamma, IL-4 and IL-10, whereas transforming growth factor-beta production was low throughout. There was a significant increase in apoptosis of antigen-specific T cells, even in the presence of IL-2, which was not found in resting T cells. Similar findings were observed using a proteasome inhibitor to block NF-kappaB. The effective downregulation of antigen-specific T-cell responses following blockade of NF-kappaB in DCs could be a useful approach for immunomodulating inflammatory T-cell responses.

Cytokine production and mRNA expression by conjunctival T-cell lines in chronic allergic eye disease.

BACKGROUND: Activated CD4+ T cells, mast cells and eosinophils are the main cytokine-producing cell-types infiltrating the conjunctiva during chronic allergic eye diseases. Interactions between these cells are thought to play an important immunopathogenic role in these disorders (giant papillary conjunctivitis; vernal keratoconjunctivitis; atopic keratoconjunctivitis). OBJECTIVE: The objective was to compare the cytokine profiles of conjunctival T-cell lines from patients with different forms of chronic allergic eye disease. METHODS: T cells were isolated from conjunctival biopsies and non-specifically expanded into lines. The lines were immunophenotyped by flow cytometry. Cytokine production was quantified by immunoassays and more sensitive molecular techniques were used to investigate cytokine mRNA expression to identify the presence of interleukin (IL) -2, IL-4 and interferon (IFN) -gamma transcripts. RESULTS: Following four to six rounds of stimulation, the conjunctival T-cell populations were CD3+ (> 93%), with variable levels of CD4 and CD8 expression. All were HLA-DR+ (> 80%) with some HLA-DQ expression. Conjunctival T-cell lines from atopic keratoconjunctivitis produced selective increases in IFN-gamma, IL-10 and IL-13 (P<0.01), those from vernal keratoconjunctivitis produced increased IL-5 (P<0.01) whereas T-cell lines from giant papillary conjunctivitis produced only low levels of cytokines. IL-4 was only detected at the mRNA level and was expressed in four out of five T-cell lines in the vernal keratoconjunctivitis group. In contrast there was moderate to strong expression of IFN-gamma in five out of six T-cell lines in atopic keratoconjunctivitis. CONCLUSION: Different patterns of T-cell cytokine profiles were observed for each disease, with low-level, non-polarized cytokine production in giant papillary conjunctivitis, a TH2-like profile in vernal keratoconjunctivitis and a shift towards a TH1-like profile in atopic keratoconjunctivitis.

Increased CD4+ expression and decreased IL-10 in the anterior chamber in idiopathic uveitis.

PURPOSE: To compare cell types and cytokines in aqueous humor from patients with uveitis either occurring in association with a systemic disease or apparently isolated and not associated with a systemic disease. METHODS: Cells were collected by centrifugation of fresh aqueous humor from uveitis and controls, and immunofluorescence techniques were performed with markers for T cells, B cells, and monocytes. Cytokines were measured in the aqueous supernatants, and serum samples were assayed for soluble interleukin-2 receptors. RESULTS: When aqueous samples from idiopathic uveitis were compared with those from uveitis associated with a systemic disease, there were increases in CD3+, CD4+ (p = 0.001), and activated CD4+ T cells (p = 0.02) and a decrease in B cells (p = 0.0013). This was not reflected in the peripheral blood where there were no differences in the cell types or in soluble interleukin-2 receptor levels. No cells were obtainable from control aqueous. Interleukins-10 and -12, interferon-gamma, and transforming growth factor-beta2 were detected in aqueous supernatants. Interleukin-10 was reduced (p = 0.024) in uveitis in comparison with controls. CONCLUSIONS: The results suggest a selective recruitment of CD4+ T cells within aqueous humor but only in idiopathic uveitis. In both disease groups there was a decrease in the immunoregulatory cytokine interleukin-10, which might enable an immune response to occur in an otherwise highly immunosuppressive microenvironment. Increases in activated CD4+ T cells combined with depressed interleukin-10 levels could partially explain why, for example, in acute anterior uveitis, the inflammatory disease is often more severe.

Characterization of phenotype and cytokine profiles of T cell lines derived from vitreous humour in ocular inflammation in man.

Intermediate uveitis (IU) and Fuchs' heterochromic cyclitis (FHC) are two chronic ocular inflammatory disorders. They differ considerably in ocular morbidity, which is higher in IU. T cell lines were derived from the vitreous humour (VH) and peripheral blood (PB) of 10 patients with IU and four patients with FHC. There was a predominance of CD8+ in all the lines. However, there was a significantly higher percentage of CD4+ T cells in the T cell lines derived from VH of IU (32.0 +/- 8.6%) compared with FHC patients (19. 2 +/- 8.9%) (P = 0.04). The VH-derived T cell lines (VDTC) produced significantly higher levels of IL-2, interferon-gamma (IFN-gamma) and IL-10, but not IL-4, compared with PB-derived T cell lines (PBDTC) in both entities. There was significantly higher IL-2 production by VDTC from IU when compared with FHC patients (1810 +/- 220 pg/ml versus 518 +/- 94 pg/ml; P = 0.009), which could account for the more aggressive clinical features of this condition. In contrast IL-10 production was significantly higher by the VDTC from FHC compared with IU patients. The high IL-10 production by T cells infiltrating VH of FHC patients could down-regulate the inflammatory responses, thereby contributing to the benign clinical course seen in these patients. The accumulation of T cells with differing cytokine profiles in the VH suggests an important role for these cytokines in the pathogenesis of these chronic uveitides.

The immunomodulatory effect of topical cyclosporin A in atopic keratoconjunctivitis.

PURPOSE: To perform a detailed examination of the immunomodulatory effects of topical cyclosporin A (CsA) in conjunctival tissue from patients with atopic keratoconjunctivitis (AKC). METHODS: Patients with active AKC were randomly allocated into two groups of four patients. For 3 months one group received 2% CsA drops, and the other group received placebo drops. Superior tarsal conjunctival biopsy specimens were harvested before and after treatment and examined by one- and two-color immunohistochemistry to compare leukocyte counts, HLA-DR+ and IL-2R+ cell counts, HLA-DR positivity of conjunctival epithelial cells, and counts of T cells expressing the cytokines interleukin (IL)-2, IL-3, IL-4, IL-5, and interferon (IFN)-gamma. RESULTS: Posttreatment values were significantly less than pretreatment values for the total number of leukocytes and in the numbers of CD3+ T cells, CD4+ cells, CD8+ cells, CD20+ B cells, neutrophils, and macrophages, and there was a decrease in the CD4-CD8 ratio (P = 0.03) in the CsA group. There was a reduction from before CsA treatment to after CsA-treatment in the numbers of HLA-DR+ and IL-2R+ cells (P = 0.03), but the reduction in the epithelial cell HLA-DR expression did not reach significance. The number of T cells staining for IL-3 and IL-5 was reduced, although not to statistical significance, but there was a significant reduction in the number of T cells expressing IL-2 and IFN-gamma (P = 0.03) after CsA treatment compared with initial values. There were no statistically significant differences between pretreatment and posttreatment values in the placebo group. There was a clinical improvement in the CsA group and a clinical worsening in the placebo group. CONCLUSIONS: The in vitro effects of CsA translate into a reduction in T cells, a normalization of the CD4-CD8 ratio, a decrease in T-cell activation, and a reduction in T-cell cytokine expression, especially IL-2 and IFN-gamma. The decrease in HLA-DR expression may be mediated by the change in IFN-gamma. There were fewer B cells but not fewer plasma cells after CsA and no change in IL-4 expression, suggesting minimal effects on type I hypersensitivity responses. There was no significant reduction in mast cell or eosinophil numbers, but direct effects of topical CsA on their function may play a role in the therapy of ocular allergic disease. These results show that the beneficial effects of topical CsA in AKC are accompanied by important changes in conjunctival immune cell profiles.

A randomized, placebo-controlled trial of topical cyclosporin A in steroid-dependent atopic keratoconjunctivitis.

OBJECTIVE: This study aimed to investigate the therapeutic effect of topical cyclosporin A (CsA) 2% in maize oil as a steroid-sparing agent in steroid-dependent atopic keratoconjunctivitis. DESIGN: Prospective, randomized, double-masked, placebo-controlled trial. PARTICIPANTS: Twenty-one patients with steroid-dependent atopic keratoconjunctivitis were studied. INTERVENTION: Patients used either topical CsA or vehicle four times daily for 3 months in addition to their usual therapy, and the clinical response was used to taper or stop topical steroids when possible. MAIN OUTCOME MEASURES: Steroid drop usage per week, ability to cease steroid use, scores for symptoms and clinical signs, drop side effects, and overall subjective rating of trial drop by patients and clinician were measured. RESULTS: Cyclosporin A had a greater steroid-sparing effect than did placebo. Nine of 12 CsA patients ceased steroids compared to 1 of 9 placebo patients (P = 0.01), the final steroid use was lower in the CsA group (2.6 +/- 1.4 vs. 27.7 +/- 17.7, P = 0.005), and the mean reduction in steroid use was greater for CsA (85.5 +/- 14.7 vs. 13.9 +/- 16.0, P = 0.005). Clinical signs and symptom scores were reduced to a greater level for CsA. Serious side effects were lid skin maceration in one patient using CsA and an allergic reaction in one placebo patient. Marked blurring of vision after drop instillation was common in both groups, but intense stinging was more common in CsA patients (9/12 vs. 1/9, P = 0.01), limiting frequency of drop use. The clinician rated the trial drops as good or excellent more frequently for CsA (11/12 vs. 0/9, P < 0.0001). CONCLUSIONS: Topical CsA is an effective and safe steroid-sparing agent in atopic keratoconjunctivitis and, despite difficulties in patient tolerance, also improves symptoms and signs.

The role of conjunctival epithelial cells in chronic ocular allergic disease.

Recent evidence suggests that mucosal epithelial cells are capable of actively participating in immune reactions via expression of surface antigens, such as adhesion molecules, and synthesis of cytokines. This appears to be important in the pathophysiology of non-ocular allergic disorders. The objectives of the experiments were to compare the expression of HLA-DR, ICAM-I and pro-allergic cytokines in conjunctival epithelial cells in the different chronic ocular allergic disorders with each other and with normal subjects. Conjunctiva from normal patients (n=10) and patients with vernal keratoconjunctivitis (VKC, n=10), atopic keratoconjunctivitis (AKC, n=10) and contact lens-associated giant papillary conjunctivitis (GPC, n=10) were examined by immunohistochemistry. Epithelial cell staining for surface antigens and cytokines was graded by one masked observer using a four point scale based on the percentage of epithelial cells staining positive. There was no expression of ICAM-1 or HLA-DR in the normal conjunctival epithelial cells, but both antigens were induced on conjunctival epithelial cells in the allergic tissue, and there was greater expression in AKC and VKC compared with GPC. Cytokines IL-6, IL-8, RANTES and TNF-alphaall localised to normal conjunctival epithelial cells. RANTES was upregulated in all the allergic disorders and IL-8 was upregulated in GPC. IL-3 and GM-CSF were not expressed in normal conjunctival epithelial cells. GM-CSF was expressed in all disorders and there was greater expression in AKC compared with GPC and VKC. IL-3 was expressed only in AKC and VKC epithelial cells. These results suggest that conjunctival epithelial cells play an important pro-inflammatory role in chronic ocular allergic diseases; ICAM-1 may allow epithelial cells to recruit, retain and locally concentrate leukocytes; the presence of HLA-DR raises the question of conjunctival epithelial cell antigen presentation. The epithelial cytokines which are upregulated are known to promote eosinophilic inflammation and are typical of allergic inflammation. The differences in cytokine patterns may be exploitable for future therapy.

Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection.

In a rat model of corneal transplantation, Fischer 344 (RT1(lv1)) rats received orthotopic corneal isografts or Wistar-Furth (RT1(u)) donor allografts. Rejection was observed in 25 of 26 allograft recipients, at a median time of 18 days, with all isografts surviving > 100 days. Flow cytometric analysis of aqueous humour identified cellular infiltration of the aqueous at the time of allograft rejection, in contrast to the acellular aqueous found in isografts at corresponding times following transplantation. A higher proportion of CD8+ than CD4+ cells was found at days 1-3 following rejection, whereas there was a higher proportion of CD4+ cells at days 5-8. No changes in peripheral blood T cell subsets were found at the time of rejection. Immunohistochemical analysis of cells infiltrating recipient iris and grafted cornea undertaken at days 1-2, 4 and 7-10 following onset of rejection, demonstrated inflammatory cells in the graft epithelium, stroma and aggregated on the endothelium. Large numbers of macrophages, T cells (CD4+ > CD8+ at all time points), natural killer (NK) cells and neutrophils were detected in graft tissue at days 1-2 and 4, diminishing after that time. Most infiltrating cells expressed MHC class II antigen, and a smaller number expressed IL-2R. Expression of the co-stimulatory marker B7 was identified in a few cells at day 4 in the region of the graft-host wound. The immune response in graft rejection was characterized at day 4 also by expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells of iris and corneal vessels, demonstration of interferon-gamma on mononuclear cells in the peripheral (recipient) cornea, and tumour necrosis factor-alpha on aggregated mononuclear cells on the graft, but not recipient, endothelium. Only sparse cellular infiltrates were found in isograft controls, with inflammation located at the graft-host wound. These findings suggest that inflammatory cells reach a corneal allograft by two routes--from vessels in the peripheral recipient cornea, and from vessels in the recipient iris via the aqueous humour. Different aqueous and intragraft T cell subset proportions were seen early in rejection, although a preponderance of CD4+ cells was found in both aqueous and graft at later times.

T-cell cytokines in chronic allergic eye disease.

BACKGROUND: The pathophysiology of chronic allergic eye disease cannot be explained by type I hypersensitivity alone, and T cell-mediated inflammation has been strongly implicated as a possible additional mechanism. Previous studies suggested that T(H2)-like T cells play an important role in one form of chronic allergic eye disease. OBJECTIVES: This study examined the cytokine profile of T cells in different clinical groups of subjects with chronic allergic eye disease (i.e., vernal keratoconjunctivitis [VKC], atopic keratoconjunctivitis [AKC], and giant papillary conjunctivitis [GPC]) and normal control subjects. METHODS: In situ hybridization was used to identify cytokine messenger RNA (mRNA), and two-color immunohistochemical analysis was used to demonstrate cytokine immunoreactivity localizing to T cells in the conjunctiva. RESULTS: Allergic tissue expressed increased levels of mRNA for IL-3, IL-4, and IL-5 when compared with normal tissue. There was significantly greater IL-2 mRNA expression in subjects with AKC than in those with VKC (p = 0.004) and those with GPC (p = 0.02). Immunoreactivity for T-cell IL-5 was present more frequently in subjects with VKC (p = 0.004), GPC (p = 0.02), and AKC (p = 0.04) than in normal control subjects. However, T-cell IFN-gamma protein expression was greater in subjects with AKC than in subjects with VKC (p = 0.01), GPC (p = 0.01), and control subjects (p = 0.005). CONCLUSIONS: These results show a T(H2)-like T-cell cytokine array in subjects with VKC and GPC but a shift in cytokine profile toward a T(H1)-like pattern, potentially because of differences in chronicity of the disorders, in subjects with AKC. These important functional T-cell variations in chronic allergic eye conditions are likely to be important in understanding differences in clinical characteristics and therapeutic responses.

SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics.

In the central nervous system the blood-brain and blood-retinal barriers (BBB and BRB respectively) are instrumental in maintaining homeostasis of the neural parenchyma and controlling leucocyte traffic. These cellular barriers are formed primarily by the vascular endothelium of the brain and retina although in the latter the pigmented epithelial cells also form part of the barrier. From primary cultures of rat brain endothelium, retinal endothelium and retinal pigment epithelium (RPE) we have generated temperature sensitive SV40 large T immortalised cell lines. Clones of brain (GP8.3) and retinal (JG2.1) endothelia and RPE (LD7.4) have been derived from parent lines that express the large T antigen at the permissive temperature. The endothelial cell (EC) lines expressed P-glycoprotein, GLUT-1, the transferrin receptor, von Willebrand factor and the RECA-1 antigen and exhibited high affinity uptake of acetylated LDL and stained positive with the lectin Griffonia simplicifolia. The RPE cell line was positive for cytokeratins and for the rat RPE antigen RET-PE2. All the cell lines expressed major histocompatibility complex (MHC) class 1 and intercellular adhesion molecule (ICAM)-1 constitutively and could be induced to express MHC class II and vascular cell adhesion molecule (VCAM)-1 following cytokine activation. The EC also expressed platelet endothelial cell adhesion molecule (PECAM)-1. Monolayers of these cells could support the migration of antigen-specific T cell lines. The generation of immortalised cell lines derived from the rat BBB and BRB should prove to be useful tools for the study of these specialised cellular barriers.

Lymphocyte adhesion and transendothelial migration in the central nervous system: the role of LFA-1, ICAM-1, VLA-4 and VCAM-1. off.

Lymphocyte adhesion to and migration across endothelial cell (EC) monolayers, derived from the rat blood-retinal barrier (BRB), were measured in vitro. The binding of concanavalin A (Con A)-activated peripheral lymph node lymphocytes and the migration of CD4+ T-cell lines could be significantly increased by treating the EC with interleukin-1 beta (IL-1 beta). To determine the role of various adhesion molecules during the processes of lymphocyte binding and transmonolayer migration (diapedesis), lymphocytes were treated with monoclonal antibody (mAb) specific for CD11a (alpha L subunit of leucocyte functional antigen-1; LFA-1), CD18 (beta 2 subunit of leucam family) and CD49d (alpha 4 subunit of very late activation antigen-4; VLA-4) and EC with mAb specific for CD54 (intercellular adhesion molecule-1; ICAM-1) and CD106 (vascular cell adhesion molecule-1; VCAM-1). Binding of the highly adhesive but non-migratory Con A-activated lymphocytes was inhibited by mAb to CD11a (reduced to 73% and 65% of control lymphocyte adhesion) and CD18 (42% and 54%) on non-activated and IL-1 beta-treated EC, respectively, but not by mAb to ICAM-1 or VCAM-1. Diapedesis of the highly migratory T-cell line lymphocytes was also blocked by antibodies to CD11a (reduced to 11% and 10% of control T-cell migration), CD18 (29% and 43%) but in addition was also inhibited by anti-ICAM-1 (17% and 53%) on non-activated and IL-1 beta treated EC, respectively. Both anti-VLA-4 and anti-VCAM-1 were also effective in producing a smaller reduction in migration, but only on IL-1 beta activated EC (66% and 58% of control migration, respectively). These studies indicate that lymphocyte adhesion to central nervous system (CNS) vascular EC is largely dependent on LFA-1 but not through its interaction with ICAM-1. In contrast, lymphocyte diapedesis is mostly supported through the LFA-1/ICAM-1 pairing, with a small proportion being mediated by VLA-4/VCAM-1 on IL-1 beta-activated EC. This latter pathway, however, also appears to be dependent on LFA-1 interacting with the EC.

Antigen presentation by rat brain and retinal endothelial cells.

Brain and retinal endothelial cells (EC) form the blood-brain and vascular blood-retinal barriers, respectively, and are believed to play a role in mediating T cell responses in the central nervous system. In this study, Lewis rat retinal and brain EC grown in vitro were capable of expressing MHC class II I-A but not I-E molecules following treatment with interferon-gamma. In the presence of their antigen, CD4+ antigen-specific T cells were able to mediate lysis of retinal EC monolayers to a similar extent as brain EC. T cell proliferation was poorly supported by confluent retinal or brain EC monolayers, but subconfluent EC monolayers supported proliferation in a MHC class II (I-A)-restricted manner (P < 0.001). Exposure of T cells to confluent retinal EC monolayers resulted in them becoming less responsive to subsequent antigen presentation by thymocytes. Conversely, pre-exposure with subconfluent EC had no such effect. These results suggest that a non-proliferating EC monolayer is able to downregulate T cell responsiveness which may have important implications during lymphocyte traffic across the blood-tissue barriers of the central nervous system.

The kinetics of cytokine mRNA expression in the retina during experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU) was induced in Lewis rats and the inflamed retinas were examined for IFN-gamma, IL-2, IL-4, and IL-10 mRNA production at serial time points using the reverse transcriptase-polymerase chain reaction. IFN-gamma, IL-2, IL-4, and IL-10 mRNAs were all detected 24 hr before the earliest time point at which histological changes have previously been detected. IFN-gamma, IL-2, and IL-4 mRNA expression peaked during the active phase of the disease and declined in parallel with lymphocyte numbers as the inflammation resolved. IL-10 mRNA levels increased more slowly, reaching a maximum at later stages of disease. The observed pattern of cytokine mRNA expression in the retina in EAU is similar to that reported in experimental autoimmune encephalomyelitis (EAE). The increase in IL-10 mRNA expression in late disease may reflect a role in disease resolution as previously proposed in EAE.

Superoxide dismutase (glu100-->gly) in a family with inherited motor neuron disease: detection of mutant superoxide dismutase activity and the presence of heterodimers.

Superoxide dismutase glu100-->gly, a mutation known to be associated with familial motor neuron disease (familial amyotrophic lateral sclerosis) has been detected in one symptomatic and five of seven asymptomatic members of a family with a history of this disease. On average, the individuals with the mutation had 75% of normal red blood cell superoxide dismutase activity. Native polyacrylamide gels stained for superoxide dismutase activity showed two abnormal bands in the family members identified as carrying the mutation. This indicates that active mutant enzyme is present in red cells and forms stable homodimers and heterodimers with the normal chain. A silent mutation in exon 4, not associated with motor neuron disease, was also detected in one family member.

Oligoclonal activation of CD4+ T lymphocytes in posterior uveitis.

Several lines of evidence support an important role for activated T lymphocytes in the perpetuation of autoimmune intraocular inflammatory disease (posterior uveitis). In this study peripheral blood lymphocytes (PBL) were examined by three-colour flow cytometry to assess the distribution of IL-2 receptors (IL-2R) among CD4+ and CD8+ T cell subsets in patients with active posterior uveitis and control subjects. Patients with uveitis (n = 70) showed a significant increase in PBL expressing the alpha-chain (Tac) of the IL-2R compared with controls (n = 28) (34.2% versus 29.6%) (P < 0.05). This increased Tac expression was present on both the CD4+ subset (25.7% versus 20.9%) (P < 0.05) and the CD8+ subset (2.5% versus 1.8%) (P < 0.05) of lymphocytes. We also examined whether the activated CD4+ PBL from uveitis patients (n = 30) showed a dominant pattern of T cell receptor (TCR) gene rearrangement, suggestive of an oligoclonal response to a small number of antigenic peptides. A significant increase in the usage of the V alpha 2.3 TCR family by activated but not by non-activated CD4+ PBL was detected in patients (3.9% versus 3.4%) (P < 0.05) compared with controls. There was evidence of oligoclonal activation of CD4+ PBL in 11/30 patients (36.7%) but in none of the controls (n = 10). However, different V alpha or V beta TCR families were selectively activated among and even within individual patients. The heterogeneity in TCR expression among patients with active intraocular inflammatory disease is discussed.

Role of tumour necrosis factor-alpha in dendritic cell-mediated primary mixed leucocyte reactions.

Tumour necrosis factor (TNF alpha) is a major inflammatory cytokine with potentiating effects on specific immune responses, including graft-versus-host disease. This study examined the contribution of TNF alpha to dendritic cell (DC)-mediated primary allogeneic T lymphocyte responses. Purified blood DC were shown to produce minimal amounts of TNF alpha mRNA but no significant TNF biological activity or secreted TNF alpha as measured by ELISA. Amplification of DC mRNA by PCR using oligonucleotide primers to CD120a (TNFRI, p55) and CD120b (TNFRII, p75) and probing with specific internal oligonucleotides, suggested that DC express the CD120b but little if any CD120a. These results were confirmed using monoclonal antibodies to the TNF receptors. Polyclonal antiserum specific for TNF alpha blocked the blood DC-stimulated allogeneic mixed leucocyte reaction (MLR). The addition of TNF alpha to suboptimal MLRs (limited DC stimulators), increased the proliferation of responding T lymphocytes. Having confirmed that T lymphocytes produce TNF alpha and express CD120b after stimulation, we sought to clarify whether the contributing effect of TNF alpha to the allogeneic MLR resulted from a TNF alpha-mediated signal stimulating DC activity, or as a result of autocrine stimulation of T lymphocytes. Pre-incubation of DC with TNF alpha did not increase DC stimulatory capacity and late addition of anti-TNF serum (up to 72 h) still had a significant inhibitory effect on the MLR. We conclude that TNF alpha is probably not involved in the initial DC-T lymphocyte interaction, but acts as an autocrine growth factor for DC induced T lymphocyte proliferation.

Differential lymphokine expression by rat antigen-specific CD4+ T cell lines with antigen and mitogen.

Retinal soluble antigen (S-Ag) and purified protein derivative (PPD)-specific T cell lines established from Lewis rats were used to study the pattern of lymphokine expression to see if it varied with the inducing stimulus. Lymphokine mRNA expression was detected by PCR combined with Southern analysis after 6-hr stimulation and protein secretion assessed by bioassays at 24 hr poststimulation. S-Ag-specific T cell lines when stimulated with antigen expressed IL-2, IFN-gamma, and IL-4 mRNA, whereas only IL-2 and IFN-gamma could be detected in the supernatants. This is in contrast to the findings after stimulation of the PPD cell lines with PPD where IL-4 could be detected in the supernatants. The time course studies (3, 6, 24, 48, and 72 hr) with one of the S-Ag-specific T cell lines showed that S-Ag activation did not induce any detectable IL-4 bioactivity. However, when the S-Ag T cell line was stimulated by Con A or PMA, IL-4 was detected in the supernatants following Con A activation, suggesting that the way in which the T cell is activated has an effect on its resultant lymphokine secretion.