Ocular Immunology: Autoimmunity and Tolerance in the Retina
A central problem in immunology is that the strategy used to generate a large variety of antigen receptors also results in the production of receptors with specificity for self. The potential for self-reactivity is balanced by compensatory/regulatory mechanisms, collectively called tolerance. Tolerance is an operational definition which refers to the lack of, inhibition of, or alteration of an immune response. There are several types of tolerance including: clonal deletion, anergy, suppression, and immunological ignorance (lack of sufficient antigen to achieve levels of receptor binding required for signaling). Some tolerance results from the "fail-safe" manner in which the immune system operates; a minimum of two signals between two cells is required for activation.
One way to study tolerance is to use examples of the loss of tolerance, such as autoimmune disease. Our lab works with an autoimmune disease, experimental autoimmune uveoretinitis (EAU), in which the target autoantigen is a protein expressed in retinal photoreceptor cells. EAU resembles several clinical diseases of the retina with possible autoimmune origins. Nervous system autoantigens provide unique challenges to tolerance. Some of these autoantigens are tissue-specific, no antigen is available to drive negative selection in the thymus. Some appear for the first time well after the immune system has started to generate mature T lymphocytes, so that these lymphocytes have escaped usual selective mechanisms. Furthermore, the nervous system is isolated behind physiological barriers, whose effect on immune function is not well understood.
Our lab concentrates on studies of the role of the nervous system environment in immune tolerance, primarily by study of the retina. The nervous system is a complex and somewhat fragile collection of tissues. Since bystander damage due to severe inflammation can do great harm, mechanisms to minimize the potential for such damage have developed. These mechanisms are collectively termed immune privilege. As new mechanisms of immune privilege continue to be revealed, the relevance of old theories comes into question. Our lab has a particular interest in the immune privilege of retina, and we wonder how much of immune privilege is due to newly-described active mechanisms of tolerance, and how much is due to older hypotheses based on the anatomy and physiology of the retina; i.e., mechanisms such as sequestration, which depend on its relative isolation. Sequestration refers to the barrier function played by the tight junctions of the retinal vascular endothelium and the RPE. The idea is that together, these features of the eye protect it from inflammation by reducing the access of inflammatory cells, and promoting immunological tolerance to existing antigens in retina.
For several years, our lab studied a retinal antigen called S-Ag (or "arrestin") to learn about retinal immunology. While this has allowed considerable progress, there are complications. Arrestin is now known to belong to an extensive family of highly related proteins that show widespread expression and cross-reactivity. There is also evidence for thymic expression of arrestin and arrestin-like molecules. As a result, we recognized a few years ago that the complications of this model system would hinder progress.
Consequently, we recently undertook a strategy that would allow direct testing of our hypotheses based on studying immune responses in transgenic mice. These mice express an antigen, beta-galactosidase (beta-gal), without homology to any known mammalian sequence. Depending on the promoters used, the tissue site and amount of bgal expression can be manipulated. We are presently using several different strains of Tg mice; one gives systemic beta-gal expression, one gives retinal expression, and one has brain and retinal expression. Non-transgenic littermates and normal mice are used as controls. Collectively, these mice are used to compare the effects of retina-restricted expression to systemic or CNS expression on tolerance. A beta-gal-specific CD4 T cell receptor transgenic mouse has been made to provide a clonal population of naive T cells with specificity for this antigen.
This strategy has yielded important preliminary results about the difference in the immune recognition of a protein in the retina as compared to the recognition of that same protein expressed elsewhere, addressing fundamental questions about immune privilege
What's Immunology Got To Do With Eye Disease?
Most of you know about immunology through the immunizations that you received to protect against infectious diseases such as measles and tetanus. You have heard how AIDS compromises the immune system with devastating consequences. You may not realize the impact of the immune system on the eye. My laboratory works on the immunology of the eye, an area upon which the success of many new and current treatments relies. These diseases are like a "Who's Who" in notoriety - macular degeneration, retinal degeneration, corneal injuries and defects, all of the inflammatory diseases with an "-itis" in their name including retinitis, uveitis, iritis, etc., infections and cancers of the eye. The immune system is the common thread through all of these, whether as the cause, or a factor that complicates or prevents treatment, or the mechanism that could be a treatment or cure.
Here are some examples. The success of cornea transplants is dependent on the surgery, AND on the suppression of the ensuing transplant rejection reaction, which is the immune system in action. Any kind of transplant, including those proposed in the future to replace defective RPE in macular degeneration will require coping successfully with rejection. Even new stem cell procedures which are hoped to regenerate retinal defects may face a rejection reaction unless the cells are prepared from the recipient. There are also the more common problems of inflammation associated with uveitis, for example. Perhaps the commonly used steroids will not work, or the side effects are overwhelming. The answer lies in finding other ways to manage the immune system. Already there are procedures that could be done, but are not, because the immune response will cause them to fail.
What is this immune system that seems to cause so much difficulty? It is the protective system that allows us to live in the world, with all its infectious agents and parasites. Without a good immune system, you would not last long. On the other hand, it is not fool-proof, and sometimes makes mistakes. It was never intended to know the difference between a transplant and a parasite, so it does its best to reject even life-saving transplants. It is also ever vigilant, resisting our efforts at manipulation and requiring powerful drugs to keep it under control.
Eye and Immune System Coordination
The eye and immune system try to coordinate their activities.
One thing the eye does surprisingly well is to alter the immune responses that take place in it. From recent research, we think that the eye intentionally tries to make immune responses be relatively non-destructive because vision is not only precious, but fragile, and inflammation can be quite destructive. This communication between the eye and the immune system is fascinating and can be demonstrated in the lab, so it is no longer a hypothesis - it really happens. The process is generally called immunoregulation, because it is a means of regulating the immune response. The mechanisms are being studied and, of course, are very complicated.
Now you are probably thinking that scientists always seem to look at things and then say that they are "complicated". And then we ask for more money to continue on studying them. But in defense of science, let me just say that if you are over 45, you are probably still here because of science. We do figure out some of these things, and use them to fight disease for the good of all of us. I dare say that the next 25 years will make the last 25 seem like the medical dark ages. If we could better understand the interaction between the eye and immune system and the resulting immunoregulation, we might have a means to use these mechanisms to preserve vision, precious to all of us. The results might even be useful in other places in the body.
What We Do
The examples and ideas described above draw attention to the breadth of issues that require manipulation of the immune system. No one lab can do it all, and we concentrate on a short list of immune processes that we hope will bring benefits to some these difficult disease processes in the eye.
Our lab philosophy is that there is particular merit in understanding the bodies' own regulatory mechanisms, and developing treatments based on those molecules and cells. Within this context endogenous regulatory mechanisms, our lab concentrates on finding the basis for novel treatments for inflammatory eye diseases. We divide our efforts into 2 strategies.
Strategy #1. Much medical research focuses on the study of diseased tissues, with the goal of finding an intervention that will reduce the severity of disease or limit the last steps in the disease process. The irreversibility of the damage that can be caused in the eye, especially the retina, calls for strategies that are more effective at much earlier stages of the disease progression. To meet this goal, we have initiated studies to understand the normal interactions of the retina and immune system, and developed the tools that allow us to examine those interactions in the normal eye and retina, before pathological changes or disease is present.
Strategy #2. On the other hand, there is the reality that many individuals already have, or will develop, disease that is blinding and irreversible with present medical practice. The greatest foreseeable hope for these individuals lies in the application of stem cells. We have exploratory studies underway showing that adult-derived stem cells can integrate into retina, and express differentiation antigens associated with retinal photoreceptor function.
Studies Part of Strategy 1
Goal: Develop methods to track and assay T lymphocytes
T lymphocytes are both the mediators of autoimmunity and potential inhibitors of autoimmune disease. We need to know if, how, and when these cells migrate into the target tissue (retina). The first step is to be able to detect them and their activity in vivo. Our first results in this area showed that normal T lymphocytes have difficulty interacting with retinal antigens. The lack of interaction seems, at first glance to possibly be a good thing. But, for the immune system to be tolerant of self-antigens, including retinal self-antigens, they must first be aware of them. This awareness seems to be inhibited in the retina. Is this an immunological "mistake"? Or, is it part of a greater immune system strategy that we have yet to discover? We are continuing these studies with transgenic T cells that should be more sensitive, and easier to track.
Goal: Antigen-presenting cells in the retina
The studies showing that an immunoregulatory response is developed to a retinal antigen imply that the antigen is made available to the lymphocytes. The cells that gather antigen for recognition by lymphocytes are called antigen presenting cells. Some results suggest that such cells may be present in the retina. However, our experiments to directly detect these cells could not find them. Instead, we just reported that the retina is capable of recruiting antigen presenting cells from the circulation.
Goal: Immune regulation
In addition to minimizing the recognition of retinal antigens by T lymphocytes, there is also evidence that recognition must have occurred at some time in development, or in another place. We learned of this by asking if mice have a spontaneous regulatory response to retinal antigens. It is clear now that they do, but that detection of this regulatory response requires different assays. This interesting new result has been published. Our new results show that an existing protective, regulatory response must be overcome before a pathogenic autoimmune response can damage the retina. Learning how to boost the potency of the regulatory response may be a means to prevent or treat autoimmunity. Learning how to limit its activity would increase the success of transplantations.
Studies Part of Strategy 2
Goal: Stem cell treatment for retinal damage
Stem cells of various kinds have received considerable attention for their potential to regenerate damaged tissues. We are particularly interested in the repair of damaged retina. Our preliminary work is based on our ability to damage retina using our model of retinal autoimmunity in mice. This strategy provides a realistic and medically relevant means to damage the retinal neurons and photoreceptor cells. These cells have no ability to regenerate in mice (or humans), so that damage is permanent and results in loss of vision.
We recently initiated studies to determine if neural stem cells have the ability to incorporate into adult retina, and give evidence of differentiation into photoreceptor cells. We made stem cells from a transgenic mouse from our lab that carries the gene for a protein that is only expressed in mature photoreceptor cells. If the stem cells, which are undifferentiated, progress substantially into the maturation process to becoming retinal photoreceptor cells, they should express this unique protein. Three months after injection of these cells into mouse eyes, we have found colonies of descendants of the stem cells that make the unique protein, and which have migrated to the region of the retina where photoreceptor cells are found. This particularly exciting result is being prepared for publication.