Murine respiratory tract dendritic cells: isolation, phenotyping and functional studies.

Respiratory tract dendritic cells (RTDC) form a contiguous subepithelial network within the nasorespiratory tract bridging innate and acquired immunity and have been implicated in nasal mucosal tolerance induction. Discrepancies exist between isolation techniques with respect to phenotype and function of RTDC. Therefore, the aim of this study was to modify previous methods to provide a consistent isolation method whilst maintaining good cell viability and enriched cell numbers so as to facilitate further phenotype and functional studies of murine RTDC. RTDCs isolated by enzyme digestion, Percoll density gradient centrifugation and overnight GM-CSF culture followed by MACS separation retain an archetypical immature dendritic cell phenotype, characterised by MHCII(low) CD40(neg) CD86(neg) CD80(neg) CD11c(low) cell surface expression. Splenic-derived DC (SDC) isolated conformed to a day 1 in vitro phenotype; MHCII(low) CD40(neg) CD86(low) CD80(neg) CD11c(low) but can further mature phenotypically in vitro. Both RTDC and SDC processed and presented antigen efficiently to T cells in vitro. Using such modified isolation procedures for RTDCs, we have developed a consistent method of RTDC enrichment, which maintains the immature phenotype and functional antigen presenting capability.

Single dose intranasal administration of retinal autoantigen generates a rapid accumulation and cell activation in draining lymph node and spleen: implications for tolerance therapy.

BACKGROUND/AIMS: A single intranasal delivery of retinal autoantigen suppresses effectively experimental autoimmune uveoretinitis (EAU). To further unravel underlying mechanisms the authors wished to determine, firstly, the kinetics of antigen delivery and, secondly, the early cellular responses involved in the initial stages of nasal mucosal tolerance induction. METHODS: Flow cytometry, cell proliferation assays, and microscopy were used to track antigen following a single, intranasal dose of Alexa-488 labelled retinal antigen. RESULTS: A rapid accumulation of antigen within both superficial cervical lymph nodes (SCLN) and spleen was observed after 30 minutes. Significant proliferative responses to IRBP were elicited by 48 hours indicating that systemic priming of naive T cells to retinal antigen had occurred. Cell activation was further confirmed by immunoprecipitation studies, which demonstrated phosphorylation of STAT4 but not STAT6 in both lymph nodes and spleen. However, at 24 hours, STAT4 heterodimerisation with STAT 3 was only observed in spleen. CONCLUSIONS: The results provide novel evidence that following a single intranasal application rapid transfer of antigen occurs. Resulting T cell proliferation develops consequent to differential cell signalling in SCLN and spleen. Further understanding of these underlying cellular mechanisms, in particular as is inferred by the results the contribution of local versus systemic tolerance induction, may assist in strategies to clinically apply mucosal tolerance therapy successfully.

CD69 expression on peripheral CD4+ T cells parallels disease activity and is reduced by mycophenolate mofetil therapy in uveitis.

PURPOSE: To assess the effects of mycophenolate mofetil (MMF) therapy on T helper cell activation status, using CD69 expression and cytokine profile with flow cytometry in relation to clinical activity in uveitis. METHODS: Patients with posterior or intermediate uveitis treated with MMF (n = 10), patients with active uveitis not treated with MMF and receiving no or minimal therapy (n = 10), and healthy volunteers (n = 21) had peripheral blood lymphocyte immunofluorescence analysis for T helper cell (CD4, CD3) markers, activation status (CD69), and intracellular cytokine (interleukin [IL]-2, interferon [IFN]-gamma, and IL-4) levels. Patients were compared before and during MMF therapy in relation to T helper cell activation and clinical activity. RESULTS: Patients with active uveitis not treated with MMF and receiving no or minimal therapy had increased frequency of CD69-positive CD4 T cells (10.5% +/- 4.6%, P = 0.0007) compared with healthy volunteers (3.3% +/- 2.7%). Of all patients receiving MMF therapy, only patients with moderate to severe uveitis activity in the pre-MMF treatment group (n = 5; 15.5% +/- 5.0%, P = 0.004) had increased frequency of CD69-positive CD4 T cells compared with healthy volunteers. During MMF therapy, a significant reduction in frequency of CD69-positive CD4 T cells occurred in patients with prior moderate to severe uveitis activity (to 8.9% +/- 3.8%, P = 0.04). Levels of CD69-positive CD4 T cells in patients who had had inactive or mildly active disease (n = 5) before and during MMF therapy were comparable with levels in healthy volunteers. No significant changes in cytokine levels were found between the patient and control groups. A significant association between changes in frequency of CD69-positive CD4 T cells and changes in visual acuity (P = 0.008) and changes in vitreal haze (binocular indirect ophthalmoscopy score; P = 0.01) was observed in MMF-treated patients with prior moderate to severe uveitis activity. CONCLUSIONS: Reduction in uveitis activity during MMF therapy correlates with reduction in frequency of peripheral blood CD69-positive CD4 cells. The frequency of CD69-positive CD4 T cells is a measure of activity in posterior uveitis and may guide adequate immunosuppression.

Immunogenetics and clinical phenotype of sympathetic ophthalmia in British and Irish patients.

BACKGROUND/AIMS: Sympathetic ophthalmia (SO) is a classic example of autoimmune disease where human leucocyte antigen (HLA) genomic associations could provide further understanding of mechanisms of disease. This study sought to assess HLA genetic polymorphism in British and Irish patients with SO, and to assess whether HLA gene variants are associated with clinical phenotype or disease severity. METHODS: High resolution DNA based HLA typing using polymerase chain reaction sequence specific primers was performed in 27 patients with SO and 51 matched healthy controls. Clinical phenotype and markers of disease severity were determined prospectively in 17 newly diagnosed patients and from medical record review and repeat clinical examination in 10 previously diagnosed patients. RESULTS: HLA-Cw*03 (p=0.008), DRB1*04 (p=0.017), and DQA1*03 (p=0.014) were significantly associated with SO. For class II alleles at higher resolution, only HLA-DRB1*0404 (relative risk (RR) = 5.6, p = 0.045) was significantly associated with SO. The highest relative risk for any of the associated haplotypes was with HLA-DRB1*0404-DQA1*0301 (RR=10.9, p=0.019). Patients with the DRB1*04-DQA1*03 associated haplotype were significantly more likely to develop SO earlier, with fewer inciting ocular trauma events, and to require more systemic steroid therapy to control inflammatory activity. CONCLUSIONS: Sympathetic ophthalmia is associated with HLA-DRB1*04 and DQA1*03 genotypes in white patients, similar to Japanese patients. Differences in DRB1*04 gene variant associations (-0404 in Britain and Ireland and -0405 in Japan) may have implications for HLA peptide binding in disease initiation. The DRB1*04-DQA1*03 haplotype is a marker of increased SO susceptibility and severity, as in Vogt-Koyanagi-Harada disease, which also has similar clinicopathological and HLA associations.

Expression of human beta-defensins in intraocular tissues.

PURPOSE: Defensins are naturally occurring antimicrobial peptides. Recently the authors published evidence of defensin production by the human ocular surface. A study was undertaken to look for intraocular defensins that may account for unexplained antimicrobial activity of intraocular fluids. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was performed on human postmortem ciliary body samples for beta defensins-1 (HBD-1) and beta defensin-2 (HBD-2), and alpha defensins 5 and 6. Induction of defensins by cytokines was analyzed in cultured human ciliary body epithelial (CBE) and retinal pigment epithelial (RPE) cells. Polyclonal antibodies were used to immunoblot aqueous and vitreous to detect HBD-1 and HBD-2 and to estimate their concentration. RESULTS: RT-PCR revealed constitutive HBD-1 message in ciliary body. HBD-2 and alpha defensin 5 and 6 messages were absent. HBD-2 message was induced by cytokine stimulation of both CBE and RPE cells. Immunoblots of vitreous and aqueous stained positively for HBD-1 but not HBD-2. The estimated aqueous concentration of HBD-1 was less than 16 ng/ml. CONCLUSIONS: This study demonstrates that HBD-1 is constitutively present in the aqueous and vitreous, probably at sub-bacteriocidal concentrations. HBD-2 was absent from aqueous, but cytokine stimulation studies suggest that it may be generated in response to inflammatory cytokines during infections. HBD-2 has a wider antibacterial spectrum, is 10-fold more potent, and may play a more significant role in antimicrobial defense than HBD-1. The use of defensins therapeutically may be indicated; however, caution is required because defensins also promote cell proliferation and fibrin formation, which are 2 key elements in ocular scarring processes such as proliferative vitreoretinopathy.

Induction or suppression of a B cell-specific response to self antigen in vivo is dependent upon dendritic cell activation via the TNF-alpha receptor at the time of antigen uptake.

In this study we show that the retinal autoantigen, S-antigen, contains a functional TNF-alpha homologous domain which stimulates maturation and differentiation of cultured dendritic cells (DC) or tissue DC via the p55 TNF-alpha receptor. Tissue DC became more dendritiform in shape, and migrated into culture supernatant. S-antigen also stimulated accumulation of cell surface MHC class II antigen with a corresponding loss of acidic intracellular vesicles, and induced IL-1beta and IL-12 mRNA expression in cultured bone marrow-derived DC. In addition, cultured splenic DC primed immune responses to S-antigen in vivo in the absence of other, exogenous cytokine sources. DC pulsed with either retinal S-antigen or another retinal autoantigen, interphotoreceptor retinoid binding protein (IRBP), were able to stimulate naive T cell proliferation in vitro, but only S-antigen-pulsed DC were able to induce an immune response in vivo and initiate antibody class switching. In contrast, IRBP-pulsed DC had no detectable in vivo priming effect and IgG antibody levels remained suppressed even after immunization with IRBP in complete Freund's adjuvant. These results indicate that DC from the same precursor population can either induce or suppress a B cell-specific response to self antigen in vivo, the outcome being dependent upon DC activation at the time of antigen uptake and presentation.

Fas-Fas ligand-mediated apoptosis within aqueous during idiopathic acute anterior uveitis.

PURPOSE: Despite ocular immune privilege, (auto)immune-mediated acute anterior uveitis (AAU) is relatively common. However, although relapses of AAU are usually self-limiting, possible regulatory mechanisms remain undefined in humans. Experimentally, Fas-Ligand (FasL)-mediated apoptosis of Fas+ inflammatory cells contributes to the immune privilege within the anterior chamber and provides an explanation for the success of corneal allograft transplantation. Therefore, whether such mechanisms regulate the immune response in AAU was investigated. METHODS: Aqueous and peripheral blood samples from consecutive patients presenting with idiopathic AAU were obtained with consent. Leukocytic phenotype was analyzed by flow cytometry, and apoptosis was determined by both flow cytometry and TdT-dUTP terminal nick-end labeling analysis. Presence of soluble Fas and FasL was determined by western blot analysis and enzyme-linked immunosorbent assay and compared with control aqueous from patients undergoing cataract surgery. The ability of the aqueous to induce apoptosis in a Fas+ Jurkat cell line was also determined. RESULTS: During AAU aqueous-infiltrating Fas+ cells included CD3+ T cells and granulocytes, whereas FasL+ cells comprised predominantly of non-CD3+ T cells. Higher levels of functional soluble FasL were found in aqueous of AAU patients than in normal aqueous, capable of inducing apoptosis in 68.9% +/- 7.6% of Fas+ lymphoid cells. Compared with peripheral blood, the CD4+ T cells infiltrate within aqueous showed significantly increased CD69 and CD25(IL-2r) expression. Flow cytometric analysis of aqueous showed that 9.32% +/- 1.2% of infiltrating non-granulocyte CD45+ cells were apoptotic, confirmed as T cells on subsequent three-color flow cytometric analysis. CONCLUSIONS: Taken together with published experimental data, the present study provides evidence for FasL-mediated apoptotic cell death contributing to the local immune regulation of ocular inflammatory disease and provides a mechanism to account for the self-limiting clinical course of AAU.

Uveitogenic epitopes of retinal S-antigen are generated in vivo via an alternative antigen-presentation pathway.

We have found that different antigen-processing pathways are involved in the induction of experimental autoimmune uveoretinitis (EAU) by the retinal autoantigens S-antigen and interphotoreceptor retinoid-binding protein (IRBP). Although in vitro T-cell proliferative responses to IRBP were completely inhibited in the presence of irreversible cysteine protease inhibitors, no significant reduction of S-antigen proliferative responses was found. Furthermore, acidic proteolysis of S-antigen by the cysteine protease cathepsin B prior to immunization radically reduced pathogenicity (disease severity). In addition, in vitro processing of S-antigen, but not IRBP, was also found to be resistant to the action of cycloheximide and lysosomotropic agents, inhibition of proliferation only occurring after extended exposure of antigen-presenting cells to methyl amine or high concentrations of chloroquine. These data indicate that an alternative pathway of antigen processing exists for S-antigen, which is independent of processing within the normal endolysosomal pathway and that uveitogenic peptides of naturally processed S-antigen bind to major histocompatibility complex class II antigens either at the cell surface or within very early endosomes where cathepsin B is inactive.

In vivo cell tracking by scanning laser ophthalmoscopy: quantification of leukocyte kinetics.

PURPOSE: To image peripheral blood leukocyte traffic in the normal retinal and choroidal vasculature and to quantify the differences in the circulation dynamics between normal and concanavalin A (ConA)-activated leukocytes. METHODS: Normal or ConA-activated splenocytes were fluorescently labeled in vitro with 6-carboxyfluorescein diacetate (CFDA) and reinfused in vivo where they were tracked in the retinal and choroidal circulations of syngeneic rats by means of a scanning laser ophthalmoscope (SLO). Simultaneous digital and video images were captured for as long as 30 minutes, and the initial 15 seconds of image sequences and leukocyte dynamics were analyzed from digitized images by recording the velocity of trafficking cells and the number of stationary cells that accumulated with time, using a customized software package. RESULTS: Mean velocity (+/-SD) was 29.8 +/- 15.3 mm/sec in the retinal arteries, 14.7 +/- 7.2 mm/sec in the retinal veins, and 3.0 +/- 3.6 mm/sec in the retinal capillaries. Mean velocity in the choroidal vessels was 6.1 +/- 6.0 mm/sec. No significant difference in leukocyte velocity was found between activated and normal leukocytes in any of the vessel systems. However, activated leukocytes were observed to accumulate more within the choroidal vasculature (P < 0.001) and the retinal capillaries (P < 0.001) than in control animals, but not in larger retinal vessels. CONCLUSIONS: A technique to measure the kinetics of circulating leukocytes in vivo has been developed. Although leukocyte activation itself is insufficient to cause slowing of leukocyte velocity, the data indicate that leukocyte adherence to endothelium can be induced in the absence of local or systemic activating stimuli.

Nitric oxide accelerates the onset and increases the severity of experimental autoimmune uveoretinitis through an IFN-gamma-dependent mechanism.

The production of large amounts of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been described as a double-edged sword eliciting pro- or anti-inflammatory effects in different immune situations. Our aim, therefore, was to investigate its role in experimental autoimmune uveoretinitis (EAU), a model of ocular inflammation, induced in the Lewis rat following a single footpad injection of retinal Ags. iNOS enzyme was not detected in the normal Lewis rat eye, but was strongly expressed by infiltrating ED1+ macrophages during the acute inflammatory stages of EAU. Treating immunized animals with L-arginine increased urinary NO metabolite (NOx) levels, accelerated the inflammatory response, and increased disease severity, whereas treatment with the NOS inhibitor, N(G)-nitro-L-arginine methyl ester, reduced NOx excretion, delayed the onset, and reduced the clinical signs of EAU. Reverse transcription-PCR analysis of ocular tissue from untreated and treated animals detected iNOS mRNA at all stages of disease, and expression was up-regulated during peak disease. L-arginine treatment enhanced cytokine mRNA expression, particularly of IFN-gamma, which was detected earlier than in control animals, corresponding with the more rapid onset of disease and increased disease severity observed in this group. N(G)-nitro-L-arginine methyl ester had little or no effect on iNOS or inflammatory cytokine mRNA expression. These results suggest NO is central to the pathogenesis of EAU and highlight the importance of the macrophage as an effector cell in what is considered a CD4+ T cell-dependent disease. Furthermore, this study demonstrates the therapeutic potential of NOS inhibitors in the treatment of inflammatory and autoimmune mediated disease.

Interphotoreceptor retinoid binding protein is a potent tolerogen in Lewis rat: suppression of experimental autoimmune uveoretinitis is retinal antigen specific.

AIMS: Administration of unfractionated retinal antigen(s) (retinal extract, RE) suppresses RE induced experimental autoimmune uveoretinitis (EAU) and offers a potential therapeutic alternative to non-specific immunosuppressive therapies for posterior uveitis and autoimmune diseases. S-Ag and interphotoreceptor retinoid binding protein (IRBP) are two major autoantigens within soluble RE. It was aimed to assess, firstly, as has previously been shown with S-Ag, if IRBP can induce intranasal tolerance and, secondly, the contribution of both these major autoantigens to tolerance induction by whole RE. METHODS: Animals were tolerised by intranasal administration with S-Ag or IRBP, either alone or in combination, or RE before immunisation with either IRBP or RE. Control animals were administered nasally either PBS or MBP. Daily clinical responses were recorded biomicroscopically and histological grades were obtained using a semiquantitative scoring system. Weekly serum antibody levels to retinal antigens were measured by ELISA and delayed hypersensitivity responses (DTH) were assessed by skin reactivity to intradermal inoculation with retinal or non-specific antigens. RESULTS: Microgram doses of IRBP successfully suppressed both clinically and histologically IRBP induced EAU. This suppression was accompanied by reduced antigen specific DTH reactivity but maintained T cell dependent (IgG2a) antibody responses. Furthermore, combined S-Ag and IRBP administration afforded equal suppression of RE induced EAU when compared with RE therapy alone. Suppression of RE induced EAU was not achieved with administration of a non-retinal specific autoantigen, MBP. Although individually, both S-Ag and IRBP suppressed RE induced EAU, whole RE was unable to protect against IRBP induced disease. CONCLUSIONS: Intranasal administration of IRBP suppressed IRBP induced EAU in the Lewis rat. S-Ag and IRBP are the major contributors to the tolerogenicity within RE, despite the known uveogenicity of other retinal antigens within RE and induction of tolerance was retinal antigen specific. Furthermore, suppression induced by single antigen administration is antigen specific although concomitant bystander suppression may also play a role. RE was unable to protect against IRBP induced disease despite tolerogenic levels of antigen within RE. Although this may be due in part to a dose effect of either tolerising or immunising antigen, further investigation into the possible antigen dominance of IRBP or mucosal processing of combinations of antigens is necessary so that the full efficacy of mucosal tolerance therapy can be assessed.

Nasal administration of retinal antigens maintains immunosuppression of uveoretinitis in cyclosporin-A-treated Lewis rats: future treatment of endogenous posterior uveoretinitis?

PURPOSE: Current treatment of autoimmune endogenous posterior uveoretinitis (EPU) is limited by drug toxicity, unpredictable relapses on dose reduction and resistance to therapy. Administration of autoantigens via gastrointestinal or respiratory mucosa prior to antigen exposure induces immune hyporesponsiveness (mucosal tolerance) to further antigen sensitisation. In this study we assessed whether mucosal tolerance induction was possible after immunisation with retinal antigens in experimental autoimmune uveoretinitis (EAU) in animals that were short-term immunosuppressed with cyclosporin A (CsA) to determine whether mucosal administration of retinal antigens can maintain immunosuppression in sensitised and immunosuppressed individuals. METHODS: Female Lewis rats were immunised with retinal extract (RE) and then treated as follows. Group 1 received no specific therapy and served as control; group 2 were fed CsA from day 7 to day 20 post-immunisation; group 3 received inhalational tolerance therapy with RE in addition to CsA; tolerance therapy was continued after day 20 when CsA was stopped. Experiments varying the timing and dosage of both tolerising and immunising antigen were also performed, the details of which are described. Incidence, day of onset and clinical activity were recorded and histopathological assessment of intraocular inflammation, in particular the extent of autoimmune target-organ damage, was graded semiquantitatively. RESULTS: Compared with controls and group 2, group 3 showed both a marked delay in disease onset and a reduction in disease severity. This effect was both dose and dose-timing dependent. Tissue damage assessed in terms of preservation of rod outer segments was significantly less in group 3. CONCLUSIONS: The success of combination therapy, clinically, remains unknown at present but these results support continuing present clinical trials of mucosal tolerance therapy and in particular have future implications for either maintaining or inducing immunosuppression in autoimmune diseases in combination with present immunosuppressive therapies.

Prostaglandin E2 and monoclonal antibody to lymphocyte function-associated antigen-1 differentially inhibit migration of T lymphocytes across microvascular retinal endothelial cells in rat.

Lymphocyte-transendothelial cell migration is a complex event, and although much is known about the receptor-ligand interactions involved, little is understood about the intracellular events which accompany these interactions, or their regulation by inflammatory mediators. In this study we have shown that activation of T lymphocytes increased the proportion of cells migrating across monolayers of cultured retinal microvascular endothelial cells by both lymphocyte function-associated antigen-1 (LFA-1)-dependent and LFA-1-independent mechanisms. In preliminary experiments, it was found that activation of T cells with mitogens such as concanavalin (Con A) increased significantly T-cell migration across the endothelial monolayers. In contrast, activation of the endothelial monolayer with interferon-gamma (IFN-gamma) (96 hr) had no effect on transendothelial migration. Investigation of adhesion molecule requirements for transendothelial migration indicated that LFA-1 was necessary (P < 0.02) but that intracellular adhesion molecule-1 did not appear to be involved. Investigation of the role of prostaglandins in transendothelial migration revealed that, while prostaglandin E2 (PGE2) did not affect adhesion molecule expression on endothelial cells or T cells, treatment of either cell significantly blocked transendothelial migration (P < 0.05). Pretreatment with PGE2 combined with LFA-1 blockade had an additive effect on inhibition of T-cell transendothelial migration, indicating that two independent mechanisms were operative. PGE2 also had a direct inhibitory effect on T-cell adhesion to the endothelium. These results highlight the importance of considering non-adhesion receptor-mediated mechanisms, perhaps involving cytoskeletal and/or motility, in the migration of T cells across endothelial monolayers.

CD59 and CD48 expressed by rat retinal pigment epithelial cells are major ligands for the CD2-mediated alternative pathway of T cell activation.

The alternative CD2-mediated pathway of T cell activation, which is independent of MHC/peptide recognition by the TCR/CD3 complex, is dependent upon two signals being received by the CD2 molecule. The natural ligand for CD2 is CD58, but controversy exists over alternative or additional ligands that could deliver the second signal in vivo. We have used rat retinal pigment epithelial cells (RPE), which lack temperature-insensitive ligands for CD2 adhesion, to study Ag-independent T cell activation. Rat RPE cells expressed high levels of CD59 and low levels of another potential CD2 ligand, CD48, both in vitro and in the in vivo model of experimental autoimmune uveoretinitis. When increasing numbers of syngeneic T cells were added to microwell cultures of rat RPE cells, the T cells, even in the absence of any exogenous stimulant in the cultures, underwent spontaneous proliferation. This effect required metabolically active RPE cells, and was IL-2 driven and enhanced in the presence of indomethacin. Proliferation was modulated by phosphatidylinositol-phospholipase C treatment of the RPE, and blocked by mAbs to CD59. Ab cross-linking of CD48 but not CD59 on the RPE was found to induce messenger RNA expression for IL-1 beta, which together with constitutively expressed IL-6 are required costimulatory factors for T cell activation through CD2. This is the first demonstration in a fully syngeneic system that bi-directional signaling involving CD59 and CD48 molecules expressed by physiologically normal, nonhematopoietic, cells can trigger T lymphocyte activation and proliferation through autocrine IL-2 production in the absence of Ag.

Peripheral CD25 positive T lymphocytes with biased T cell receptor Vbeta gene usage in autoimmune endogenous posterior uveitis.

Aims-To determine T cell receptor (TCR) Vbeta gene usage in peripheral blood T lymphocytes of patients with endogenous posterior uveitis (EPU). If biased TCR variable (V) gene usage occurs in this autoimmune disease, it should be detectable in immune activated peripheral blood T cells in vivo.Methods-Relative proportions of each Vbeta gene family expressed in total peripheral blood lymphocytes (PBL) and in vivo activated (CD25+) T cells from patients with EPU and controls were determined using the anchored polymerase chain reaction (anchored PCR) in conjunction with a novel hybridisation assay. The TCR Vbeta repertoires seen in these cell populations were then compared.Results-Vbeta1 usage within the CD25+ lymphocytes of patients with EPU was substantially elevated (mean +/- SD 15 +/-9%) compared with control CD25+ cells (3.3 +/-2.4%).Conclusions-By contrasting the repertoires of these cell populations, biased TCR Vbeta gene usage was detected in patients with EPU, namely increased usage of Vbeta1 in CD25+ T cells from peripheral blood of these patients. This approach of directly analysing the activated T cells in blood, using bulk PBL as an internal control, has wide applicability where specific T cell subpopulations are thought to play an important aetiopathological role.

Rat retinal pigment epithelial cells express an inducible form of nitric oxide synthase and produce nitric oxide in response to inflammatory cytokines and activated T cells.

In this report we show that rat retinal pigment epithelial (RPE) cells express an inducible form of nitric oxide synthase (iNOS) and secrete high levels of nitric oxide (NO.) when co-cultured with activated lymphocytes. We have previously shown that cultured rat RPE cells suppress syngeneic lymphocyte proliferation, an effect attributed to prostaglandin E2 (PGE2) secretion by the RPE cells. However supernatants from such co-cultures were also found to contain high levels of nitrite (NO2-), the stable end-product of NO. synthesis. RPE cell secretion of NO. was stimulated by the cytokines interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), an effect enhanced by endotoxin [lipopolysaccharide (LPS)], reduced by the competitive inhibitor of L-arginine metabolism, NG-monomethyl-L-arginine (L-NMMA) and inhibited by cycloheximide. These effects were dose dependent. Using reverse transcription (RT)/PCR a product of 1398 bp was amplified which showed sequence identity with iNOS cloned from rat vascular smooth muscle. Northern blot analysis of total RNA extracted from rat RPE before and after cytokine stimulation showed induction of a 4.5 kb (kilobase) transcript which hybridized with a 1398 bp (base pair) polymerase chain reaction (PCR)-generated cDNA probe derived from the sequence of rat RPE cell iNOS. These results indicate RPE cells express an inducible form of nitric oxide synthase (NOS) and that high levels of NO. may be produced locally in the eye by the RPE in the presence of activated lymphocytes. Given the cytostatic and cytotoxic properties of this molecule, NO. may play an important role as an inducible mediator of immunosuppressive mechanisms within the microenvironment of the eye at the site of lymphocyte activation.

ICAM-1/LFA-1 interactions in T-lymphocyte activation and adhesion to cells of the blood-retina barrier in the rat.

To identify the signals involved in the adhesion and subsequent migration of lymphocytes across the endothelium (REC) and pigment epithelium (RPE) of the blood-retina barrier we have studied the effects of monoclonal antibodies (mAb) to rat adhesion/accessory molecules on the binding of normal and concanavalin A (Con A)-activated rat spleen lymphocytes to cultured unstimulated and interferon-gamma (IFN-gamma)-stimulated RPE and REC. Forty to 48% of unactivated T cells were found to bind to normal REC or RPE by leucocyte function-associated antigen-1/intercellular adhesion molecule-1 (LFA-1/ICAM-1)-independent mechanisms, despite constitutive expression of ICAM-1 by the RPE cells and LFA-1 by the T cells. Con A-activated lymphocytes showed an enhanced adhesion to both RPE and REC. However, IFN-gamma-stimulated RPE and REC did not demonstrate a significant increase in adhesiveness for normal lymphocytes highlighting the importance of lymphocyte integrin activation from low-affinity to high-affinity state. Activated lymphocyte adhesion to unstimulated RPE and REC was significantly blocked by LFA-1 mAb (35%, P < 0.0001) and ICAM-1 mAb (20%, P < 0.001). Inhibition of adhesion by antibody to CD2 was not significant. Both ICAM-1 and LFA-1 mAb also significantly (P < 0.05) blocked antigen presentation following retinal extract stimulation of lymphocytes from immunized rats in proliferation assay. These data suggest that the ICAM-1/LFA-1 system is important in lymphocyte trafficking into the eye only after lymphocyte activation.

Intranasal administration of retinal antigens suppresses retinal antigen-induced experimental autoimmune uveoretinitis.

Bovine retinal extract (RE) is a heterologous mixture of highly uveitogenic proteins including S-Antigen (S-Ag), interphotoreceptor retinol binding protein (IRBP) and rhodopsin, and is a potent inducer of experimental autoimmune uveoretinitis (EAU). Intranasal inoculation of Lewis rats with RE performed daily for 10 days prior to immunization with RE suppresses both the severity and the incidence of the clinical response and histopathological changes in EAU. Significant suppression of the disease in treated animals could be achieved with a total (cumulative) intranasal inoculum of 42 micrograms of antigen. Animals which were treated with extract exhibited a normal total antibody response to S-Ag, IRBP and retinal extract when compared with controls [phosphate-buffered saline (PBS) treated] animals. The antibody response in tolerized animals was predominantly anti-S-Ag IgG2a with suppression of anti-S-Ag IgM response. Treated animals had a significantly suppressed delayed-type hypersensitivity (DTH) response to retinal extract but normal response to purified protein derivative (PPD) compared to control animals. Adoptive transfer of splenocytes from treated animals also demonstrated some protection against RE-induced EAU. These results demonstrate that tolerance induction impairs the onset and severity of EAU by inhibiting the DTH response to heterologous mixture of retinal antigens.