I & i Antigens (Cold Agglutinin Disease)
Autoantibodies that produce this effect like to work in the cold. Can you guess what they are binding to on the surface of these red cells?
These carbohydrate antigens are present on red cells as well as on many other types of cells. The fetal version (i or “little i”) matures to the adult version (I or “big I”) sometime after birth. Cold agglutinins are IgM autoantibodies to either (or both) of these and they bind to red cells at temperatures lower than 37oC. Although agglutination (as shown in the image) may confound automated hematology analyzers in vitro and cause skin discoloration in vivo, the usual presentation is anemia due to chronic hemolysis. Most cases are idiopathic but cold agglutinins have also been associated with other disorders including Mycoplasma infection, infectious mononucleosis and lymphoma.
Antibodies to this molecule (and autoantibodies to the enzyme that degrades it in your intestine) may make you want to pass on the dinner rolls this Thanksgiving. Can you guess what it is?
Wheat gluten is a mixture of polypeptides (monomeric gliadins and polymeric glutenins) that are rich in glutamine residues. Digested peptides reach the intestinal mucosa where the enzyme transglutaminase removes an amino group (either allowing cross-linking with another polypeptide or creating a glutamate residue). If you happen to be unlucky enough to have the type of major histocompatibility complex that can present small peptides with glutamate residues, the antigen-presenting cells in the lamina propria of the small intestine may activate CD4 T cells. The ensuing inflammation causes diarrhea and malabsorption and, eventually, villous atrophy. Autoantibodies to the tissue transglutaminase are a marker of this disorder (called celiac disease).
The white spaghetti-like strands of spectrin form the infrastructure of the membrane cytoskeleton and a fragment of a related molecule may be the target of an autoantibody in Sjogren’s syndrome.
Sjogren’s syndrome is a common autoimmune disorder combining a major systemic rheumatic disease (such as rheumatoid arthritis or systemic lupus erythematosus) with the sicca complex of xerostomia (dry mouth) and keratoconjunctivitis (due to dry eyes). Diagnostic criteria include biopsy evidence of lymphocytic infiltration of either salivary or lacrimal glands and the presence of an autoantibody to the ribonucleoprotein complex Ro and La.
In fact, these autoantigens were once called SS-A and SS-B for “Sjogren’s syndrome” (see Autoantibody Target of the Month for July, 2006). Recent studies have indicated that an autoantibody to fodrin, a component of the membrane cytoskeleton, may also be involved in Sjogren’s syndrome. Although fodrin shares some homology with spectrin, it has distinct epitopes and the one targeted by anti-fodrin autoantibodies may be related to apoptosis.
Anti-thyroid peroxidase (TPO)
One of the two anti-thyroid antibodies depicted in this cartoon of indirect immunofluorescence (using thyroid substrate) is more important in diagnosing autoimmune thyroid disease than the other.
The cartoon depicts the indirect immunofluorescence pattern seen with anti-thyroglobulin antibody (left, with colloid brightly stained) and anti-thyroid peroxidase (TPO) antibody (right, with the epithelial cell cytoplasm brightly stained). Both assays are now routinely performed using immunoassay. In autoimmune thyroid disease, anti-thyroglobulin antibodies do not persist and may be negative in a significant number of patients when the disorder is diagnosed. Many physicians do not even believe that it is worthwhile to order anti-thyroglobulin antibody in such situations, and rely exclusively on the assay anti-TPO antibody. Note that the cytoplasmic distribution of anti-TPO antibody reflects its former name (anti-microsomal antibody).
Lens Epithelium-Derived Growth Factor
At a symposium at this year’s AACC meeting, Dr. Luis Andrade described finding autoantibodies to the transcription co-activator shown in red as the major cause of positive ANA tests in patients with no evidence of rheumatic disease.
While not likely to be a specific anti-nuclear antibody test in our clinical laboratories anytime soon, recognizing the indirect immunofluorescence pattern associated with this antibody may be of some utility. The cartoon clue refers to its function as a co-activator of transcription in the nucleus. In his presentation at the 2006 AACC meeting, Dr. Andrade described how the “dense fine speckled” ANA pattern associated with this autoantibody was a marker of ANA-positive individuals unlikely to have significant rheumatic autoimmune disease.
Ro and La
Like the stars and stripes, autoantibodies to the protein antigens depicted in red and blue usually go together.
These two proteins both bind to small RNA molecules. Ro (shown in red) has two major components (60 and 52kd) while La (shown in blue) has only one. They probably have very different functions (and probably seldom both bind to the same RNA molecule as shown in the cartoon) but autoantibodies to Ro and La usually appear in the same patients. Originally discovered by two groups of investigators, the antigens Ro and La (to which patients with systemic lupus erythematosus, or SLE, made antibodies) were shown to be identical to SS-A and SS-B, respectively. Antibodies to these latter antigens were found in patients with Sjogren’s syndrome. The alternative nomenclature persists to this day. Antibodies to Ro may appear without anti-La, but the converse is not true. Anti-Ro antibodies have also been associated with a variety of odd disorders including congenital heart block (in infants born to mothers with anti-Ro antibodies), neonatal lupus and subacute (primarily cutaneous) lupus.
A rare autoantibody to this molecule can mimic the effect of its regular ligand and cause symptoms that are quite common in many people this month.
There are two receptors for IgE on the surface of mast cells. The one that binds soluble IgE (and holds onto it) is the high-affinity IgE receptor (called FcERI). These are the receptor that signal for the mast cell to degranulate (and release histamine) when cross-linked by allergen. The other IgE receptor is the low-affinity one (formerly called FcERII, but now more commonly referred to as CD23). This receptor appears to bind IgE-antigen immune complexes, an activity that enhances the mast cell’s ability to degranulate. Some patients with chronic urticaria (hives) have an autoantibody against the high-affinity IgE receptor (especially against the alpha sub-unit shown in red). Some think that these autoantibodies may be contributing to the mast cell degranulation in these patients. However, the assay is rather insensitive (present in less than half of patients with chronic urticaria in most studies) and not well-standardized (performed by different techniques including Western blot, basophil mediator release and flow cytometry). Also, there does not seem to be any good correlation between the presence of the antibody and disease activity.
These two autoantibodies are reacting with the same autoantigen but they “see” different parts of it.
Antibodies to deoxyribonucleic acid (DNA) recognize two major types of epitopes. The first is the sugar-phosphate backbone of the molecule (shown to the left, in blue); these antibodies probably bind to both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA), as well as possibly other nucleic acids (such as RNA). The second major type of epitope is the nucleotide bases; these are not exposed in dsDNA but are especially prevalent in denatured ssDNA (shown to the right, in red). The epitopes of the sugar-phosphate backbone are very tolerogenic; it is difficult to make these antibodies even in experimental animals. Only disorders in which there is a major defect in tolerance, such as systemic lupus erythematosus (SLE), will result in the production of such autoantibodies. This is why their presence is very predictive of SLE. On the other hand, antibodies to nucleotide bases are very common and may be seen in a variety of rheumatic diseases. Anti-ssDNA antibodies are no longer even used clinically.
Autoantibodies to the small proteins associated with this molecule are often measured together and one of them is a specific marker for an important disease.
Autoantibodies against the protein components of uridine-rich ribonucleoprotein complexes are common in systemic lupus erythematosus. These complexes are important parts of the cellular machinery that splices transcribed messenger RNA. The hair-pin shaped RNA molecule (5’ at top; 3’ at bottom) shown is decorated with two types of protein. The purple-colored proteins to the left are core components of all forms of these RNP complexes and are referred to as “Sm” proteins (based on the first two initials of the last name of a patient in whom the autoantibodies were first discovered). Anti-Sm antibodies are only found in a small proportion of SLE patients but apparently are not found in any other disorder, making them very disease-specific. Autoantibodies to the other ribonucleoproteins (colored yellow and which vary, depending on the type of complex) are associated with SLE as well as other autoimmune disorders.
Glomerular Basement Membrane
Autoantibodies against this molecule may produce this immunofluorescence appearance when the kidney from a patient with rapidly progressive renal failure is biopsied.
Basement membranes are complex structures composed of collagen, adhesion molecules and sulfated polysaccharides. Both epithelial and endothelial cells use them as structural supports and guides to cellular organization. The collagen used is Type 4 collagen but there are a variety of type 4 collagen genes. Consequently, autoantibodies that develop against basement membrane antigens can show organ specificity. Ernest Goodpasture originally described a patient with explosive lung hemorrhage and acute renal failure and we now know that Goodpasture’s syndrome may be caused by autoantibodies against particular regions of type 4 collagen found in glomerular and alveolar capillary basement membrane. The appearance of the renal glomerular damage includes the presence of linear glomerular staining for IgG when direct immunofluorescence is used.
This molecule is an important target of typing antisera in the Microbiology laboratory but cross-reactive autoantibodies in infected patients may attack the heart.
M-proteins of Streptococcus pyogenes (an important gram-positive pathogen) extend from the cell membrane through the capsule (as pairs of coiled proteins) and prevent phagocytosis by neutrophils and macrophages. Therefore, the production of antibodies against them is an important part of protection from serious streptococcal infection. There is a great deal of heterogeneity (antibodies are used to “type” streptococci) and some anti-M-protein antibodies may cause disease instead of providing protection. Some M-proteins contain epitope that apparently resemble myosin (or other muscle proteins) and this molecular mimicry results in autoantibodies that react with myocardium and heart valves. Especially if the infection is not properly treated (with antibiotics), acute rheumatic fever may develop with chronic valvular disease (especially involving the mitral valve).
A molecule that turns the substance on the left into the substance on the right may be the target of an autoantibody that causes vasculitis.
The granules in both polymorphonuclear leukocytes and macrophages contain a number of enzymes that generate toxic oxygen species when fused with the phagosome containing an engulfed microorganism. One of these is hydrogen peroxide (pictured on the left). The enzyme myeloperoxidase forms hypochlorous acid (essentially chlorine bleach, pictured on the right) from hydrogen peroxide and chloride. This adds even greater killing power to the inside of the fused phagolysosome. Myeloperoxidase (which is being investigated as a marker of inflammation in acute coronary syndrome) is a target for one of the two major autoantibodies that are responsible for ANCA (anti-neutrophil cytoplasmic antibodies). ANCA are associated with small vessel vasculitis. Using indirect immunofluorescence, antibodies to myeloperoxidase are associated with the “p-ANCA” pattern (for “peri-nuclear” because one type of fixation causes the granules to coalesce around the nucleus).