Dendritic cells play no significant role in the laser-induced choroidal neovascularization model.

Age-related macular degeneration (AMD) is genetically associated with complement. Dendritic cells (DCs) play key roles during innate and adaptive immunity, and express complement components and their receptors. We investigated ocular DC heterogeneity and the role of DCs in the laser-induced choroidal neovascularization (CNV) model. In order to determine the function of DCs, we used two models of DC deficiency: the Flt3-/- and Flt3l-/- mouse. We identified three types of ocular DCs: plasmacytoid DC, classical DC-1, and classical DC-2. At steady-state, classical DCs were found in the iris and choroid but were not detectable in the retina. Plasmacytoid DCs existed at very low levels in iris, choroid, and retina. After laser injury, the number of each DC subset was up-regulated in the choroid and retina. In Flt3-/- mice, we found reduced numbers of classical DCs at steady-state, but each DC subset equally increased after laser injury between wildtype and Flt3-/- mice. In Flt3l-/- mice, each DC subsets was severely reduced after laser injury. Neither Flt3-/- or Flt3l-/- mice demonstrated reduced CNV area compared to wildtype mice. DCs do not play any significant role during the laser-induced CNV model of neovascular AMD.

Zika Virus Infection of Human Iris Pigment Epithelial Cells.

During recent Zika epidemics, adults infected with Zika virus (ZIKV) have developed organ-specific inflammatory complications. The most serious Zika-associated inflammatory eye disease is uveitis, which is commonly anterior in type, affecting both eyes and responding to corticosteroid eye drops. Mechanisms of Zika-associated anterior uveitis are unknown, but ZIKV has been identified in the aqueous humor of affected individuals. The iris pigment epithelium is a target cell population in viral anterior uveitis, and it acts to maintain immune privilege within the anterior eye. Interactions between ZIKV and human iris pigment epithelial cells were investigated with infectivity assays and RNA-sequencing. Primary cell isolates were prepared from eyes of 20 cadaveric donors, and infected for 24 hours with PRVABC59 strain ZIKV or incubated uninfected as control. Cytoimmunofluorescence, RT-qPCR on total cellular RNA, and focus-forming assays of culture supernatant showed cell isolates were permissive to infection, and supported replication and release of infectious ZIKV. To explore molecular responses of cell isolates to ZIKV infection at the whole transcriptome level, RNA was sequenced on the Illumina NextSeq 500 platform, and results were aligned to the human GRCh38 genome. Multidimensional scaling showed clear separation between transcriptomes of infected and uninfected cell isolates. Differential expression analysis indicated a vigorous molecular response of the cell to ZIKV: 7,935 genes were differentially expressed between ZIKV-infected and uninfected cells (FDR < 0.05), and 99% of 613 genes that changed at least two-fold were up-regulated. Reactome and KEGG pathway and Gene Ontology enrichment analyses indicated strong activation of viral recognition and defense, in addition to biosynthesis processes. A CHAT network included 6275 molecular nodes and 24 contextual hubs in the cell response to ZIKV infection. Receptor-interacting serine/threonine kinase 1 (RIPK1) was the most significantly connected contextual hub. Correlation of gene expression with read counts assigned to the ZIKV genome identified a negative correlation between interferon signaling and viral load across isolates. This work represents the first investigation of mechanisms of Zika-associated anterior uveitis using an in vitro human cell model. The results suggest the iris pigment epithelium mounts a molecular response that limits intraocular pathology in most individuals.

Plasmacytoid dendritic cells in the eye.

Plasmacytoid dendritic cells (pDCs) are a unique subpopulation of immune cells, distinct from classical dendritic cells. pDCs are generated in the bone marrow and following development, they typically home to secondary lymphoid tissues. While peripheral tissues are generally devoid of pDCs during steady state, few tissues, including the lung, kidney, vagina, and in particular ocular tissues harbor resident pDCs. pDCs were originally appreciated for their potential to produce large quantities of type I interferons in viral immunity. Subsequent studies have now unraveled their pivotal role in mediating immune responses, in particular in the induction of tolerance. In this review, we summarize our current knowledge on pDCs in ocular tissues in both mice and humans, in particular in the cornea, limbus, conjunctiva, choroid, retina, and lacrimal gland. Further, we will review our current understanding on the significance of pDCs in ameliorating inflammatory responses during herpes simplex virus keratitis, sterile inflammation, and corneal transplantation. Moreover, we describe their novel and pivotal neuroprotective role, their key function in preserving corneal angiogenic privilege, as well as their potential application as a cell-based therapy for ocular diseases.

Transcriptomic and proteomic analysis of iris tissue and aqueous humor in juvenile idiopathic arthritis-associated uveitis.

Gene and protein expression profiles of iris biopsies, aqueous humor (AqH), and sera in patients with juvenile idiopathic arthritis-associated uveitis (JIAU) in comparison to control patients with primary open-angle glaucoma (POAG) and HLA-B27-positive acute anterior uveitis (AAU) were investigated. Via RNA Sequencing (RNA-Seq) and mass spectrometry-based protein expression analyses 136 genes and 56 proteins could be identified as being significantly differentially expressed (DE) between the JIAU and POAG group. Gene expression of different immunoglobulin (Ig) components as well as of the B cell-associated factors ID3, ID1, and EBF1 was significantly upregulated in the JIAU group as compared to POAG patients. qRT-PCR analysis showed a significantly higher gene expression of the B cell-related genes CD19, CD20, CD27, CD138, and MZB1 in the JIAU group. At the protein level, a significantly higher expression of Ig components in JIAU than in POAG was confirmed. The B cell-associated protein MZB1 showed a higher expression in JIAU patients than in POAG which was confirmed by western blot analysis. Using bead-based immunoassay analysis we were able to detect a significantly higher concentration of the B cell-activating and survival factors BAFF, APRIL, and IL-6 in the AqH of JIAU and AAU patients than in POAG patients. The intraocularly upregulated B cell-specific genes and proteins in iris tissue suggest that B cells participate in the immunopathology of JIAU. The intracameral environment in JIAU may facilitate local effector and survival functions of B cells, leading to disease course typical for anterior uveitis.

[HLA-B27 Positive Acute Anterior Uveitis - A Translational Perspective]. / HLA-B27-positive akute anteriore Uveitis ­ eine translationale Perspektive.

Background HLA-B27 positive acute anterior uveitis is the most common type of uveitis, and it is an autoimmune disease that can be triggered by infections. The precise mechanism of the interaction between involved microbes (mostly gram negative bacteria) and the host immune system is not clear. The disease probably results from an imbalance between pro- and anti-inflammatory components. Project description This article gives a compact overview about the current knowledge of the clinic and the etiopathogenesis of acute anterior uveitis as a basis for future research approaches. The goal of the current research is to classify the cellular and molecular pathogenetic factors in acute anterior uveitis. In this regard, a project on uveitis within the clinical research unit FOR 2240 "(Lymph)Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye", examines the hypothesis that dysregulation of regulatory cell populations and anti-inflammatory cytokines, such as interleukin-10 (IL-10), might contribute to the development of ocular autoimmunity following infections. The goal is to establish new markers for individual susceptibility in the risk group of the HLA-B27 positive population, because only about 1% of the HLA-B27 positive population will eventually develop acute anterior uveitis. Conclusions Translational research approaches to identify predisposed risk groups from the HLA-B27 population could improve patient care both on a prophylactic and a therapeutic level.

Immunological Characterization of Intraocular Lymphoid Follicles in a Spontaneous Recurrent Uveitis Model.

PURPOSE: Recently, formation of tertiary lymphoid structures was demonstrated and further characterized in the R161H mouse model of spontaneous autoimmune uveitis. In the horse model of spontaneous recurrent uveitis, intraocular lymphoid follicle formation is highly characteristic, and found in all stages and scores of disease, but in depth analyses of immunologic features of these structures are lacking to date. METHODS: Paraffin-embedded eye sections of cases with equine spontaneous recurrent uveitis (ERU) were characterized with immunohistochemistry to gain insight into the distribution, localization, and signaling of immune cells in intraocular tertiary lymphoid tissues. RESULTS: Ectopic lymphoid tissues were located preferentially in the iris, ciliary body, and retina at the ora serrata of horses with naturally-occurring ERU. The majority of cells in the tertiary lymphoid follicles were T cells with a scattered distribution of B cells and PNA+ cells interspersed. A fraction of T cells was additionally positive for memory cell marker CD45RO. Almost all cells coexpressed CD166, a molecule associated with activation and transmigration of T cells into inflamed tissues. Several transcription factors that govern immune cell responses were detectable in the tertiary lymphoid follicles, among them Zap70, TFIIB, GATA3, and IRF4. A high expression of the phosphorylated signal transducers and activators of transcription (STAT) proteins 1 and 5 were found at the margin of the structures. CONCLUSIONS: Cellular composition and structural organization of these inflammation-associated tertiary lymphoid tissue structures and the expression of markers of matured T and B cells point to highly organized adaptive immune responses in these follicles in spontaneous recurrent uveitis.

Expression of MicroRNAs in the Eyes of Lewis Rats with Experimental Autoimmune Anterior Uveitis.

Purpose. This study aimed to determine the dynamic changes of NF-κB-related microRNAs (miRNAs) and cytokines over the course of experimental autoimmune anterior uveitis (EAAU) and elucidate the possible immunopathogenesis. Materials and Methods. Uveitis was induced in Lewis rats using bovine melanin-associated antigen. The inflammatory activity of the anterior chamber was clinically scored, and leukocytes in the aqueous humor were quantified. RNA was extracted from the iris/ciliary bodies and popliteal lymph nodes to reveal the dynamic changes of eight target miRNAs (miR-155-5p, miR-146a-5p, miR-182-5p, miR-183-5p, miR-147b, miR-21-5p, miR-9-3p, and miR-223-3p) and six cytokine mRNAs (IFN-γ, IL-17, IL-12A, IL-1ß, IL-6, and IL-10). In situ hybridization of miRNA and enzyme-linked immunosorbent assay quantification of cytokines were performed to confirm the results. Results. Disease activity and leukocyte quantification were maximum at day 15 after immunization. The profiling of miRNA revealed downregulation of miR-146a-5p, miR-155-5p, miR-223-3p, and miR-147b and upregulation of miR-182-5p, miR-183-5p, and miR-9-3p. Cytokine analysis revealed IFN-γ, IL-17, IL-12A, IL-1ß, and IL-6 overexpression, with IL-10 downregulation. Conclusions. Dynamic changes of miRNAs were observed over the course of EAAU. By initiating NF-κB signaling, the expressions of downstream cytokines and effector cells from the Th17 and Th1 lineages were sequentially activated, contributing to the disease.

Infiltration of Plasma Cells in the Iris of Children With ANA-Positive Anterior Uveitis.

PURPOSE: We investigated inflammatory cell infiltrates in iris biopsies in uveitis associated with juvenile idiopathic arthritis (JIA) in comparison with other pediatric uveitis entities and noninflammatory pediatric controls. METHODS: Iridectomy specimens were obtained during elective trabeculectomy from 31 eyes of 25 patients: 12 eyes with JIA-associated uveitis, 13 eyes with other uveitis entities, and 6 eyes with open angle nonuveitic juvenile glaucoma. Histopathologic and immunohistochemical analyses were performed. A semiquantitative scoring system was used with a scale ranging from 0 to 4 depending on the number of stained cells. RESULTS: An inflammatory infiltrate was present in 8/12 (67%) specimens with JIA-associated uveitis. The cellular infiltrate in JIA specimens was characterized by the presence of CD138+ plasma cells and CD68+ macrophages, while the presence of CD20+, CD4+, and CD8+ cells was variable. Presence of plasma cells in the inflammatory infiltrates in anterior uveitis correlated with antinuclear autoantibody (ANA) positivity regardless of the diagnosis of JIA. CD4+ and CD8+ T cells were not always detectable in the iris biopsies of all childhood uveitis patients, although a slight predominance of CD4+ cells was noted. CONCLUSIONS: Children with ANA-positive anterior uveitis often show an infiltrate of plasma cells, regardless of the diagnosis of JIA. The iris of JIA-associated uveitis patients is additionally characterized by the presence of various numbers of macrophages.

Human cytokines activate JAK-STAT signaling pathway in porcine ocular tissue.

BACKGROUND: The JAK/STAT (Janus Tyrosine Kinase, Signal Transducers and Activators of Transcription) pathway is associated with cytokine or growth factor receptors and it is critical for growth control, developmental regulation and homeostasis. The use of porcine ocular cells as putative xenotransplants appears theoretically possible. The aim of this study was to investigate the response of various porcine ocular cells in vitro to human cytokines in regard to the activation of JAK-STAT signaling pathways. METHODS: Porcine lens epithelial cells, pigmented iris epithelial cells and pigmented ciliary body cells were used in this study. These cells were isolated from freshly enucleated porcine eyes by enzymatic digestion. Cultured cells between passages 3-8 were used in all experiments. Electromobility shift assay (EMSA), proliferation assay, immunofluorescence staining and flow cytometry were used to evaluate the JAK-STAT signaling pathway in these cells. RESULTS: JAK/STAT signaling pathways could be activated in porcine pigmented epithelial ciliary body cells, in pigmented iris epithelial cells and in lens epithelial cells in response to porcine and human interferons and cytokines. All cells showed very strong STAT1 activation upon stimulation with porcine interferon-gamma. Porcine ocular cells also respond to human cytokines; IFN-alpha induced strong activation of STAT1 in EMSA, flow cytometry and immunofluorescence experiments whereas activation of STAT3 was less strong in EMSA, but strong in flow cytometry and immunofluorescence. Human recombinant IL-6 activated STAT3 and human IL-4 activated STAT6. With the help of immunofluorescence assay and flow cytometry we observed nuclear localization of STAT proteins after activation of porcine ocular cells with cytokines and interferons. Human IFN-α had an inhibitory effect on porcine ocular cells in proliferation assays. CONCLUSION: Our study demonstrated that some types of human cytokines and interferon activate intracellular JAK-STAT signaling pathways in porcine ocular cells. We hypothesize that direct stimulation of the JAK-STAT pathway in porcine cells in response to human cytokines will lead to complications or failure, if pig-to-human ocular tissue xenotransplantation were to be carried out. For successful xenotransplantation among other obstacles there must be new approaches developed to regulate signaling pathways.

Anti-inflammatory effect of the alpha-melanocyte stimulating hormone in animal eyes undergoing extracapsular lens extraction.

In this study anti-inflammatory effects of the alpha-melanocyte stimulating hormone (alpha-MSH) on ocular inflammation caused by extracapsular lens extraction (ECLE) have been investigated and the potential mechanism of an anti-inflammatory effect is discussed. Pigmented rabbit eyes after ECLE were treated locally with alpha-MSH, dexamethasone, diclofenac, or saline 4 times a day (q.i.d.) for 4 weeks. The inhibitory effect of alpha-MSH on infiltrating cells in the aqueous humor (AqH) was almost twice as good as that of dexamethasone or diclofenac for 3 days, 1 week, and 2 weeks after the operation. The eyes of Sprague-Dawley rats were treated with an intravenous injection of alpha-MSH or saline immediately after ECLE. Six hours postoperatively, the iris/ciliary body exhibited increased expression of TNF-alpha and IL-6 mRNAs, which were significantly decreased after alpha-MSH treatment. The number of activated NF-kappa B (NFkappaB)-positive cells in the iris/ciliary body was also significantly reduced by the alpha-MSH treatment. These results suggested that alpha-MSH could effectively reduce ocular inflammation after ECLE, and the potential mechanism for this is by down-regulating the expression of proinflammatory cytokines and inhibiting the NFkappaB-dependent signaling pathway.

Ocular immune privilege sites.

The eye is one of the immune privilege sites of the body that is consequently protected from the detrimental and potentially blinding influences of immunologic inflammation. Within the eye, the anterior chamber has been recognized for its immune privilege property for many years now; however, a similar property detectable in the subretinal space has only recently been appreciated. These ocular sites are not only equipped with specialized mechanisms that barricade local inflammatory responses, but also induce systemic regulatory immune response. Numerous studies have characterized molecular and cellular mechanisms involved in conferring both these sites with an immune privilege status. Pigmented epithelial cells lining the anterior chamber in the iris and ciliary body area as well as those in the retina are endowed with immunomodulatory properties that contribute to ocular immune privilege. These cells, via expression of either soluble factors or membrane molecules, inhibit inflammatory T cell activation and promote the generation of regulatory T cells. In the anterior chamber resident antigen-presenting cells, influenced by the various immunosuppressive factors present in the aqueous humor, capture ocular antigens and present them in the spleen to T cells in association with NKT cells and marginal zone B cells. Immunomodulatory microenvironment created by these cells helps generate regulatory T cells, capable of interrupting the induction as well as expression of inflammatory responses. Furthermore, neural regulation of both intraocular and systemic regulatory mechanisms also contributes to ocular immune privilege.

Canine pre-iridal fibrovascular membranes: morphologic and immunohistochemical investigations.

OBJECTIVE: Pathologic intraocular neovascularization is a key component of many canine ophthalmic diseases such as uveitis, retinal detachment, intraocular neoplasms, and corneal perforation. The purpose of this study was to evaluate the structure of pre-iridal fibrovascular membranes (PIFMs) associated with several different disease processes and to identify specific factors associated with their development in the canine eye. PROCEDURE: This study examined 36 enucleated canine eyes with the diagnosis of PIFM and one of the following: lens-induced uveitis, retinal detachment, iridociliary adenoma, corneal perforation, severe hyphema, or vitreal gliovascular membranes (canine ocular gliovascular syndrome, COGS). Three histologic stains and six immunohistochemical stains were performed in all 36 PIFM eyes and four histologically normal eyes, including: hematoxylin and eosin, alcian blue periodic acid schiff (PAS), Masson's trichrome, platelet endothelial cell adhesion molecule-1 (CD31), smooth muscle actin, vimentin, laminin, vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2). RESULTS: Pre-iridal fibrovascular membrane extracellular matrix staining was consistent with collagen and mucins in all cases and positive for laminin in most cases. All PIFMs contained CD31-positive vessels and predominantly lymphoplasmacytic inflammation. Both PIFM vessels and spindle cells were positive for laminin, vimentin, smooth muscle actin, VEGF, and COX-2. Secondary intraocular pathology and immunohistochemical staining of other intraocular structures are also reported. CONCLUSIONS: Pre-iridal fibrovascular membrane morphology and immunohistochemical characteristics were similar across six canine disease processes, suggesting analogous pathophysiologic mechanisms. COX-2 and VEGF were identified using immunohistochemistry and may play a role in PIFM development.

Human iris pigment epithelium suppresses activation of bystander T cells via TGFbeta-TGFbeta receptor interaction.

Iris pigment epithelial (IPE) cells from the anterior segment in the eye are able to suppress activation of bystander responder T cells in vitro. The cultured IPE cells fully suppress proliferation and cytokine production by responder T cells via direct cell-to-cell contact. We have now investigated whether primary cultured human iris pigment epithelial (h-IPE) cells that were established from fresh iris tissues can also inhibit the activation of T cells in vitro. We found that cultured h-IPE cells significantly inhibited T cell proliferation and the IFN-gamma production by the target T cells from both the allogeneic and autogeneic peripheral blood mononuclear cells (PBMCs). The h-IPE cells also inhibited the activation of CD4(+) T cells from patients with active uveitis. The suppression by h-IPE occurred in a completely contact-dependent manner. The h-IPE constitutively expressed transforming growth factor beta (TGFbeta) and the receptors, and the T cells exposed to h-IPE greatly expressed Smad transcripts. In addition, TGFbeta2-siRNA transfected h-IPE failed to inhibit activation of responder T cells. Similarly, h-IPE cells in the presence of anti-TGFbeta neutralizing antibodies or recombinant TGFbeta receptor blocking proteins failed to inhibit the T-cell activation. In conclusion, cultured human iris pigment epithelium fully inhibits T cell activation in vitro. Our data support the hypothesis that the ocular resident cells play a critical role in immunosuppression in the eye.

Human iris pigment epithelial cells suppress T-cell activation via direct cell contact.

The purposes of the present study were to investigate whether cultured human iris pigment epithelial (hIPE) cells acquire the ability to modify T-cell activation, and if so, to identify the mechanism. Human IPE cells were prepared from patients who underwent glaucoma surgery, and were cultured in RPMI 1640 medium containing 10% fetal calf serum for 4-7 days. Expression of MHC molecules and co-stimulatory molecules on cultured hIPE cells either unstimulated or stimulated with IFN-gamma was examined by FACS. In addition, peripheral blood T cells were incubated with cultured hIPE cells prepared from the same patients and anti-CD3 antibody in a transwell culture system, or in the presence of anti-PD-L1 and PD-L2 antibodies, and T cell proliferation was assessed by [3H]-thymidine incorporation. The hIPE cells inhibited anti-CD3-driven T-cell activation but the inhibition was diminished when tested in the transwell culture system, indicating that a contact-dependent mechanism is important in the immunoregulatory roles of hIPE. Although cultured hIPE cells expressed Class I and PD-L1 but not Class II or PD-L2, all these molecules were observed on hIPE cells cultured in the presence of IFN-gamma. Blocking antibodies against both PD-L1 and PD-L2 reduced the immunoregulatory activity of hIPE cells. Our data indicates that cultured hIPE cells inhibit T-cell activation by T-cell receptor ligation, which is mediated by cell-to-cell contact in part via the PD-L1 and PD-L2 pathways.

Effects of the immunomodulator, VGX-1027, in endotoxin-induced uveitis in Lewis rats.

BACKGROUND AND PURPOSE: VGX-1027 is a novel, low molecular weight, immunomodulatory compound that has shown efficacy against a variety of immuno-inflammatory disease models in animals including autoimmune diabetes in NOD mice, collagen-induced arthritis and chemically induced inflammatory colitis. Here, we have studied the effects of VGX-1027 on the development of endotoxin-induced uveitis (EIU) in male Lewis rats, as a model of inflammatory ocular diseases in humans. EXPERIMENTAL APPROACH: EIU was induced by a single footpad injection of 200 microg lipopolysaccharide (LPS). Groups of rats were treated with either VGX-1027 (25 mg kg(-1)) or its vehicle at different time points (30 min, 6 h or 12 h) after the challenge with LPS or, as positive control, with dexamethasone. The rats were killed within 16 h after LPS challenge, and the eyes and aqueous humor were collected to study serological, immunological and histological signs of EIU. KEY RESULTS: The rats treated with VGX-1027 within 6 h after LPS challenge exhibited milder clinical, histological and laboratory signs of EIU than those treated with vehicle. CONCLUSION AND IMPLICATIONS: This study provides the first evidence that systemic treatment with VGX-1027 counteracts the uveitis-inducing effect of LPS in rats and suggests that this drug may have potential in the treatment of immuno-inflammatory conditions of the eye in humans.

Immunohistochemical study of chronic nongranulomatous anterior uveitis in juvenile idiopathic arthritis.

PURPOSE: To provide a detailed immunohistochemical analysis of juvenile idiopathic arthritis (JIA)-associated anterior uveitis. DESIGN: Interventional case report. PARTICIPANT: One patient. INTERVENTION: A 12-year-old patient had recurrent pauciarticular JIA and smoldering anterior uveitis in the right eye. Despite treatment with local and systemic corticosteroids and an anti-tumor necrosis factor agent, the right eye became hypotonous and painful and eventually was enucleated. The clinical history and histopathologic and immunohistochemical analyses of the enucleated globe were reviewed. MAIN OUTCOME MEASURES: Histopathologic and immunohistochemical features of JIA-associated anterior uveitis. RESULTS: The iris and ciliary body showed nongranulomatous chronic inflammation predominantly made up of plasma cells, Russell bodies, and plasmacytoid lymphocytes. The ciliary processes and pars plana ciliaris showed focal aggregates of CD20-positive cells with several CD3- and CD8-positive cells and occasional CD4- and CD68-positive cells. Pancytokeratin stain showed ciliary epithelial proliferation admixed with lymphocytes. The iris revealed kappa-positive cells within the stroma and lambda-positive cells on the surface. The iris infiltrate primarily was made up of immunoglobulin (Ig) G-positive cells with occasional IgA- and IgM-positive cells. The anterior chamber exudate was mainly positive for IgG and IgA. CONCLUSIONS: The immunohistochemical findings suggest that JIA-associated nongranulomatous iridocyclitis is a primarily B-cell-infiltrative process.

Characterizing extravascular neutrophil migration in vivo in the iris.

Extravascular neutrophil migration is poorly characterized in vivo. To test the hypothesis that this migration is a non-random process, we used videomicroscopy to monitor neutrophils in irises of living mice with endotoxin-induced uveitis (EIU). Paths of individual cells were analyzed. Nearly all of these cells were moving in divergent directions, and mean displacement plots indicated that the predominant movement was random. The paths of some cells were fit to bivariate autoregressive integrated moving average models that revealed at least two modes of movement: random search and linear trend. Cell speed was significantly reduced by the actin inhibitor, cytochalasin D. The pattern of migration for neutrophils is in marked contrast to what we previously described for antigen-presenting cells in the iris, but somewhat resembles recent descriptions for T cells within a lymph node. Characterization of extravascular migration of neutrophils has important implications for understanding infection and immunity.

Transforming growth factor beta-producing Foxp3(+)CD8(+)CD25(+) T cells induced by iris pigment epithelial cells display regulatory phenotype and acquire regulatory functions.

The ocular pigment epithelial (PE) cells convert T cells into T regulators (Tregs) in vitro. The PE-induced Tregs fully suppress activation of bystander responder T cells. Iris PE (IPE) cells from anterior segment in the eye produce costimulatory molecules and transforming growth factor beta (TGFbeta) that is delivered to CD8(+) Tregs. We have now examined whether T cells exposed to cultured IPE express CD25 and Foxp3, and to determine if the CD25(+) IPE-exposed T cells display regulatory functions in vitro. We have found that cultured B7-2(+) IPE converted CTLA-4(+) T cells into CD25(+) Tregs that suppress the activation of bystander T cells. The CD8(+) IPE-induced Tregs constitutively expressed CD25. Through TGFbeta-TGFbeta receptor interactions, the IPE converted these T cells into CD25(+) Tregs that express Foxp3 transcripts. The CD8(+) IPE-induced Tregs produced immunoregulatory cytokines, e.g., interleukin-10 and TGFbeta. In addition, IPE-exposed T cells that downregulated Foxp3 mRNA failed to acquire the regulatory function. In conclusion, ocular pigment epithelial cells convert CD8(+) T cells into CD25(+) Tregs by inducing the transcription factor Foxp3. Thus, T cells that encounter ocular parenchymal cells participate in the T-cell suppression.

Regeneration, tissue injury and the immune response.

The involvement of the immune system in the response to tissue injury has raised the possibility that it might influence tissue, organ or appendage regeneration following injury. One hypothesis that has been discussed is that inflammatory aspects may preclude the occurrence of regeneration, but there is also evidence for more positive roles of immune components. The vertebrate eye is an immunoprivileged site where inflammatory aspects are inhibited by several immunomodulatory mechanisms. In various newt species the ocular tissues such as the lens are regenerative and it has recently been shown that the response to local injury of the lens involves activation of antigen-presenting cells which traffic to the spleen and return to displace and engulf the lens, thereby inducing regeneration from the dorsal iris. The activation of thrombin from prothrombin in the dorsal iris is one aspect of the injury response that is important in the initiation of regeneration. The possible relationships between the immune response and the regenerative response are considered with respect to phylogenetic variation of regeneration in general, and lens regeneration in particular.

Phenotypic and immunoregulatory characteristics of monocytic iris cells.

The introduction of antigen into the anterior chamber of an eye induces the antigen-specific suppression of cell-mediated immunity and the antigen-induced production of immunoglobulin G2 antibodies. To define further the role of iris monocytic cells in the systemic suppression of cell-mediated immunity that follows the entry of foreign antigen into the anterior chamber, murine iris wholemounts or cell suspensions of iris cells were stained with fluorescent anti-F4/80 and/or anti-CD11c, anti-CD11b antibodies and examined by confocal microscopy or flow cytometry, respectively. Monocytic cells in iris cell suspensions were recovered from mice receiving an injection of trinitrophenylated bovine serum albumin (TNP-BSA) into an anterior chamber and Percoll-enriched iris cells separated into cells expressing F4/80 or CD11c were injected intravenously into TNP-BSA-immunized or naive recipients. The recipients were challenged to induce delayed-type hypersensitivity (DTH) or were provided with splenocytes or thymocytes that transfer the suppression of DTH. The homing of monocytic bone marrow cells to the iris was determined by the intravenous injection of bone marrow cells from green fluorescent protein (GFP)-transgenic donors into C57 mice, and the staining of recipient iris wholemounts with anti-F4/80 antibodies. Iris cells with a dendritic morphology expressing both F4/80 and/or CD11c and CD11b, some cells expressing only F4/80 or CD11c, were detected. The irides of irradiated GFP- mice that received intravenous GFP+ bone marrow cells contained GFP+ F4/80+ cells. F4/80+ and CD11c+ cells from the irides of donors that received intracameral TNP-BSA transferred the suppression of DTH when injected intravenously into TNP-BSA-immunized recipients, activated immunoregulatory thymocytes and activated antigen-specific splenic regulatory effector cells. These results support the hypothesis that iris monocytic cells may participate in the systemic induction of regulatory T cells.