Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

T-regulatory cells in a process requiring the spleen, which helps to reestablish homeostasis and limits recurrence of disease (reviewed in Caspi 2006b).

Susceptibility to Experimental Autoimmune Uveoretinitis Starts from Defects in T Cell Repertoire Selection

Self tolerance to tissue antigens, including uveitogenic retinal antigens such as S-Ag and IRBP, is first established through thymic selection (Kyewski and Klein, 2006). Lymphocytes passing through the thymus during their natural maturation process are selected for survival or death according to their ability to recognize self molecules. Many if not most tissue-specific antigens, including the retinal antigens S-Ag and IRBP, are expressed by specialized thymic epithelial cells. Immature lymphocytes that fail to recognize any self antigens undergo death “by neglect”; lymphocytes that recognize self with low affinity are selected for survival (positive selection), and lymphocytes possessing highaffinity antigen receptors for a self antigen are eliminated or tolerized (negative selection). Egwuagu et al. (1997) demonstrated that mice express relatively high levels of S-Ag in the thymus but low levels of IRBP, which correlates with their relative resistance to uveitis induced by S-Ag and susceptibility to IRBP-induced EAU. The lowest expression of IRBP was seen in B10.RIII mice, the mouse strain most susceptible to IRBP-induced EAU. These findings support the notion that inefficient thymic elimination of uveitogenic cells due to low expression of the selecting antigen predisposes to uveitis. (However, this does not explain the susceptibility to S-Ag-EAU of HLA-DR3 transgenic mice, unless their thymic expression of S-Ag is in some way altered by expression of human, instead of mouse, class II MHC molecules!)

Direct evidence that insufficient thymic elimination of retinal antigen-specific cells predisposes to autoimmune uveitis comes from the observation that T cells originating from IRBP-deficient mice are much more highly uveitogenic than T cells from wild-type mice, suggesting that a repertoire that has not been properly depleted of IRBP-reactive T cells indeed predisposes to autoimmunity (Avichezer et al., 2003). The two models of spontaneous uveitis described in the previous section lend further support to this notion (it should be noted that these models develop also other tissue-specific autoimmune manifestations, which are disregarded for the purposes of this discussion). Antiretina antibodies and cellular responses develop in mice deficient in the AIRE molecule, a transcription factor controlling ectopic expression of tissue antigens in the thymus. These mice fail to express retinal antigens in the thymus, and therefore, similarly to IRBP knockouts, cannot cull their lymphocyte repertoire of high-affinity uveitogenic T cells (Anderson et al., 2002; Devoss et al., 2006). This is reminiscent of the spontaneous uveitis that develops in athymic (nude) mice implanted

with neonatal rat thymus. Although there is proper expression of retinal antigens in the thymus, they apparently cannot be presented to the mouse T cells because of an incompatibility between the participating thymic elements of the donor and the immune system of the recipient (Ichikawa et al., 1991).

Of note, in both these spontaneous models the antiretina response that develops targets IRBP, even though negative selection fails to other retinal antigens as well. Even in the absence of IRBP (double knockouts for AIRE and for IRBP), no other retinal antigens are recognized as surrogate targets for autoimmunity. This supports the concept of a speciesspecific primary autoantigen, which may help to explain why S-Ag is by far the most frequent antigen recognized by uveitis patients.

The thymus also selects “natural” regulatory T cells, which characteristically coexpress the molecules CD4 and CD25 (IL-2Rα chain, gene symbol Il2ra), which have a role in raising the threshold of susceptibility to EAU by keeping the potentially uveitogenic T cells in check (Avichezer et al., 2003; Grajewski et al., 2006). Interestingly, although IRBP expression in the thymus is required to select natural T- regulatory cells specific to IRBP, “polyclonal” T-regulatory cells specific to other antigens can also be harnessed to inhibit induction of uveitogenic effector T cells by innate stimuli (Grajewski et al., 2006), which might overlap with the innate stimuli necessary to trigger the induction of effector T cells.

Deficient Peripheral Tolerance as a Factor in Susceptibility to Experimental Autoimmune Uveoretinitis

Negative selection in the thymus is never fully efficient and, as discussed previously, is affected by the quantitative aspects of self-antigen expression in the thymus. Potentially autoreactive T cells that exit the thymus into the periphery normally have a “second chance” at tolerance as they recirculate through the tissues and encounter self antigens under noninflammatory conditions. However, unlike other organs, the eye becomes separated from the immune system early in ontogeny by an efficient blood-organ barrier that restricts free entry of lymphocytes into the eye, and also limits free exit of antigenic molecules from the eye, especially from the posterior segment. This separation from the immune system results in a circulating pool of uveitogenic lymphocytes that are not truly tolerant but merely “ignorant” of retinal antigens, and that can potentially be triggered into action by an (in)appropriate stimulus.

The notion that inadequate peripheral tolerance contributes to susceptibility is borne out by the finding that if these circulating, potentially uveitogenic lymphocytes are tolerized by expressing the uveitogenic retinal antigen outside of the eye, thus making it accessible to peripheral tolerance mechanisms, such mice become profoundly resistant to

EAU. Mice transgenic for β-gal on the S-Ag (arrestin) promoter express β-gal as a neo-self antigen in the retina, where it can serve as a target for EAU. Mice that also express β-gal in the periphery (ROSA26, JAX strain ID 004847) are tolerant and resist EAU induction by immunization with β-gal (Gregerson et al., 1999). In adult animals, infusion of genetically compatible B cells retrovirally transduced to express a uveitogenic fragment of IRBP also results in resistance to EAU (Agarwal et al., 2000). Peripheral tolerance thus constitutes a “weak link” in homeostasis of tolerance to retinal antigens, which might be successfully targeted therapeutically once the inciting retinal antigens in humans are positively identified.

Antigen Presentation to Uveitogenic Lymphocytes: What, Where, and by Whom? It is believed that the triggering event for uveitis is exposure of the circulating “ignorant” retina-specific T cells to their cognate antigen. A prototypic autoimmune disease of the eye that is believed to be triggered in this way is sympathetic ophthalmia, where a penetrating wound to one eye is followed after weeks or months by a destructive inflammation in the contralateral, “sympathizing” eye, apparently as a result of autoimmunization to retinal or uveal proteins (Boyd et al., 2001). However, for most types of autoimmune uveitis an exposure to antigen originating from the eye cannot be demonstrated, and it is believed that cross-reactive microbial antigens may provide such a stimulus through antigenic mimicry. A number of antigenic substances have been identified that cross-react with retinal S-Ag and induce EAU in rats, supporting the antigenic mimicry hypothesis (Wildner and Diedrichs-Mohring, 2004), but there are no reports as yet of molecular mimics that induce EAU in mice.

Where does the exposure to retinal antigens occur? The retina resides behind an efficient blood-organ barrier that restricts passage of molecules and of cells. Those lymphocytes that do enter the eye encounter a hostile environment composed of a variety of cell-bound and soluble inhibitory factors, as well as a paucity of professional antigen-present- ing cells (APCs). Therefore, it is unlikely that priming of errant lymphocytes in the eye could occur and be a cause of uveitis. Rather, it is believed that lymphocyte priming takes place in the periphery, that is, in the lymph nodes, likely in conjunction with an adjuvant effect provided by a concomitant infection. Although the inside of the healthy eye lacks lymphatic drainage, antigen from an injured eye escapes into the subconjunctival, scleral, and periocular space, which is drained by the submandibular and cervical lymph nodes. In support of this notion, Camelo et al. (2006) demonstrated that injected antigen from the anterior chamber of the eye initially travels in a soluble form to secondary lymphoid organs via lymphatic and vascular routes. A microbial mimic antigen encountered through a wound or infection else-

where in the body would drain to the relevant local lymph node, and would in addition provide its own adjuvant effect.

Who are the antigen-presenting cells? In order to present antigen for acquisition of effector function, the APC must express appropriate costimulatory molecules, which are induced by microbial or endogenous “danger” signals. Hence the need for adjuvant, whose role is discussed in more detail in the next section. In the draining lymph nodes, resident and migrating DCs serve as APCs to naive T cells, and the ability to induce EAU with antigen-pulsed DCs (Tang et al., 2007) is in line with this being a mechanism of priming in EAU. However, experimental evidence indicates that although the uveitogenic cells have been primed in the periphery, they must recognize their antigen within the eye for EAU to be induced (Prendergast et al., 1998; Chen et al., 2004). Although in the healthy eye there are few or no functional APCs in the retina, it is conceivable that those initial effector T cells that infiltrate the eye might induce a microenvironment through local cytokine secretion that would result in “arming” of resident cells such as microglia to acquire APC function. However, resident APCs may not be essential in the case of peripherally primed T cells, as APCs recruited from the circulation are sufficient to support EAU induction with activated retina-specific T cells (Gregerson and Kawashima, 2004).

Interestingly, and arguably of functional relevance to induction of uveitis, retinal antigens may facilitate their own presentation to uveitogenic T and B cells. We recently reported that both S-Ag and IRBP induce migration of immature DCs, as well as T and B lymphocytes, by binding to the chemotactic receptors CXCR3 and CXCR5 (Howard et al., 2005). This would accomplish a dual role, on one hand attracting immunocompetent cells to the site where antigen is present, and on the other hand promoting antigen presentation, as association of antigens with cells’ surface receptors strongly enhances their uptake by APCs and their immunogenicity (Biragyn et al., 2004; Bonifaz et al., 2004).

Innate Immune Signals Trigger Autoimmunity: Toll Receptors and Other Danger Signals EAU is elicited by immunization with the retinal antigen in emulsion with CFA. The role of the adjuvant, through its mycobacterial component, is to provide innate immune danger signals to DCs, which then become activated and direct the ensuing adaptive T cell response toward a pro-inflammatory, tissuedestructive, effector phenotype. In the past decade much attention was focused on Toll-like receptors (TLRs) that recognize conserved pathogen-associated molecular patterns (PAMPs) and activate cells of the innate immune system, including macrophages, DCs, and granulocytes, although TLRs are by no means the only receptors that activate

innate immune responses. The antigen-pulsed DCs used to elicit EAU in the model described earlier must be activated (“matured”) in vitro using LPS (a TLR4 agonist) and at the same time crosslinking the CD40 molecules (costimulatory tnfsf5) by a monoclonal antibody, to acquire the ability to prime uveitogenic T cells for effector function (Tang et al., 2007). Antigen-pulsed DCs that have not been matured prime retinal antigen-specific T cells into a nonpathogenic, or even overtly regulatory, phenotype, and protect mice from a uveitogenic challenge with IRBP in CFA, presumably through induction of regulatory T cells (Jiang et al., 2003; Siepmann et al., 2007). It is currently not clear whether such immature DCs could convert to regulatory function T cells that had already acquired effector function, or whether the T-regulatory cells they induce are capable of inhibiting already primed effector cells, which would be important from the clinical point of view.

CFA provides its innate stimulatory effect not only through TLRs. The extent of dependence of EAU induction on TLR stimulation currently appears somewhat contradictory. In a transgenic uveitis model using mice that express HEL in their retina and receive an infusion of TCR transgenic T cells that express a HEL-specific TCR, coinjection of HEL with any one of several TLR ligands is sufficient to induce EAU (Fujimoto et al., 2006). Interestingly, PT, which in addition to being a TLR4 ligand also has other biological activities, was superior to “pure” TLR ligands in supporting induction of disease. In some situations, TLR signaling may be redundant with other innate signals. For example, in EAU induced with IRBP in CFA, single knockout mice deficient in TLR4, TLR9 or TLR2 (as well as double knockout mice lacking any two of these receptors) remain fully susceptible (Su et al., 2005). However, abrogation of IL-1 signaling (which, like the TLRs, also uses the MyD88 signaling pathway) prevented induction of EAU, indicating that whereas IL-1 signaling is necessary and nonredundant for effector T cell generation in EAU, signaling through TLRs may be replaced by other innate stimuli.

Recent data indicate that innate signaling is also important in activating natural T-regulatory cells (nT-regs) that protect from EAU. nT-regs can be activated by mycobacterial components present in CFA (Grajewski et al., 2006) and limit induction of uveitogenic effector T cells, though it is obviously the latter that “win out” when EAU is induced. Preliminary data implicate TLR signaling in nT-reg activation (Grajewski, 2006), but it is currently not clear whether the effect is direct or indirect.

Pathogenic Effectors: Th1 or Th17? Retinal antigenspecific T cells that have encountered a uveitogenic stimulus acquire a pro-inflammatory, tissue-destructive effector phenotype. This depends strictly on the presence of the innate danger signals discussed earlier, many of which are

still probably unidentified, that induce appropriate cytokine production and costimulatory molecule expression on APCs. It has long been known that Th1-like cells, which are induced in the presence of IL-12 and whose hallmark is the production of IFN-γ, are a pathogenic effector phenotype (Xu et al., 1997). EAU can be transferred with a few million primary IRBP-specific IFN-γ-producing effector T cells, or less than a million cells from a polarized IRBP-specific Th1 cell line (Silver et al., 1999). IRBP-specific T cells that do not produce IFN-γ and are not pathogenic can be converted in the presence of IL-12 into a pathogenic, IFN-γ-producing phenotype (Tarrant et al., 1998).

Recently a new effector phenotype has been described and shown to be centrally involved in several autoimmune and inflammatory diseases. Its induction and maintenance depend, respectively, on TGF-β + IL-6 and on IL-23, and its hallmark cytokine is IL-17 (Bowman et al., 2006). Some studies have gone so far as to suggest that it is the IL-17- producing “Th17” effectors that are the main players in tissue destructive autoimmunity, and that Th1 cells play at best only a minor role. However, recent data in the EAU model support a distinct role for the Th1 effector phenotype.

In murine EAU, both Th1 and Th17 antigen-specific effectors are induced in mice by uveitogenic immunization with IRBP. Studies aimed at elucidating their relative roles suggest that both participate in pathogenesis, and conceivably complement each other. On one hand, a small number of cells from an IRBP-specific Th1 cell line, which is unable to produce IL-17, elicits severe EAU. This speaks strongly in favor of an important role for Th1 effector T cells. On the other hand, polarized Th17 cell lines unable to produce IFN-γ elicit EAU, and neutralization of IL-17 in IRBP/ CFA-immunized hosts aborts EAU even when started 7 days after immunization, implicating IL-17 as an effector cytokine (Luger et al., in press). The question of whether IFN-γ is an effector cytokine or just a hallmark of Th1 cells is more complex to answer, as neutralization of IFN-γ in immunized mice exacerbates EAU, and IFN-γ knockout mice develop severe disease. This appears to be due at least in part to inhibition of Th1 effector cell generation by a feedback effect of IFN-γ in a process involving induction of nitric oxide and apoptosis (Tarrant et al., 1999). However, when EAU is induced by adoptive transfer of polarized IFN-γ-producing Th1 effector lymphocytes, treatment with anti-IFN-γ inhibits disease, suggesting that IFN-γ does have an effector function(s) (Luger et al., in press). Additionally, antigenpulsed mature DCs from wild-type mice fail to induce EAU in IFN-γ knockout recipients, even though these mice produce large amounts of IL-17, suggesting that an IFN-γ- producing antigen-specific effector is important (Tang et al., 2007). IFN-γ is thus a double-edged sword, and its effects on disease depend on where and when it is produced.

Effector Cell Migration, Infiltration, and Inflammatory Cell Recruitment Activated antigen-specific T cells that had been primed in peripheral lymphoid tissues must migrate and find their way to the eye if uveitis is to occur. Although often we refer to this as “homing” to the eye, the term is probably not correct, as it is unlikely that immune cells are able to detect retinal antigens on the other side of the blood-retinal barrier in a healthy, unmanipulated eye (Gregerson et al., 1999). It is likely that extravasation of antigen-specific T cells primed elsewhere into the eye occurs at random (Prendergast et al., 1998; Caspi, 2006b). Because of the small size of the retina it is possible to retrieve essentially all the cells that have entered the target tissue. By a simple calculation of how many cells were injected versus how many can be retrieved from the retina it appears that only a few such cells (fewer than 15) are needed to trigger the sequence of events leading to EAU (Caspi, 2006b). Release of inflammatory mediators by these initial cells “activates” the vascular endothelium of the retina and uvea, promoting adhesion of passing cells and facilitating recruitment of all types of leukocytes from the circulation. Since the great majority of the infiltrating cells found in a uveitic eye are the recruited leukocytes (Pennesi et al., 2003; Chen et al., 2006), it is clear that this recruitment provides a massive amplification mechanism.

When antigen-specific donor T cells are transferred into a naive host with a healthy eye, there is a period of several days after the initial antigen-specific donor T cells enter the retina when nothing much seems to happen in the eye. However, changes undetectable by current methods must be taking place in the local microenvironment, including changes in the blood-retinal barrier and the production of chemotactic substances in the target tissue, that will facilitate subsequent massive recruitment of leukocytes from the periphery. This is evident from the finding that only transfer of retinal antigen-specific T cells, but not of activated T cells that do not recognize retinal antigens, is followed by a massive entry of additional donor T cells and of recruited host cells into the eye on the fourth day after transfer (Prendergast et al., 1998; Chen et al., 2004, 2006). Studies in a transgenic EAU model showed that during this seemingly quiescent period, the bulk of the uveitogenic T cells accumulate in the recipient’s spleen, where they proliferate and activate nonspecific host CD4+ T cells. They also undergo (and induce) changes in chemokine and adhesion molecule expression, which culminates on day 4 in a mass emigration of donor and host cells from the spleen to the eye and EAU induction (Chen et al., 2004, 2006).

Owing to the transparency of ocular media, the eye offers special advantages as a model for analyzing cellular interactions, leukocyte flow, adhesion, and extravasation of cells by intravital microscopy. Unlike in other tissues, intravital microscopy in the eye does not require invasive procedures,

which by themselves may modify cellular interactions and behavior. Thus, it is possible to easily visualize sticking, rolling, and extravasation of leukocytes, and to track the T cell infiltration into the eye and the cells’ interactions with putative APCs there (Rosenbaum et al., 2002). Similar techniques are being applied to the human eye and should yield important information about the pathogenic processes in human uveitis.

Natural Resolution Mechanisms In humans, uveitis can remit spontaneously even without treatment, and the disease in animal models is self-limiting without necessarily proceeding to destruction of the entire retina and elimination of the source of inciting antigen. The mechanisms that bring about remission are not completely understood. Unraveling natural resolution mechanisms of disease can give us clues to the types of processes that we would wish to promote via immunotherapy.

An important mechanism that is being actively studied in mouse models is regulatory T cells. Several types of T-regulatory cells related to uveitis have been described (Stein-Streilein and Taylor, 2007), but their origin and the relationships between them remain unclear. As mentioned earlier, CD4+CD25+ thymic-derived nT-regs protect from induction of EAU (Grajewski et al., 2006), and although in disease their threshold of protection has obviously been overcome, they may proliferate and constitute a homeostatic mechanism that would help to bring about remission. T- regulatory cells can also be induced or converted from CD25− populations, which also give rise to effector T lymphocytes, and under some conditions functional effectors may also convert to acquire T-regulatory properties. As an example, IRBP-specific T cells may be converted from effectors to regulators by aqueous humor, a phenomenon that has been attributed to the effects of TGF-β and inhibitory neuropeptides (Stein-Streilein and Taylor, 2007). Natural T-regulatory cells as well as some types of induced T- regulatory cells express the transcription factor FoxP3, but although expression of this molecule is generally recognized to be indicative of T-regulatory cell function, it does not follow that the regulatory cells that express it are necessarily descended from CD4+CD25+ nT-regs (Ziegler, 2006).

The uniquely eye-related phenomenon of anterior chamber–associated immune deviation (ACAID) has been well studied. It is induced by injection of antigen into the anterior chamber of the eye and results in development of regulatory CD4+ and CD8+ T cells that inhibit, respectively, the induction and the expression of antigen-specific immune responses (Stein-Streilein and Streilein, 2002). Although elicitation of ACAID to IRBP protects mice from EAU (Hara et al., 1992), it is difficult to conclude that ACAID represents a natural mechanism of protection from autoimmunity used by the intact eye. This is because ACAID

results from injection of an antigen into the anterior chamber of the eye, necessarily perturbing its integrity. It is possible, in fact, that such mechanisms come into play as a result of damage to the eye. IRBP-specific T-regulatory cells, though apparently distinct from ACAID-induced T-regulatory cells, are induced as a result of EAU. Such cells are found in mice that have recovered from EAU and limit reinduction of disease (Kitaichi et al., 2005). Interestingly, their induction is dependent on the presence of the eye, indicating that something originating in the eye is crucial to their development. The eye could be necessary as a source of antigen, or of inhibitory factors such as α-MSH, which has been shown to convert primed IRBP-specific T cells to a regulatory phenotype (Stein-Streilein and Taylor, 2007).

Other mechanisms of suppression could conceivably include production of inhibitory cytokines, such as IL-10 and TGF-β, or inhibition by contact-driven mechanisms. IL-10 is detected in the retina of mice recovering from EAU, and can inhibit activation and function of already immune effector T cells, which are impervious to the inhibitory effects of TGF-β (Xu et al., 1997; Rizzo et al., 1998; Xu et al., 2003). They could also include contact with ocular resident cells, such as retinal glial Müller cells, which proliferate during uveitis as part of the process of healing and retinal scar formation, and whose proliferation in vitro is induced by activated lymphocyte/monocyte products (Caspi et al., 1987). Müller cells are able to inhibit activation and function of T cells, even if these T cells have previously been activated, and similar inhibitory effects are exerted by retinal pigment epithelial cells and iris/ciliary body epithelial cells (Caspi et al., 1987; Yoshida et al., 2000). In some cases interaction with ocular resident cells additionally induces conversion of T cells to a regulatory phenotype (Ishida et al., 2003; Sugita et al., 2006). Only some of the molecular interactions involved in these phenomena have been characterized.

Translational potential

Many of critical checkpoints in pathogenesis of uveitis, as defined in mouse models and depicted in figure 42.3, can serve as targets for immunotherapeutic intervention. To mimic the clinical situation in which the patient already has acquired immunity to retinal autoantigens, ideally the experimental approaches should be able not only to prevent but also to reverse disease. However, approaches that target only acquisition of immunity may also be of value, since chronic autoimmunity involves constant priming of new lymphocytes and their entry into the effector pool. This section provides some examples of possible immunotherapeutic approaches, drawing on what we have learned about disease pathogenesis, but it is by no means an exhaustive discussion of the translation of experimental approaches to the clinic.

Deficits in peripheral tolerance could be corrected in the adult animal by expressing the retinal antigen outside the eye in a tolerance-inducing form. Agarwal et al. showed that infusion of B cells, made to express a uveitogenic fragment of IRBP into susceptible mice, resulted in a profound and long-term induction of resistance to EAU, and moreover was able not only to prevent but also to reverse the disease process (Agarwal et al., 2000). Adapting this regimen to autologous human B cells using S-Ag, which appears to be the major retinal antigen recognized by humans, could represent a viable approach to therapy of uveitis, a notion that is supported by the ability of S-Ag-transduced B cells to inhibit EAU in HLA-DR3 transgenic mice (Liang et al., 2006). Another antigen-based approach to prevent EAU that has been successful in animal models is induction of oral tolerance by feeding retinal antigens. In uveitis patients, oral administration of S-Ag showed promise in a double-blind, placebo-controlled Phase I/II clinical trial; however, larger trials are required to establish efficacy (Nussenblatt, 2002).

A limitation of antigen-based therapy is that it requires knowledge of the inciting retinal antigen. Although uveitisassociated antigens are increasingly being identified, there are still many unknowns. Furthermore, the phenomenon of epitope and antigen spreading, which can accompany chronic autoimmune disease, could make identification of the antigen(s) driving pathology at a given time a moving target. Finally, there are concerns about introducing the putative disease-inciting antigen into an already immune individual.

Immunotherapeutic approaches that target common pathways of lymphocyte activation do not require knowledge of the inciting antigen, but are not as selective, and therefore have the potential to also inhibit immunity to pathogens. Several such approaches have been examined in mouse models of uveitis, and some appear to have clinical potential. For example, costimulatory molecule blockade targeting the B7 or CD40 pathways inhibits induction of EAU pathology in mice (Silver et al., 2000; Bagenstose et al., 2005). However, it does not induce long-term tolerance, contrary to reports in transplantation models. Thus, in theory, continuing treatment would be needed to maintain protection. A related approach is blockade of the IL-2 receptors, which are present on all activated T cells. This approach was shown to inhibit EAU in animals, and IL-2 receptor blockade with humanized anti-CD25 antibodies (Daclizumab or Zenapax) showed efficacy in clinical trials (Nussenblatt et al., 1999). Surprisingly, the mechanism of action of this treatment in humans appears to involve CD56high regulatory natural killer (NK) cells rather than elimination of the CD25+ effector T cells, as had initially been assumed (Li et al., 2005).

Migration and infiltration of recruited cells into the eye, which constitutes a necessary mechanism for disease

induction and progression, could be targeted by blockade of adhesion molecules or of chemokines and chemokine receptors. Blockade of the adhesion molecules ICAM-1 and LFA-1 in mice inhibited EAU, presumably by blocking inflammatory cell recruitment (Whitcup et al., 1993), as does blockade of the chemokine receptor CXCR3, which is important for migration of uveitogenic and recruited cells into the eye (Chen et al., 2004).

Finally, the effector cytokines produced by pathogenic T cells and the inflammatory leukocytes recruited by them may serve as targets for inhibition or neutralization. Neutralization of IL-17 by monoclonal antibodies in mice aborts disease even when instituted after the uveitogenic effectors have already been generated (Luger et al., in press). Similarly, TNF-α neutralization, which had been shown in animal models to be protective, is showing efficacy in clinical trials for some types of uveitis (Greiner et al., 2004).

These examples underscore the contribution of research in animal models to understanding the fundamental mechanisms driving ocular autoimmune and inflammatory disease, and their importance in devising novel therapies to combat sight-threatening uveitic diseases.

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Norrie disease is a severe X-linked recessive trait with the hallmark features of congenital blindness, deafness, and mental retardation. The disorder is caused by mutations in a gene encoding norrin, a small extracellular protein. To characterize the function of norrin and to study the pathophysiology of Norrie disease, a knockout mouse model was generated and examined. The results of these studies showed that abnormal retinal angiogenesis during development is one of the most prominent observations in mice lacking norrin. This causes severe retinal hypoxia, which leads to profound tissue damage. The disease phenotype was rescued by breeding knockout mice with transgenic animals with ectopic norrin expression in the lens. In addition, transgenic lenses induced proliferation of microvascular endothelial cells in coculture. These and other findings identified norrin as a key regulator of angiogenic processes in the retina. Most of the ocular symptoms in human patients may also be attributed to oxygen deficiency during retinal development, and the mouse lines significantly contributed to a better understanding of the primary events of this severe neurological disorder.

Clinical background

Norrie disease was named after Gordon Norrie, a consultant ophthalmologist at the Eye Clinic of the Institute for the Blind in Copenhagen. Norrie was the first to describe this trait as a nosological entity, in the late 1920s (Norrie, 1927). The medical condition was given its name by Mette Warburg, another Danish ophthalmologist, who reported the clinical picture and X-linked inheritance in a comprehensive monograph (Warburg, 1966). She decided to use this eponym in part to avoid a descriptive name that might reflect only secondary clinical features. Norrie realized that the disease was familial and that only boys were affected. Warburg then provided evidence that the disease is inherited in a sex chromosome–linked fashion (Andersen and Warburg, 1961; Warburg et al., 1965). In an X-linked recessive mode of inheritance, only males are affected, but a few exceptions exist (Chen et al., 1993b; Woodruff, et al., 1993; Kellner et al., 1996; Sims et al., 1997; Yamada et al., 2001).

The most prominent clinical finding in affected patients is early-onset bilateral blindness. Soon after birth, leukokoria is observed, a white pupillary reflex due to vascularized retrolental membranes and masses. This is a cardinal symptom

but not pathognomonic, as it is present in a number of other eye diseases (Francois, 1978). Additional ocular manifestations consist of vitreoretinal hemorrhages, retinal folding and detachment, bilateral and congenital pseudotumor of the retina (pseudoglioma), and atrophy of the bulbus (phthisis bulbi) late in the disease. The appearance of the vitreal cavity alludes to persistent primary vitreous and hyaloid vessels. The lens becomes cataractous during the first months or years of life. Also, the cornea, iris, ciliary body, and retinal pigment epithelium may be affected by the disease.

The extraocular features of Norrie disease include deafness and mental retardation. The latter also occurs early in childhood in one-third to one-half of patients. The onset of progressive hearing loss was observed in the second or third decade of life. Initially, deafness was reported in about onethird of patients (Warburg, 1966), but recent data suggest an almost 100% occurrence of hearing loss in patients (Halpin et al., 2005). Some histological studies of affected tissues (eye, ear, brain) available in the literature describe advanced stages of the disease. Hypotheses regarding the pathophysiology of Norrie disease involve an early arrest in neuroectodermal development, as well as abnormal vasoproliferative processes (Warburg, 1966; Parsons et al., 1992).

The molecular basis of Norrie disease

With the advent of molecular genetic mapping in humans, the responsible locus has been localized to the short arm of the human X chromosome (Xp11.4). Linkage mapping was consistent with deletions of the same chromosomal segment in affected male patients (L. M. Bleeker-Wagemakers et al., 1985; Gal et al., 1985, 1986; E. M. Bleeker-Wagemakers et al., 1988; Diergaarde et al., 1989). Detailed analysis of the corresponding genetic interval led to the identification of the mutation-carrying gene by positional cloning (Berger, Meindl, et al., 1992; Chen et al., 1992). The official gene symbol is NDP (Norrie disease pseudoglioma), and the protein was designated norrin. The human gene spans a genomic region of approximately 25 kb (24,729 bps) and consists of three exons. The length of the transcript is 1,833 nucleotides. It codes for a protein of 133 residues, including a signal peptide of 24 amino acids. The small size of the gene allows rapid diagnostic testing. Around 100 mutations have been described so far. Point mutations in NDP represent missense and nonsense mutations, as well as frameshifting