SCURFY MICE: A MODEL FOR AUTOIMMUNE DISEASE
   
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   AUTOIMMUNE DISEASE
   
   The condition in which the body attacks its own tissue has been an
   object of public concern recently. Former President George Bush and his
   wife Barbara both are afflicted with Graves' disease in which the body's
   own immune system attacks the thyroid gland. The safety of breast
   implants was called into question because of evidence that some
   recipients had developed autoimmune disorders such as rheumatoid
   arthritis, systemic lupus erythematosus, and scleroderma. Women, the
   media pointed out, have a higher-than-average incidence of many
   autoimmune disorders. These events suggest the need to know more about
   what makes the immune system work so well and what makes it go awry.  
   At ORNL's Biology Division, we have made progress in understanding the
   underlying causes of immune disease by studying mice having a disease
   that causes them to be underdeveloped; to have scaly skin, small ears,
   and large spleens; to open their eyes late; and to die early. These
   "scurfy" mice are helping us better understand the role of the thymus
   gland in autoimmune disease. 
   
   
   B AND T LYMPHOCYTES    
   
   Our immune system protects us from diseases by its unique ability to
   distinguish our own "self" molecules from those of invading
   microorganisms. To make this distinction, the immune system must first
   learn to recognize and tolerate self molecules during growth and
   development. This critical function is performed by
   lymphocytes--circulating white blood cells that search for foreign cells
   or microorganisms and directly or indirectly cause chemical attacks on
   these invaders to inactivate them. Each invader carries
   antigens--proteins or carbohydrates that, when introduced into the body,
   stimulate the production of a specific immune response. Foreign antigens
   are recognized by lymphocytes.    
   
   Lymphocytes are divided into two major categories based on their
   development pathways and functions. B lymphocytes recognize a foreign
   antigen by their cell surface antibody receptors. When stimulated by the
   appropriate foreign antigen, B lymphocytes can be transformed into 
   antibody-producing factory cells called plasma cells that secrete
   antibodies specific for that antigen. Like a protective cover for an
   electrical outlet, an antibody attaches to the foreign antigen. This
   antigen-antibody complex then attracts scavenger-type white blood cells
   called macrophages that ingest and destroy the offending microorganisms. 
   
   T lymphocytes are so named because they develop and mature within the
   thymus, a bi-lobed gland next to the heart. They recognize an antigen by
   their cell surface protein called the T cell receptor. T lymphocytes
   develop into two types of cells having different functions: killer cells
   and helper cells. Killer T cells carry the CD8 cell surface protein
   associated with their T cell receptors and act to destroy virus-infected
   cells or cells with foreign tissue antigens (as in graft rejection).
   Helper T cells are the master coordinators of the immune system.
   Carrying the CD4 cell surface protein next to their T cell receptors,
   helper T cells respond to foreign antigens by releasing a host of
   chemical signals called interleukins. These interleukin molecules call
   lymphocytes and other white blood cells to the site of the immune
   response, enable other lymphocytes to proliferate, and help B
   lymphocytes transform into antibody-producing plasma cells. The CD8 and
   CD4 cell surface proteins are often referred to as "markers" for the Tc
   and Th subclasses of T cells because monoclonal antibodies to these
   proteins can be used to count these cells using fluorescent staining.  
   
   Obviously, anything that goes wrong within this complex, interlocking
   system can have profound effects on the individual. For example, the
   human immunodeficiency virus (HIV) that causes acquired immunodeficiency
   syndrome (AIDS) attacks and destroys CD4 helper T cells. As a result,
   the immune system is devastated and the AIDS patient is left vulnerable
   to a host of life-threatening infectious diseases. Persons or animals
   born without a thymus (such as the nude mouse) lack T lymphocytes
   entirely and are, therefore, severely immune deficient. Other inherited
   immunodeficiencies cause the inability to make some or all types of
   antibodies.
   
   On the other hand, excessive or incorrect functions of the immune system
   can have equally harmful effects. These may range from the discomfort of
   allergies (overreaction to pollens, animal dander, etc.) to
   life-threatening autoimmune diseases such as juvenile diabetes, systemic
   lupus erythematosus, or scleroderma. The immune system's mistaken
   attacks on the body's own normal tissues may be limited to very specific
   tissues, such as the pancreatic islet cells in juvenile diabetes, or
   they may involve entire organs or organ systems. Currently, the causes
   of autoimmune disease are unknown. Treatments consist of powerful
   immuno-suppressive drugs such as those given to recipients of organ
   transplants. These drugs have severe side effects and only inhibit the
   symptoms of autoimmune disease, not the root causes.
   
   
   MUTANT MICE
   
   In 1987, I joined Richard Griesemer's laboratory in ORNL's Biology
   Division to investigate several mutant mouse stocks as potential animal
   models of human disease. We quickly settled upon an animal of historic
   importance in the genetics community-the scurfy mouse.    
   
   The scurfy mutant mouse was discovered by William L. Russell in 1949.
   Russell, who was scientific director of the Mammalian Genetics Section
   in the Biology Division, noticed a litter of young mice that contained
   several runted animals with scaly skin. Thinking that these animals were
   sick, he promptly discarded them! Happily, when similar offspring
   appeared in a litter from a closely related female, he immediately
   recognized their problem as a genetic disease and began to investigate
   its inheritance pattern. It soon became apparent that only male
   offspring were affected, whereas female mice were unaffected carriers of
   the trait, suggesting that the scurfy gene was located on the mouse X
   chromosome-the first sex-linked mutation to be discovered in the mouse.
   Because the mode of sex determination had not yet been established for
   mammals, this conclusion could not yet be a firm one.     
   
   However, a few years later, scurfy mice provided the means for
   demonstrating how sex determination takes place. Occasional female
   offspring were found that had the scurfy disease. Although they were
   doomed to die before they were old enough to reproduce, their genetic
   constitution could be tested by William Russell by transplanting their
   ovaries to healthy host females. The results of these genetic tests of
   scurfy females, in conjuction with genetic data and chromosome counts
   obtained by W. J. Welshons and Liane B. Russell (now head of the Biology
   Division's Mammalian Genetics and Development Section) for another
   mutation, enabled the team to prove that the exceptional females
   possessed only a single X chromosome (XO) instead of the normal two and
   that the Y chromosome is male-determining in mammals. These findings
   contributed to Liane Russell's subsequent hypothesis that one of the X
   chromosomes in females is genetically inactive.    
   
   My initial examinations of scurfy mice revealed startling results.
   Scurfy males had massively enlarged lymph nodes and spleen, a shrunken
   thymus, severe anemia, and thickened scaly skin. Histologic sections
   revealed a massive activation and proliferation of lymphoreticular cells
   affecting lymphoid organs, liver, and skin. These lesions suggested that
   the scurfy mouse immune system was reacting uncontrollably, possibly
   against its own tissues.     
   
   I attempted to control this immune reaction by surgically removing the
   thymus gland from scurfy mice on the first day after birth. Thymectomy
   almost doubled the lifespan of scurfy mice (from 24 to 45 days), but the
   mice still developed disease and died. This experiment suggested that
   the thymus gland and its T lymphocytes were important in scurfy disease
   and that sufficient disease-producing T cells formed in the embryonic
   stage to cause disease after birth.
   
   
   CD4 HELPER T CELLS     
   
   My subsequent work on identifying the disease-producing cell in scurfy
   mice has largely consisted of crossing the scurfy trait onto various
   lines of mice having other mutations affecting the immune system. For
   example, I mated scurfy carrier females with nude mice, which lack a
   thymus gland (and T lymphocytes). We found that the scurfy-nude mice are
   free of scurfy disease. Similarly, I crossed scurfy carriers with SCID
   mice, immunodeficient mice that are unable to form mature B or T cells.
   I observed that mice bred to be scurfy-SCID have T cells that fail to
   mature and, thus, these animals are also free of disease. I was also
   able to transfer scurfy disease to T cell-deficient nude or SCID mice by
   giving them a transplanted scurfy thymus. These experiments proved that
   mature T lymphocytes were the critical mediators of scurfy disease.    
   
   We are now testing scurfy on several lines of "knockout" mice--mice
   created with specific genetic defects by use of the techniques of
   homologous recombination and embryonal stem cell transfers. In this
   technique, an artificially disrupted mouse gene is created in the
   laboratory and inserted into cultured mouse embryo cells. Some of the
   mouse embryo cells accept this nonfunctional artificial gene into their
   genetic material in place of the homologous natural gene. Replacement of
   the natural gene with the artificial, nonfunctional gene causes the cell
   to lose the ability to produce the gene product; hence, a "knockout" of
   the gene function results. The mouse embryo cells containing the
   knockout gene are injected into developing mouse embryos that are
   implanted and brought to term in host mothers.    
   
   If the procedure is successful, mice born to these host mothers will
   carry one knockout gene and one normal gene. These mice are then bred
   together to create offspring that have two copies of the knockout gene
   (and no normal gene). Such mice are, therefore, unable to produce the
   targeted gene product. Many lines of knockout mice that lack various
   cells or molecules important to the immune system have been produced.
   For example, mice with knockouts of the CD4 gene lack CD4 T helper
   cells, whereas mice lacking the á-2 microglobulin gene fail to develop
   CD8 killer T cells. We have crossed the scurfy gene onto both of these
   knockout lines. Scurfy mice lacking CD8 killer T cells still have
   disease, but some scurfy mice lacking CD4 helper T cells appear to be
   cured, whereas others have a prolonged lifespan but eventually succumb
   to scurfy disease. This finding correlates well with recent in vitro
   studies showing that helper T lymphocytes from scurfy mice produced up
   to 100 times the amount of interleukins made by the helper T cells of
   their normal siblings. It appears that the culprit cells in scurfy
   disease are CD4 helper T cells that react abnormally to self-antigens
   and produce enormous amounts of interleukins. The excess interleukins
   are then responsible for the symptoms of scurfy such as weight loss,
   anemia, inflammation of skin and organs, and chronic diarrhea.
   
   
   THYMUS FAILS TO "TEACH"    
   
   Although we may have identified the disease-producing cell type in
   scurfy mice, we still do not know how or why it is formed. Patrick
   Blair, a University of Tennessee graduate student in my laboratory, has
   shown that precursors of disease-producing scurfy T cells are already
   present in the thymus glands of 14-day-old scurfy fetuses. Thus, scurfy
   T cells are committed to their abnormal phenotype at the earliest stages
   of the gland's development.     
   
   In contrast to other mouse models of autoimmune disease, scurfy is not
   a stem-cell defect--that is, the bone marrow precursor cells for T
   lymphocytes are normal, suggesting that the helper T cells acquire their
   defective behavior during development in the thymus. It could be that
   the scurfy fetal thymus fails to "teach" its T cells to tolerate
   self-antigens, allowing subsequent CD4 cells to react abnormally after
   the mouse is born. Current dogma suggests that self-tolerance occurs in
   the thymus by two mechanisms: clonal deletion (killing selfreactive T
   cells) and clonal anergy (rendering self-reactive T cells unresponsive
   to stimulation). Defects in either process could account for the T
   cell-mediated disease in the scurfy mice. In recent experiments, my
   students have found that scurfy mice do appear to have a defect in
   clonal deletion; clonal anergy in scurfy mice is still under
   investigation.     
   
   If we thoroughly understood these defects, we might discover the basic
   mechanisms that underlie many autoimmune diseases in both humans and
   animals. Understanding the autoimmune disease of scurfy mice will lead
   to greater insights into basic immunologic functions and may ultimately
   provide better diagnosis and treatment of autoimmune disease.
   
   
   BIOGRAPHICAL SKETCH    
   
   Virginia L. Godfrey, a board-certified veterinary pathologist, became
   manager of laboratory animal resources at ORNL's Biology Division in
   1989. She received her D.V.M. degree from Auburn University in 1982 and
   completed a Ph.D. degree in comparative and experimental medicine at the
   University of Tennessee in 1990. Her research interests include animal
   models of human disease. For her research on scurfy mice, Godfrey, along
   with Eugene Rinchik and Liane B. Russell, received a 1992 Technical
   Achievement Award from Martin Marietta Energy Systems, Inc., for
   demonstrating the role of the thymus in "educating" T lymphocytes and
   providing a genetic mouse model for autoimmune diseases. 
   
   Virginia L. Godfrey
   
   (keywords: autoimmune disease, immune system)
   
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   Date Posted:  1/11/94  (ktb)