Autoimmunity PDF

4466_Ch15_233-262 30/08/16 5:05 PM Page 234 Anergy Celiac disease Molecular mimicry Thyroglobulin (Tg) Anticentromere antibodies Central tolerance Multiple sclerosis (MS) Thyroid pero...

4466_Ch15_233-262 30/08/16 5:05 PM Page 234 Anergy Celiac disease Molecular mimicry Thyroglobulin (Tg) Anticentromere antibodies Central tolerance Multiple sclerosis (MS) Thyroid peroxidase (TPO) Anticyclic citrullinated CREST syndrome Myasthenia gravis (MG) Thyroid-stimulating peptide (anti-CCP or Double-stranded DNA Nucleolus hormone ( TSH) ACPA) (dsDNA) antibodies Thyroid-stimulating Nucleosome antibodies Antihistone antibodies Epigenetics hormone receptor Peripheral tolerance Antineutrophil cytoplasmic antibodies ( TRAbs) Epitope spreading Primary biliary cirrhosis (PBC) antibody (ANCA) Thyrotoxicosis Extractable nuclear antigens Rheumatoid arthritis (RA) Antinuclear antibodies (ANA) (ENAs) Thyrotropin-releasing Rheumatoid factor (RF) hormone ( TRH) Antiphospholipid antibodies Fluorescent antinuclear Self-tolerance Tissue transglutaminase Anti-RNP antibody antibody (FANA) testing Sm antigen (tTG) Autoantibodies Goodpasture’s syndrome Superantigens Type 1 diabetes mellitus Autoimmune diseases Granulomatosis with SS-A/Ro (T1D) Autoimmune hepatitis (AIH) polyangiitis (PGA) SS-B/La Wegener’s granulomatosis Autoimmune liver disease Graves disease (WG) Hashimoto’s thyroiditis Systemic lupus Autoimmune thyroid erythematosus (SLE) diseases (AITDs) Immunologic tolerance In the early 1900s, Paul Ehrlich noted that the immune system specific antigen, in this case, a self-antigen. In order for self- could attack the very host it was intended to protect, a phenom- tolerance to develop, lymphocytes must be “educated” so enon he referred to as “horror autotoxicus,” or “fear of self- they can distinguish between self-antigens and foreign anti- poisoning.” Conditions in which this phenomenon occurred gens. This education takes place at two levels: central and later became known as autoimmune diseases. Autoimmune dis- peripheral. eases are disorders in which immune responses are targeted to- Central tolerance occurs in the central or primary lym- ward self-antigens and result in damage to organs and tissues in phoid organs, the thymus, and the bone marrow.1,4 As T cells the body. These harmful effects can be caused by T-cell–mediated mature in the thymus, they encounter self-antigens that are immune responses or autoantibodies that are directed against normally present on the surface of the thymic epithelial cells. host antigens. More than 100 autoimmune diseases have been In a process called negative selection, T cells that express discovered, and these can involve various organ systems. T-cell receptors (TCRs) with a strong affinity for these self- Autoimmune diseases are a leading cause of chronic illness and antigens are deleted by apoptosis, a physiological form of cell death, affecting about 5% of the world’s population, including death (see Chapters 4 and 17 and Figure 15–1). Negative se- 50 million people in the United States alone.1-3 This chapter will lection occurs with both the immature, double-positive begin by discussing the factors that are thought to contribute to CD4+/CD8+ cells in the cortex and with the more mature, the development of autoimmunity so that you can gain a better single-positive CD4+ or CD8+ cells in the medulla. During understanding of the underlying pathology of autoimmune dis- this process, some of the self-reactive CD4+ T cells are not ease. The discussion will then proceed to the clinical manifesta- deleted, but instead differentiate into T regulatory (Treg) tions and immunopathology of specific autoimmune diseases, cells that can specifically inhibit immune responses to self- as well as the laboratory tests that are used in their diagnosis. antigens. Similarly, as B cells mature in the bone marrow, those with receptors having a strong affinity for self-antigens Etiology of Autoimmune Disease are eliminated by apoptosis. Some self-reactive B cells are not deleted: rather, they are stimulated to rearrange their im- munoglobulin genes so that their B-cell receptors are no Self-Tolerance longer antigen specific. This process is known as receptor ed- Under normal circumstances, the immune system is able to iting. B cells that possess receptors that only weakly recognize differentiate between “self” and “nonself” or “foreign,” so that self-antigens are induced to downregulate the expression of self-antigens are not destroyed. This fundamental concept was their receptors and develop a specific state of unresponsive- introduced in Chapter 2. Central to this phenomenon is self- ness to the antigens known as anergy. tolerance, or the ability of the immune system to accept self- Thus, there are several ways in which the central tolerance antigens and not initiate a response against them. Autoimmune of T and B cells can be achieved. This process is not totally ef- disease is thought to result from a loss of self-tolerance. fective, however, and some self-reactive lymphocytes manage Self-tolerance is a type of immunologic tolerance, or a to escape to the secondary lymphoid organs such as the lymph state of immune unresponsiveness that is directed against a nodes and spleen. Therefore, a second level of protection is 4466_Ch15_233-262 30/08/16 5:05 PM Page 235 Th Central (bone marrow) B Tc Central (thymus) Receptor editing B Negative selection Anergy Differentiation to Treg B Apoptosis Peripheral Peripheral (lymph nodes, spleen) Treg (lymph nodes, spleen) Tc Th Anergy B Inhibition by Treg or lack of costimulation Apoptosis Apoptosis Th Escape of Tc B self-reactive cells Autoimmune disease Mechanisms of central and peripheral tolerance. needed. In peripheral tolerance, lymphocytes that recognize be caused by complex interactions between genetics, exposure self-antigens in the secondary lymphoid organs are rendered to environmental factors, and defects in immune regulation. incapable of reacting with those antigens.1,4 Peripheral toler- Some of the major factors that are believed to contribute to ance of T cells can result from anergy caused by the absence autoimmunity will be discussed in the text that follows. of a costimulatory signal from an antigen-presenting cell (APC) or binding of inhibitory receptors such as CTLA-4 (a molecule that prevents T-cell activation). Peripheral T-cell Genetics tolerance can also result from inhibition by Tregs or death by There is much evidence supporting a genetic basis for autoim- apoptosis. Self-reactive B cells in the periphery can be deleted mune disease.5,6 Autoimmune diseases are often more preva- by apoptosis, be rendered anergic after repeated stimulation lent among family members than among unrelated individuals with self-antigens, or receive inhibitory signals through recep- and are more prevalent among monozygotic (genetically iden- tors such as CD22. tical) twins than dizygotic (non-identical) twins or siblings. Re- In some individuals, self-tolerance can fail even after this searchers conducting molecular studies continue to identify second layer of protection; if this happens, autoimmunity can specific genetic polymorphisms or mutations that are associ- arise. The development of autoimmune disease is thought to ated with autoimmune diseases. 4466_Ch15_233-262 30/08/16 5:05 PM Page 236 Most of the research concerning humans has focused on autoimmunity at an earlier age and have a higher risk for ac- genes in the major histocompatibility complex (MHC) (see quiring more than one autoimmune disease as compared with Chapter 3). Investigators have found that there is an association men. Furthermore, females have been found to have higher ab- between the presence of certain human leukocyte antigen solute CD4+ T-cell counts and higher levels of circulating anti- (HLA) types and the risk of developing a particular autoim- bodies than men.8 These observations suggest that there is a mune disorder. The strongest link found is between the HLA- hormonal influence on the development of autoimmunity. Stud- B27 allele and the development of ankylosing spondylitis, an ies on the effects of hormones have shown that estrogens tend autoinflammatory disease that affects the spine. Individuals to direct the immune system in favor of a type 2 helper cell who possess HLA-B27 have about a 100 times greater chance (Th2) response, resulting in more B-cell activation and antibody of developing the disease than individuals who do not have production, whereas androgens favor a type 1 helper cell that allele.4,6 Other associations with specific MHC genes are (Th1) response with activation of CD8+ T cells. Prolactin, a discussed in the sections on particular autoimmune diseases hormone that stimulates production of breast milk in pregnant in this chapter. Differences in MHC genes are thought to in- and nursing women, can stimulate both humoral and cell- fluence the development of autoimmune disease because the mediated immune responses.8 The stimulatory effects of female specific structure of the MHC molecule can determine whether hormones may place women at a greater risk for developing or not a self-antigen can attach to the peptide-binding cleft of autoimmune disease. the molecule and subsequently be processed and presented to T cells.4 In addition, class II MHC molecules can sometimes be abnormally expressed on cells where they are not typically When immunologic tolerance to self-antigens occurs during found, resulting in the presentation of self-antigens for which the early development of lymphocytes in the thymus and bone no tolerance has been established. marrow, some self-antigens may be cryptic, or hidden within Genome-wide association studies are revealing that poly- the tissues of the host. T and B lymphocytes are shielded from morphisms in some non-MHC genes can also be associated these sequestered antigens and are not educated to become tol- with development of autoimmune disease. Many of these genes erant to them. At a later time in life, inflammation or tissue influence the development and regulation of immune re- trauma could cause the cryptic antigens to be released and to sponses. Examples include the PTPN22 gene, which has a role suddenly be accessible to the uneducated lymphocytes, trig- in T- and B-cell receptor signaling; the IL2RA gene, which is gering an immune response.1,4,9 Tissue damage could be involved in T-cell activation and maintenance of Tregs; the caused by factors such as infections, contact with environmen- CTLA4 gene, which has an inhibitory effect on T-cell activation; tal toxins, or physical injury from exposure to ultraviolet (UV) the BLK gene, which is involved in B-cell activation and devel- radiation. This concept has also been referred to as immunologic opment; and the AIRE (autoimmune regulator) gene, which ignorance and may be responsible for the production of autoan- promotes the development of T-cell tolerance in the thymus.4 tibodies to the lens of the eye following an ocular injury, au- Although most autoimmune diseases involve multiple genes toantibodies to sperm after a vasectomy, and autoantibodies to (~20 to 30), single-gene mutations that can be inherited in a DNA following damage to skin cells by overexposure to UV Mendelian fashion have been associated with rare autoimmune rays from the sun. disorders. Inheritance of specific genes may make an individual more Scientists have been very interested in the association of mi- susceptible to a particular autoimmune disease, but genetic crobial infections with the development of autoimmune dis- makeup is not totally responsible because the majority of peo- ease. Bacteria, viruses, and other infectious pathogens may be ple with a particular gene will not develop autoimmunity. In able to trigger autoimmune responses in a variety of ways. A addition, the concordance rate among monozygotic twins principal means by which microbes are thought to accomplish (i.e., the presence of autoimmune disease in both members of this is through molecular mimicry. Molecular mimicry refers a pair of identical twins) is only between 20% and 30% for most to the fact that many bacterial or viral agents contain antigens autoimmune diseases.7 Furthermore, the prevalence and sever- that closely resemble the structure or amino acid sequence of ity of many autoimmune diseases vary in different geographic self-antigens. Exposure to such foreign antigens may trigger locations.5 This variance suggests that environmental and other immune responses that cross-react with similar self-antigens. factors also play a role in the development of autoimmune dis- Molecular mimicry has been postulated as a mechanism for a ease. A discussion of some of the major factors that are believed number of human autoimmune diseases.9-11 The best known to trigger autoimmune responses follows. example involves the association between the gram-positive bacterium Streptococcus pyogenes and rheumatic fever, an au- Other Endogenous and Environmental toimmune disorder that primarily affects the joints and the Factors heart. Some patients who have acquired scarlet fever or pharyngitis as a result of infection with S pyogenes will proceed to develop rheumatic fever if they are not treated adequately Women are 2.7 times more likely to acquire an autoimmune dis- with antibiotics (see Chapter 20). Symptoms develop 2 to ease than men; in fact, about 78% of patients with autoimmune 4 weeks after the infection and are thought to be caused by diseases are of female gender.8 Women also tend to develop the production of antibodies to the M protein and N-acetyl 4466_Ch15_233-262 30/08/16 5:05 PM Page 237 glucosamine components of the bacteria, which cross-react underexpression of certain genes in the immune system may with cardiac myosin, causing damage to the heart.10 result in homeostatic imbalances and a breakdown of self- A second way that microbes might trigger autoimmunity is tolerance, leading to autoimmunity.15,16 through a bystander effect.4,9,11 In this mechanism, the microbial Sometimes, exposure to environmental factors can lead organism does not have to share structurally similar antigens to changes at the protein level. The changes are known as with the host. Instead, the microorganism can induce a local post-translational modifications and may involve biochemical inflammatory response that recruits leukocytes and stimulates processes such as acetylation, lipidation, citrullination, and APCs to release cytokines that nonspecifically activate T cells. glycosylation.6 These modifications can alter the immuno- Some of the T cells that are activated may have specificity for genicity of an antigen, affecting its ability to be processed self-antigens. This expansion of the immune response to unre- by APCs and presented to T cells. Such alterations of self- lated antigens has also been termed “epitope spreading.” antigens can make them more immunogenic, leading to au- A third way that microorganisms might induce autoim- toimmune responses. For example, citrullination of collagen munity is through superantigens. Superantigens are pro- might play a role in the pathogenesis of rheumatoid arthri- teins that are produced by various microbes that have the tis (RA) and glycosylation of myelin may be involved in the ability to bind to both class II MHC molecules and TCRs, re- pathology of multiple sclerosis (see the Rheumatoid Arthritis gardless of their antigen specificity.12 Examples are the and Multiple Sclerosis sections in the text that follows). staphylococcal enterotoxins that cause food poisoning and toxic shock syndrome. These superantigens can act as potent T-cell mitogens by activating a large number of T cells with Although the precise etiology of autoimmunity is unknown, different antigen specificities. If some of these T cells possess there is much evidence that suggests that this heterogeneous specificity for a self-antigen, autoimmune responses might disease entity is caused by complex interactions between result.9 Likewise, some viruses, including the Epstein-Barr genetic and environmental factors (Fig. 15–2).1,4,6,15 Certain virus (EBV) and cytomegalovirus (CMV), can cause poly- genes are thought to make individuals more susceptible to clonal activation of B cells.1 immune responses against self-antigens, but are not suffi- Scientists have also been very interested in the complex re- cient by themselves to cause autoimmune disease. Gender lationship between microbiota, or normal flora, and the im- of the individual, tissue injury, and exposure to infectious mune system. Research has shown that the presence of certain microorganisms or other environmental agents are all be- strains of endogenous bacteria may be associated with a greater lieved to have significant effects on the immune system that risk for autoimmune disease.11 These strains, as well as path- can trigger autoimmune responses in susceptible individuals. ogenic microorganisms, may stimulate innate immune re- As a result of this break in immunologic tolerance, autore- sponses through interaction with pattern recognition receptors active T cells recognize and proliferate in response to self- such as the Toll-like receptors (TLRs). This interaction triggers antigens and B cells develop into plasma cells that secrete cell-signaling pathways that result in the production of cy- autoantibodies. This can result in the release of proinflam- tokines such as IFN α, which can stimulate cells of the adaptive matory cytokines, which, when coupled with dysfunctions immune system, some of which are directed toward self- in immune-regulatory cells, perpetuate the autoimmune re- antigens. A decrease in the number and function of Tregs can sponses. If these responses are not held in check, they can perpetuate the activity of autoreactive cytotoxic T cells and culminate in autoimmune disease.1,7,14 Tissue injury in these hyperactive B cells that produce autoantibodies. The activated disorders results from hypersensitivity reactions that involve lymphocytes, in turn, produce proinflammatory cytokines that autoantibodies to cell-surface receptors, deposition of im- provide signals to stimulate cells of the innate system, thus pro- mune complexes that contain self-antigens, and cell-mediated ducing a vicious cycle that amplifies the immune response and cytotoxicity.1 The immunopathological mechanisms vary sustains autoimmunity.7 with specific autoimmune diseases and will be discussed in the text that follows. Investigators have done much research in the area of epige- netics and how it may relate to the development of autoim- Systemic Autoimmune Diseases munity. Epigenetics refers to modifications in gene expression that are not caused by changes in the original DNA se- Autoimmune diseases can be classified as systemic or organ- quence.13,14 These alterations are stable and can be inherited. specific, depending on the extent of the pathology. There They are thought to be triggered by exposure to environmen- is often a good bit of overlap between the two categories tal toxins, ingestion of harmful foods or drugs, or the aging because some diseases may start out affecting a single process. These factors can induce epigenetic changes by in- organ, but progress later to affect other locations in the body. creasing or decreasing methylation of cytosine bases, modify- Table 15–1 lists some of the systemic autoimmune diseases ing histones, and causing abnormal regulation by microRNAs. along with their corresponding target tissues and associated These modifications can result in changes in the level at which autoantibodies, whereas Table 15–3 later provides this in- genes are expressed by affecting their ability to be transcribed formation for some of the organ-specific diseases. The sec- into mRNA, which is subsequently translated into proteins tions that follow will discuss three systemic autoimmune that will influence the phenotype of an individual. Over- or diseases and the laboratory tests that are essential to their 4466_Ch15_233-262 30/08/16 5:05 PM Page 238 Environmental and Genetically Loss of Immunologic Autoimmune disease Endogenous Triggers susceptible Tolerance individual Activation of self-reactive T Female hormones (possesses cells and B cells and Tissue injury and release of certain HLA or development of immune self-antigens other genes) responses to self-antigens Microbial infections (molecular mimicry, epitope spreading, superantigens, stimulation of immune responses) Epigenetic factors (toxins, foods, drugs, aging) and post-translational modifications Interactions between genetic and environmental factors in the development of autoimmunity. Table 15–1 Systemic Autoimmune Diseases DISEASE TARGET CELLS AND TISSUES ASSOCIATED AUTOANTIBODIES Systemic lupus Multiple cells and organs through- Antibodies to double-stranded DNA and other nuclear erythematosus out the body, including the skin, components, such as Sm (ANAs) (SLE) joints, kidneys, brain, heart, lungs Phospholipid antibodies Antibody to RBCs Antibody to platelets Antibody to lymphocytes Antibody to ribosomal components Antibody to endothelium Rheumatoid factor Rheumatoid Joints, bone; other tissues in Anti-CCP (cyclic citrullinated proteins) arthritis (RA) some cases Rheumatoid factor Antinuclear antibodies (ANAs) Granulomatosis Upper respiratory system, lungs, Antineutrophil cytoplasmic antibodies (ANCA); c-ANCA pattern with polyangiitis blood vessels Rheumatoid factor (Wegener’s ANAs granulomatosis) diagnosis: systemic lupus erythematosus, RA, and granulo- within the immune system.21 Environmental factors thought matosis with polyangiitis (Wegener’s granulomatosis). to play a role in SLE include UV light, certain medications, and possibly infectious agents.17,21,22 Exposure to sunlight is a well- Systemic Lupus Erythematosus (SLE) known trigger of the photosensitive skin rashes seen in many lupus patients. Certain drugs, such as procainamide (used to Systemic lupus erythematosus (SLE) is a chronic systemic treat abnormal heart rhythms), hydralazine (used for high inflammatory disease that affects between 40 and more than blood pressure), and the tuberculosis drug isoniazid, can in- 200 persons per 100,000, depending on the population.17 The duce a transient lupus-like syndrome that resolves once the peak age of onset is usually between 20 and 40 years. Women drug is stopped.23 Hormones are also important as indicated are much more likely to be affected than men, by a ratio of by the significantly higher incidence of lupus in females and about 9 to 1.18 SLE is also more common in African Americans an increased risk of developing lupus in women that have used and Hispanics than in Caucasians.17,19 With earlier diagnosis estrogen-containing contraceptives or hormone replacement and improved treatments, the 5-year survival rate has increased therapy.19 Hormones may be important because they may help from 50% in the 1950s to greater than 90% today.17,18,20 regulate the transcription of genes that are central to the ex- pression of SLE.22 SLE appears to originate from complex interactions between The majority of individuals exposed to the environmental environmental factors, genetic susceptibility, and abnormalities factors mentioned previously do not develop lupus, and genetic 4466_Ch15_233-262 30/08/16 5:05 PM Page 239 makeup is believed to play an important role in susceptibility Connections to SLE. More than 20 genetic loci associated with lupus in humans have been reported.18,21 People with certain HLA Type III Hypersensitivity types, especially HLA-A1, B8, and DR3—have an increased The immune complexes generated in SLE activate complement, chance of developing lupus.17 Another group of genes that inducing the generation of chemotaxins such as C5a. The have been associated with increased susceptibility to SLE plays activation of complement results in recruitment of neutrophils, a role in the clearance of immune complexes (see the text that which release lysosomal enzymes that cause injury to the sur- follows).21,24 Other lupus-associated genes include polymor- rounding tissues. This is an example of a type III hypersensitivity phisms in genes associated with immune function, genes cod- response, which was discussed in Chapter 14. ing for various cytokines, and genes involved in signaling of innate immune responses.25 These defects are thought to result in uncontrolled autoreactivity of T and B cells, which leads to antibodies to red blood cells (RBCs) can cause hemolytic ane- the production of numerous autoantibodies. mia and antibodies to platelets can cause thrombocytopenia by antibody-mediated cytotoxic (type II) hypersensitivity.17 An- tibodies to endothelial cells can cause inflammation of the Over 100 autoantibodies associated with SLE have been dis- blood vessels and vascular damage in lupus, which may be re- covered.25 These include antibodies to double-stranded DNA sponsible for the vasculitis and neuropsychiatric symptoms (dsDNA), histones, and other nuclear components, as well seen in some SLE patients.26 Phospholipid antibodies are as- as autoantibodies to lymphocytes, erythrocytes, platelets, sociated with increased miscarriage, stillbirth, and preterm de- phospholipids, ribosomal components, and endothelium.25,26 livery in pregnant women with lupus.30,31 Neonatal lupus, The typical patient has an average of three circulating which occurs in up to 8% of babies born to pregnant women autoantibodies.19 with SLE, is associated with antibodies to the nuclear antigens, B cells and the autoantibodies they produce are believed SS-A/Ro and SS-B/La.30 Symptoms are transient and resolve to play a central role in the pathogenic mechanisms that are at 6 to 8 months of age when the maternal antibodies have responsible for this complex disease. In fact, the presence cleared from the infant’s circulation. In utero heart block is a of autoantibodies can precede the onset of disease by 9 to serious complication that occurs in 2% of fetuses whose moth- 10 years.26 Abnormal apoptosis of certain types of cells may ers have anti–SS-A antibodies.30 occur, releasing excess amounts of cellular constituents such as DNA and ribonucleic acid (RNA). Dysfunctional removal of cellular debris by phagocytes may allow these cellular com- The clinical signs of SLE are extremely diverse; nonspecific ponents to persist, increasing the chances for autoantibody symptoms such as fatigue, weight loss, malaise, fever, and production.17,27 anorexia are often the first to appear.31 The disease is marked Antibodies to dsDNA are present in 70% of patients with by alternating relapses or flares and periods of remission.18 Joint lupus and are highly specific for the disease.17 Anti-dsDNA involvement seems to be the most frequently reported manifes- and complement proteins have been found in immune com- tation because over 90% of patients with SLE are subject to pol- plexes that are deposited in organs such as the kidneys and yarthralgias or arthritis.19,32 Typically, the arthritis is symmetric skin and are thought to play a major role in the pathogenesis and involves the small joints of the hands, wrists, and knees. of SLE. It is not clear whether preformed immune complexes After joint involvement, the next most common signs are circulate in the bloodstream and settle in various organs in the skin manifestations. These can present in various forms and are body, whether the autoantibodies cross-react with protein experienced by about 80% of patients with lupus.19 An erythe- components of the tissues, or whether nuclear antigens are at- matous rash may appear on any area of the body exposed to tracted to the tissues by charge–charge interactions and then UV light. Less common but perhaps more dramatic is the classic stimulate the production of autoantibodies.17,28,29 Accumula- butterfly rash across the nose and cheeks that appears in some tion of IgG to dsDNA seems to be the most pathogenic be- SLE patients (Fig. 15–3). This rash is responsible for the name cause it forms complexes of an intermediate size that become lupus, derived from the Latin term meaning “wolf-like.” In dis- deposited in the glomerular basement membrane (GBM). coid lupus, skin lesions have central atrophy and scarring. Once immune complexes are formed, they cannot be Evidence of renal involvement is present in about half of all cleared efficiently because of other possible deficiencies in patients with lupus; nephritis is a major cause of illness and lupus patients. These include defects in complement receptors death.28 There are several types of lesions, but the most dan- on phagocytic cells; defects in receptors for the FC portion of gerous is diffuse proliferative glomerulonephritis, characterized immunoglobulins; or rarely, deficiencies of early complement by cell proliferation in the glomeruli that can lead to end-stage components such as C1q, C2, or C4 (see Chapter 7).17,19,22 renal disease.19,28,33 Other conditions involving the kidneys The immune complexes activate complement and initiate an may include deposition of immune complexes in the suben- inflammatory response. Leukocytes are attracted to the sites of dothelial tissue and thickening of the basement membrane, all inflammation and release cytokines that perpetuate the re- of which can lead to renal failure. sponse, resulting in tissue damage.17,28 Other systemic effects may include cardiac involvement with Autoantibodies to nuclear and nonnuclear antigens can also pericarditis, tachycardia, or ventricular enlargement; pleuritis cause cellular destruction by other mechanisms. For example, with chest pain; and neuropsychiatric manifestations such as 4466_Ch15_233-262 30/08/16 5:05 PM Page 240 The type of treatment used depends on the severity of the dis- ease. For mild symptoms, a high dose of aspirin or other anti- inflammatory drug may bring relief. For skin manifestations, antimalarials such as hydroxychloroquine or chloroquine and topical steroids are often prescribed.19 The antimalarial drugs are thought to inhibit signaling of TLR 7, 8, and 9. Systemic corticosteroids are used for acute fulminant (severe and sudden) lupus, lupus nephritis, or central nervous system (CNS) com- plications because these suppress the immune response and lower antibody titers.19,35 Other drugs have also been used; however, any immunosuppressive drug may have serious side effects and patients must be monitored closely. Monoclonal an- tibodies and other biological agents that target components of the immune system thought to be central to pathogenesis of lupus are being evaluated for their clinical effectiveness.17,35 The most common cause of death in lupus patients is in- Butterfly rash in SLE. Characteristic rash over the fection, followed by heart disease.31 Renal involvement is also cheekbones and forehead is diagnostic of SLE. The disease often a source of significant morbidity and mortality in this patient begins in young adulthood and may eventually involve many organ group. The key to successful treatment is to prevent organ systems. (From Steinman L. Autoimmune disease. Sci Am. 1993;269:107, damage and achieve remission. Overall, the treatments of today with permission.) have come a long way in achieving this goal, as the 5-year survival rate has increased to more than 90%.17,18,20 seizures, mild cognitive dysfunction, psychoses, or depression. Hematologic abnormalities such as anemia, leukopenia, throm- bocytopenia, or lymphopenia can also be present.32,33 General laboratory tests that can be used in the initial evalua- Drug-induced lupus differs from the more chronic form of tion of patients include a complete blood count (CBC), a the disease, in that symptoms usually disappear once the drug platelet count, and urinalysis.19 Some of the first laboratory is discontinued. The most common drugs implicated are pro- findings in lupus patients are leukopenia and possible anemia cainamide, hydralazine, chlorpromazine, isoniazid, quinidine, and thrombocytopenia.31 In addition, the erythrocyte sedimen- anticonvulsants such as methyldopa, and possibly oral contra- tation rate (ESR) may be elevated even though the C-reactive ceptives.19,31 Typically, this is a milder form of the disease and protein (CRP) level tends to be low or normal. is usually manifested as fever, arthritis, or rashes; rarely are the More specific laboratory tests include the quantification of kidneys involved.19,23 complement proteins and the detection of specific autoanti- In 1982, the American College of Rheumatology (ACR) es- bodies. C3 is the most commonly measured complement tablished a set of clinical and immunologic criteria that could protein.19 Serum complement levels may be low during disease be used to define SLE for the purposes of research and surveil- flares as a result of complement consumption by immune com- lance; an update was published in 1997.18 In 2012, the Sys- plexes. Thus, complement levels can be helpful not only in the temic Lupus International Collaborating Clinics validated and initial diagnosis, but also for monitoring patients over time.19,31 further revised these criteria.34 There are now 11 clinical cri- When SLE is suspected, the first test typically done is a teria and 6 immunologic criteria. The clinical criteria are acute screening test for antinuclear antibodies (ANAs) because cutaneous lupus, chronic cutaneous lupus, oral ulcers, non- these are present in the majority of patients with the disease. scarring alopecia (thinning or fragility of the hair), synovitis, These antibodies and the methods used to detect them are dis- serositis, renal involvement, neurological symptoms, hemolytic cussed in more detail in the section that follows. Phospholipid anemia, leukopenia, and thrombocytopenia34 The six immuno- antibodies are present in some patients with lupus and are logic criteria are elevated antinuclear antibody titer, elevated discussed in a later section. anti-dsDNA titer, presence of antibody to the Sm nuclear anti- gen, presence of antiphospholipid antibody, low complement Antinuclear Antibodies (ANAs) levels, and positive direct Coombs’ test in the absence of he- molytic anemia.34 In most cases, a patient must satisfy at least 4 of the 17 criteria, including at least one clinical criterion and ANAs are autoantibodies that are directed against antigens in one immunologic criterion, to be classified as having SLE. Al- the nuclei of mammalian cells. ANAs are present in over 95% though these criteria are not meant to be used for diagnosis, of patients with active lupus and are used as a major marker for they reflect the major clinical and laboratory features that are the disease.19,25,29,36 However, ANAs are not specific for SLE associated with SLE. Some of the main laboratory tests that are because they can also be detected in a significant percentage of helpful in diagnosis are discussed in Laboratory Diagnosis of patients with other connective tissue diseases, including mixed Systemic Lupus Erythematosus section later in this chapter. connective tissue disease, Sjögren’s syndrome, scleroderma, 4466_Ch15_233-262 30/08/16 5:05 PM Page 241 polymyositis-dermatomyositis, and RA.37 They can also be presence of these antibodies is considered diagnostic for SLE, found in some individuals with other conditions, including especially when they are found in combination with low lev- chronic infections, cancer, and pregnancy.36 Furthermore, up els of the complement component C3.39,40 Antibodies to to 5% of healthy persons and up to 30% of elderly individuals dsDNA typically produce a peripheral or a homogeneous are ANA-positive.36 staining pattern on indirect immunofluorescence (IIF).25,36,41 ANAs are a heterogeneous group of antibodies that have dif- See the Indirect Immunofluorescence (IIF) section for additional ferent antigen specificities. The nuclear antigens they are directed information. against include double-stranded (ds) and single-stranded (ss) Antihistone antibodies can also be found in lupus patients. DNA (deoxyribonucleic acid), histones, nucleosomes (DNA- Histones are nucleoproteins that are essential components of histone complexes), centromere proteins, and extractable nu- chromatin. There are five major classes of histones: H1, H2A, clear antigens (ENAs).26 ENAs are a group of nuclear antigens H2B, H3, and H4. Antibodies to H2A and H2B can be detected that were so named because they were isolated in saline extracts in almost all patients with drug-induced lupus. Presence of an- of mammalian tissues.38 These antigens represent a family of tihistone antibody alone or combined with antibody to ssDNA small nuclear proteins that are associated with uridine-rich RNA. supports the diagnosis of drug-induced lupus.32,36 About 70% The ENAs include ribonucleoproteins (RNP), the Sm antigen, of other patients with SLE have elevated levels of antihistone the SS-A/Ro and SS-B/La antigens, Scl-70, Jo-1, and PM-1.38 antibodies, but the titers are usually fairly low.19,25 High levels Some of the more common ANAs and their associated features of antihistone antibodies tend to be associated with more active are discussed in the text that follows and listed in Table 15–2. and severe SLE.32 Antihistone antibodies are also found in RA, Double-stranded DNA (dsDNA) antibodies are the most Felty’s syndrome, Sjögren’s syndrome, systemic sclerosis, and specific for SLE because they are mainly seen in patients with primary biliary cirrhosis, but the levels are usually lower.25 lupus and their levels correlate with disease activity.25,32,36 Antihistone antibodies typically produce a homogeneous pattern Although they are found in only 40% to 70% of patients, the in the IIF assay.25,32,41 Table 15–2 Common Antinuclear Antibodies CHARACTERISTICS IMMUNOFLUORESCENT AUTOANTIBODY OF ANTIGEN PATTERN DISEASE ASSOCIATION Anti-dsDNA dsDNA Peripheral or homogeneous SLE Anti-ssDNA Related to purines and pyrimidines Not detected on routine SLE, many other diseases screen Antihistone Different classes of histones Homogeneous Drug-induced SLE, other diseases Anti-DNP DNA-histone complex Homogeneous SLE, drug-induced SLE (nucleosomes) Anti-Sm Extractable nuclear antigen Coarse speckled Diagnostic for SLE (uridine-rich RNA component) Anti-RNP Proteins complexed with small Coarse speckled SLE, mixed connective nuclear RNA tissue diseases Anti–SS-A/Ro Proteins complexed to RNA Finely speckled SLE, Sjögren’s syndrome, others Anti–SS-B/La Phosphoprotein complexed to Finely speckled SLE, Sjögren’s syndrome, RNA polymerase others Antinucleolar RNA polymerase, fibrillarin, PM-1 Prominent staining of SLE, systemic sclerosis nucleoli (can be smooth, clumpy, or speckled) Anti–Scl-70 DNA topoisomerase I Atypical speckled Systemic sclerosis, scleroderma Anti–Jo-1 Histidyl-tRNA synthetase Fine cytoplasmic speckling Polymyositis Autoantibody– Characteristics of Antigen-Antigens Immunofluorescent Disease Association- Anti-Centromere in the chromosome centromeres pattern-Discrete speckled CREST syndrome Adapted from Bradwell AR, Hughes RG, Karim AR. Immunofluorescent antinuclear antibody tests. In: Detrick B, Hamilton RG, Folds, JD, eds. Manual of Molecular and Clinical Laboratory Immunology. 7th ed. Washington, DC: ASM Press; 2006:996–997. DNA = deoxyribonucleic acid; DNP = deoxyribonucleoprotein; RNA = ribonucleic acid; RNP = ribonucleoprotein; SLE = systemic lupus erythematosus. 4466_Ch15_233-262 30/08/16 5:05 PM Page 242 Nucleosome antibodies are stimulated by DNA-histone components: fibrillarin, RNA polymerase I, and PM-1.43 Anti- complexes, known as nucleosomes, or deoxyribonucleoprotein body to fibrillarin is common in systemic sclerosis (also known (DNP). These antibodies are directed only against the com- as scleroderma) and is indicated by clumpy nucleolar fluores- plexes and not against DNA or the individual histones. Nucle- cence in the IIF assay.36,40,43 Scleroderma is an autoimmune osome antibodies are found in about 85% of patients with SLE disease that primarily involves the skin and the blood vessels. and their levels correlate with disease severity.25 They typically Antibodies to RNA polymerase are also associated with sclero- produce a homogeneous pattern in the IIF assay.41 derma, but produce a speckled nucleolar pattern in IIF. Antibody to the Sm antigen is specific for lupus because it Homogeneous staining of the nucleolus is associated with is not found in other autoimmune diseases. However, it is antibodies to the PM-1 antigen (also known as PM/Scl) and is found in only 20% to 40% of patients with SLE, depending on found in polymyositis and systemic sclerosis.36,43 the race of the population.25 It is unclear whether titers corre- Anticentromere antibodies bind to proteins in the middle late with disease activity. Antibody to a preparation of this ENA region of a chromosome where the sister chromatids are joined. was first described in a patient named Smith, hence the name These antibodies are directed against three centromere antigens anti-Sm antibody. The anti-Sm antibody produces a coarse of molecular weights 16kDa, 80kDa, and 120kDa.32 They are speckled pattern of nuclear fluorescence on IIF.41 found in 50% to 80% of patients with the CREST syndrome, Anti-RNP antibody is directed against RNP, which consists a subset of scleroderma named after its five major features: cal- of several nonhistone proteins complexed to a small nuclear cinosis, Raynaud’s phenomenon, esophageal dysmotility, scle- RNA called U1-nRNP (U for “uridine-rich”). RNP forms com- rodactyly, and telangiectasia.32 In the IIF assay, centromere plexes with the Sm antigen in the nucleus, and antisera to these antibodies produce discrete speckled staining in the nuclei of antigens produce a pattern of partial identity when they are the cells.42,43 reacted in the Ouchterlony double immunodiffusion test (see discussion and Figure 15–7 in the text that follows). In the IIF A variety of methods have been developed to detect ANAs in assay, anti-nRNP produces a coarse speckled pattern.41 Anti- patient serum. These include IIF, immunoperoxidase staining, bodies to RNP are detected in 20% to 30% of patients with enzyme-linked immunosorbent assay (ELISA), microsphere SLE, but are also found at a high titer in individuals with mixed multiplex immunoassays (MIA), radioimmunoassay (RIA), im- connective tissue disease and in lower levels in patients with munodiffusion, immunoblotting (Western blot), dot blot, im- other autoimmune rheumatic diseases such as systemic scle- munoelectrophoresis, and microarray.32,38 Some of the more rosis, Sjögren’s syndrome, and RA.25,32,36 commonly used assays are discussed in the text that follows. Lupus patients can also produce antibodies to another fam- ily of ENAs called SS-A/Ro and SS-B/La. These antigens consist of small, uridine-rich RNAs complexed to cellular proteins and Fluorescent antinuclear antibody (FANA) testing has been were given the prefix of SS- because a large percentage of pa- the most widely used and accepted test because it is highly sen- tients with Sjögren’s syndrome (~70%) possess antibodies to sitive, detects a wide range of antibodies, and is inexpensive the antigens.36 Anti–SS-A/Ro also appears in approximately and easy to perform.36,38 In addition, the antigens are in their 24% to 60% of patients with SLE and has been closely associ- original form and location within the cells used in the test.25 ated with the presence of nephritis, vasculitis, lymphadenopa- This test has several applications. It has been used as a screen- thy, photosensitivity, and hematologic manifestations such as ing test to identify patients who have ANAs as well as patients leukopenia.25,32 Antibodies to SS-B/La are found in only 9% to who are negative for ANAs to provide guidance in selection of 35% of patients with SLE and all of these have anti–SS-A/Ro.40 follow-up assays based on immunofluorescence patterns and The SS-B/La antibody is most often found in patients who have to monitor ANA titers in patients during treatment.36 cutaneous manifestations of SLE, especially photosensitivity The IIF test uses a commercially prepared microscope slide dermatitis.32 Antibodies to both SS-A/Ro and SS-B/La can cross onto which nucleated cells have been fixed. The human ep- the placenta and have been associated with neonatal lupus.25 ithelial cell line, HEp-2, is the standard substrate for clinical Newborns that have anti–SS-A/Ro are more likely to develop laboratories worldwide.25,36,38 HEp-2 cells are used because cardiac manifestations, whereas those who have anti–SS-B/La they have large nuclei with high antigen expression, allowing are more likely to have other symptoms such as skin lesions. for high sensitivity and facilitating visualization of results.36 To detect the presence of these antibodies on IIF, human tissue Patient serum is incubated with the HEp-2 cell-coated slide, culture cells such as HEp-2 (human epithelial) must be used washed to remove unbound antibodies, and then allowed to because SS-A/Ro and SS-B/La antigens are not found in mouse react with an anti-human immunoglobulin labeled with a flu- or rat liver and kidney. A finely speckled pattern is evident.41-43 orescent tag to detect bound IgG or total immunoglobulins. Antibodies to the SS-A/Ro antigen are best detected on IIF if Following a second incubation and wash, the slide is mounted a special cell line, HEp-2000®, is used; these cells have been and viewed under a fluorescent microscope using 400X mag- genetically transfected so that they hyperexpress the antigen. nification (40X objective and 10X eyepiece). The principle of The nucleolus is a prominent structure within the nucleus this assay is shown in Figure 15–4. where transcription and processing of ribosomal RNA and as- The screening test is commonly performed with a 1:40 or sembly of the ribosomes takes place. Staining of the nucleolus 1:80 dilution of patient serum in order to avoid detecting low in IIF is mainly caused by antibodies to one of three nucleolar positive titers that may be seen in healthy persons, although 4466_Ch15_233-262 30/08/16 5:05 PM Page 243 Homogeneous A Rim B Speckled Fluorescent ANA test principle. (A) Microscope slide coated with HEp-2 cells (the Nucleolar plasma and nuclear membranes have been perme- abilized). (B) Patient serum (containing antinuclear antibodies) is applied. (C) After washing, fluorescent- labeled anti-human Ig is added to detect bound C Centromere autoantibodies. After a final wash, the slide is read under a fluorescent microscope. Five typical patterns in nondividing cells are shown on the right. the exact dilution used for screening may vary with the labo- cells. Staining is absent in the nucleolus and in the chro- ratory and population being tested.36 A titer of 160 is gener- matin region in dividing cells. The specks can be fine or ally considered to be clinically significant.38 Patient samples coarse, depending on the autoantibody present. The that are positive on the ANA screen are serially diluted and speckled pattern is associated with antibodies to ENAs tested to determine the antibody titer, specified as the highest and can be found in patients with SLE, Sjögren’s syn- dilution to show nuclear fluorescence. Inclusion of a 1+ end- drome, systemic sclerosis, and other systemic autoim- point control serum can help to standardize the readings by mune rheumatic diseases. setting the minimum level of fluorescence that is considered Nucleolar—Prominent staining of the nucleoli within the positive. nuclei of interphase cells is seen in this pattern. The size, In addition to the antibody titer, the pattern of fluorescence shape, and number of the nucleoli per cell are variable and is also reported because it can provide clues about the autoan- staining can be smooth, clumpy, or speckled, depending tibody present and associated diseases. Fluorescence can be on the type of antibody present. Staining may or may not within the nucleus, cytoplasm, or mitotic structures of the be present in the dividing cells. The nucleolar pattern is cell.38 Although about 40 patterns of immunofluorescence are primarily caused by antibodies to RNA and RNP and is possible, some of the most common nuclear patterns are ho- seen mainly in patients with scleroderma, but can also be mogeneous, peripheral, speckled, nucleolar, and centromere present in patients with other connective tissue diseases. (Fig. 15–5).36,42-44 Centromere—Numerous discrete speckles are seen in the Homogeneous (also known as diffuse)—This pattern is nuclei of interphase cells and the chromatin of dividing characterized by uniform staining of the entire nucleus cells. Most cells have 46 speckles, representing the num- in interphase cells and of the condensed chromosomal ber of chromosomes. This pattern is caused by antibodies region in metaphase cells. It is associated with antibodies to proteins in the centromeres of the chromosomes and to dsDNA (also known as native or nDNA), histones, and is found mainly in patients with the CREST syndrome. deoxyribonuclear protein (DNP). The homogenous pat- Mixed patterns can also be observed; in some cases, one pat- tern is found in patients with SLE, drug-induced lupus, tern may totally or partially obscure another (for example, a and many other autoimmune diseases. homogeneous pattern might cover up a speckled pattern). In Peripheral (rim or outline)—In this pattern, diffuse stain- these cases, titration of the patient serum can help to distin- ing is seen throughout the nucleus, but there is a greater guish between the separate patterns and an antibody titer staining intensity around the outer circle surrounding the would be reported for each one. If the FANA test is negative, nucleus in interphase cells. Dividing cells show strong no clearly discernable fluorescent pattern is observed in the staining of the condensed chromatin. This pattern is nuclei of the cells. Up to 5% of SLE patients test negative, so primarily caused by antibodies to dsDNA and is highly this test cannot be used to absolutely rule out SLE. specific for SLE. Although the IIF method is considered the gold standard Speckled—This pattern is characterized by discrete, flu- for ANA testing, it also has some important limitations. The orescent specks throughout the nuclei of interphase test is time-consuming and requires a significant amount of 4466_Ch15_233-262 30/08/16 5:06 PM Page 244 volume testing laboratories, because they are automated, easy to perform, and yield objective results.38,45 These assays can test for a broad range of antibodies if multiple nuclear antigens are coated onto a single test well, or for specific ANAs if each well is coated with a single antigen. The antigens used in com- mercial kits are derived from tissue extracts or are produced by recombinant technology. Because of their advantages, many laboratories are using ELISA methods to screen for the pres- ence of ANAs in addition to identifying specific ANAs. How- ever, there is a large variation in the performance of tests produced by different manufacturers, which is influenced by Homogeneous pattern the antigen preparation used. For example, one study found sensitivities of ELISA assays ranging from 69% to 98%, and specificities from 81% to 98% when they were compared with the IIF ANA method.38 These findings suggest that immunoas- says may miss a significant proportion of ANA-positive patients and also yield a significant number of false-positive results. Based on such studies, the ACR has recommended that the IIF test remain the gold standard for ANA testing and that clinical laboratories should specify the method they used when they are reporting results.46 MIAs are also very popular because they are amenable to automated, high throughput testing with objective results. In these assays, the Speckled pattern patient serum is incubated in a microtiter plate well containing a suspension of polystyrene microspheres that are coated with individual nuclear antigens or with a HEp-2 extract. Beads con- taining specific antigens can be differentiated by their unique shade of red created by a specific combination of infrared and red fluorescent dyes. Antibodies in the patient serum will bind only to the beads containing their specified antigens. Following a washing step to remove unbound antibodies, a phycoerythrin- labeled anti-human IgG is added. The conjugate will bind only to the beads that have bound patient antibodies and excess con- jugate is removed by washing. The bead suspension is analyzed for fluorescence by a flow cytometer that has two lasers, one Nucleolar pattern that identifies each bead and another that detects the amount Photomicrographs showing three patterns of of fluorescent conjugate attached. MIAs are more efficient than immunofluorescent staining for antinuclear antibodies. Examples ELISAs because they allow testing for multiple antibody speci- of predominant staining patterns obtained are homogeneous— ficities to be performed in a single tube. Studies have shown staining of the entire nucleus; speckled pattern—staining through- that MIAs, like ELISAs can be variable in terms of their sensi- out the nucleus; and nucleolar pattern—staining of the nucleolus. tivity and specificity when compared with IIF and that test per- (Courtesy of DiaSorin, Inc., with permission.) formance varies with the specific ANA detected.47,48 Hopefully, the future will bring improvement in assays such as the MIA and ELISA that will allow laboratories to report accurate results technical expertise to correctly identify the fluorescent pat- while reaping the full benefits of automation. terns. Automated IIF ANA assays have been developed that This IIF assay may help to reduce these problems and also allow for storage is used to detect antibodies to dsDNA. In testing for these anti- and retrieval of the fluorescent images.41 These assays need to bodies, a purified antigen preparation that is free from single- be validated further but hold promise for the future. However, stranded DNA (ssDNA) must be used because antibodies to the autoantibodies present cannot be precisely identified on ssDNA occur in many individuals with other autoimmune or the basis of the fluorescent patterns and additional tests are inflammatory diseases. One particularly sensitive assay for needed.38,41 Some of the more common tests used to charac- dsDNA is an IIF test using a hemoflagellate organism called terize ANAs are described in the section that follows. Crithidia luciliae as the substrate.49 This trypanosome has a cir- cular organelle called a kinetoplast that is composed mainly of ELISAs and chemiluminescence immunoas- dsDNA (Fig. 15–6). In this test, patient serum is incubated on says for ANAs have become very popular, especially for high a microscope slide coated with C luciliae organisms and binding 4466_Ch15_233-262 30/08/16 5:06 PM Page 245 Anti-Sm antibody Nucleus Kinetoplast Flagellum A ENA B An illustration of Crithidia luciliae fixed to a microscope slide. A positive test for dsDNA will show green fluorescence in the nucleus and kinetoplast. A B is detected with a fluorescent-labeled anti-Ig conjugate. Wash- ing of the slide is performed after each step to remove unbound antibody. A positive test is indicated by a brightly stained kine- toplast. This test has a high degree of specificity, although it is less sensitive than other FANA tests.36,45 Anti-RNP antibody ANAs can also be detected by immunodif- Extractable nuclear antibody immunodiffusion pat- fusion. Typically, this method is used to determine the immuno- tern. A mixture of extractable nuclear antigens (ENA), including RNP, logic specificity of a positive FANA test, particularly when a Sm, and other soluble nuclear antigens, is placed in a central well in speckled pattern is observed. Ouchterlony double diffusion de- an agarose gel. Sm antibody and RNP antibody are run as positive tects antibody to several of the small nuclear proteins, or ENA. controls and patient samples are placed between the controls. The These include antibodies to Sm, RNP, SS-A/Ro, SS-B/La, Scl-70 pattern of precipitin lines formed indicates the antibodies present in (DNA topoisomerase I), and Jo-1 (see the previous descriptions patient serum. The arc of serological identity formed between Sm and Table 15–2). A solution containing ENA antigens is placed and patient A indicates that serum A contains anti-Sm antibodies. in a central well of an agarose plate and patient samples and The arc of partial identity formed between serum A and RNP occurs controls are placed in the surrounding wells, as indicated in because RNP is always found complexed to Sm antigen. RNP Figure 15–7. A visible precipitate is formed between the ENA antibodies are not present. Serum B contains neither Sm nor RNP antibody. well and each surrounding well that contains antibodies to any of the ENAs present (e.g., anti-Sm, anti-RNP, or antibodies to other ENAs). If an outer well does not contain antibodies to any of the ENAs, no precipitate is formed between that well and the The lupus anticoagulant, one of several types of antiphos- center well. Samples in the outer wells are identified as contain- pholipid antibodies, was so named because it produces a pro- ing antibody to a particular ENA by comparing their reactivity longed activated partial thromboplastin time (APTT) and patterns of identity, nonidentity, or partial identity to control prothrombin time (PT). In patients with antiphospholipid an- sera containing specific ENA antibodies. A positive reaction is tibodies, the APTT may be prolonged, but it is not corrected indicated by immunoprecipitation lines of serological identity. by mixing with normal plasma.31 Ironically, patients with this (Refer to Chapter 10 for a discussion of Ouchterlony test prin- antibody have an increased risk of clotting and spontaneous ciples.) Although this type of testing is not as sensitive as some abortion. Platelet function may also be affected and thrombo- other techniques, it is highly specific.40 cytopenia may be present. In addition to determining the APTT and PT, there are several EIAs for antiphospholipid antibodies Antiphospholipid Antibodies that are sensitive and relatively simple to perform.25,50-52 If these antibodies are suspected, factor assays may also need Antiphospholipid antibodies are a heterogeneous group of to be performed to rule out any factor deficiencies or factor- antibodies that bind to phospholipids alone or phospholipids specific inhibitors. complexed with protein. They can affect every organ in the body, but they are especially associated with deep-vein and ar- terial thrombosis and with recurrent pregnancy loss.25,50,51 An- Rheumatoid Arthritis (RA) tiphospholipid antibodies have been found in up to 60% of RA is another example of a systemic autoimmune disorder. It patients with lupus, but they are also associated with several affects about 0.5% to 1.0% of the adult population, but preva- other disease states.25,32,52 They can be identified by their abil- lence varies with ethnicity and geographic location.53,54 Typi- ity to cause false-positive results in nontreponemal tests for cally, it strikes individuals between the ages of 25 and 55.54 syphilis, the lupus anticoagulant assay, and immunoassays for Women are three times as likely to be affected as men; in ad- antibodies to cardiolipin or other phospholipids.32 dition, the prevalence of the disease is highest in women who 4466_Ch15_233-262 30/08/16 5:06 PM Page 246 are more than 65 years of age.53,55 RA can be characterized as osteoclasts and inhibits bone formation. Significant local bone a chronic, symmetric, and erosive arthritis of the peripheral destruction occurs and there is also generalized osteoporosis joints that can also affect multiple organs such as the heart and throughout the body.54,58 the lungs.32,54 Progress of the disease varies because there may It is not known what role autoantibodies play in the ini- be spontaneous remissions or an increasingly active disease in tiation of the inflammatory response. Two key antibodies some individuals that rapidly progresses to joint deformity and found in the disease are RF and anti-CCP. RF is an antibody disability.56 In addition to a decline in functional ability, there that is most often of the IgM class and is directed against is a reduced life expectancy.54,57 In recent years, RA patients the FC portion of IgG. It has been postulated that RFs may have experienced less disability and a better quality of life be- play a role in the pathogenesis of RA by increasing cause of advances in therapy and earlier treatment (see the macrophage activity and enhancing antigen presentation to Treatment section in the text that follows). T cells by APCs.59 Antibodies to cyclic citrullinated proteins (anticyclic cit- rullinated peptide antibody [anti-CCP or ACPA]) are a sec- Associations of RA with more than 30 genetic regions have ond major type of antibody associated with RA. Citrullinated been discovered. The strongest associations have been between proteins contain an atypical amino acid called citrulline, which a subset of patients with RA and specific HLA-DRB1 alleles or is generated when the enzyme peptidyl arginine deiminase PTPN22 gene polymorphisms.53,54,58 These patients are posi- (PAD) modifies the amino acid arginine by replacing an NH2 tive for rheumatoid factor (RF) or antibodies to CCP (see the group with a neutral oxygen.60,61 This enzyme is associated section on Laboratory Diagnosis). The strongest environmental with granulocytes, monocytes, and macrophages, as well as risk factor for RA is believed to be cigarette smoking, which other types of cells. Death of granulocytes and macrophages doubles the risk of developing the disease.53,55 Other factors triggers production of citrullinated proteins; overexpression of have also been implicated, but their associations are these antigens may provoke an immune response in individu- weaker.53,54 Numerous infectious agents have been proposed als with certain HLA-DRB1 alleles.61,62 These antibodies can as possible triggering antigens for RA, but a cause-effect rela- react with citrulline-containing components of the matrix, in- tionship has not been proven.54,55 cluding filaggrin, keratin, fibrinogen, and vimentin, and are thought to correlate with the pathogenesis of RA.54 Antinuclear antibodies are also present in some RA patients (see the previ- The pathology of RA is caused by an inflammatory process ous Antinuclear Antibodies section). that results in the destruction of bone and cartilage. The le- In RA, autoantibodies such as RF and anti-CCP are sions in rheumatoid joints show an increase in cells lining the thought to combine with their specified antigen, and the re- synovial membrane and formation of a pannus, a sheet of in- sulting immune complexes become deposited in the joints, flammatory granulation tissue that grows into the joint space resulting in a type III (or immune complex) hypersensitivity and invades the cartilage. Infiltration of the inflamed syn- reaction. The complement protein C1 binds to the immune ovium with T and B lymphocytes, plasma cells, dendritic cells, complexes, activating the classical complement cascade. mast cells, and granulocytes is evidence of immunologic ac- During this process, C3a and C5a are generated, which act tivity within the joint.54 as chemotactic factors for neutrophils and macrophages. The The balance between proinflammatory and anti-inflammatory continual presence of these cells and their associated cy- cytokines in RA appears to be tipped toward continual inflam- tokines leads to chronic inflammation, which damages the mation. Proinflammatory cytokines found in synovial fluid synovium itself.54 that contribute to inflammation include IL-1, IL-6, IL-17, and TNF-α (see Chapter 6).56,58 TNF-α plays a key role in the inflammatory process by stimulating the production of other The initial symptoms of RA involve the joints, tendons, and cytokines and facilitating the transport of white blood cells bursae.54 The RA patient commonly experiences nonspecific (WBCs) to the affected areas.54,56 The proinflammatory cy- symptoms such as malaise, fatigue, fever, weight loss, and tran- tokines trigger the release of matrix metalloproteinases from sient joint pain that begin in the small joints of the hands and fibroblasts and macrophages; these enzymes degrade important feet. The joints are typically affected in a symmetric fashion. structural proteins in the cartilage.58 Joint stiffness and pain are usually present in the morning and Local bone erosion is another feature that is characteristic improve during the day. The disease can progress to the larger of the pathology in RA. Multinucleated giant cells called osteo- joints, often affecting the knees, hips, elbows, shoulders, and clasts are central to the structural damage that is seen in the cervical spine. Joint pain can lead to muscle spasms and limi- bones. Osteoclasts absorb bone as part of the normal bone re- tation of motion. The ongoing inflammation, if left untreated, modeling process that occurs in the body in response to growth results in permanent joint dysfunction and deformity. Osteo- and repair of damaged bone. Normally, there is a good balance porosis (bone erosion) occurs in about 20% to 30% of RA pa- between bone production and destruction. However, in RA, tients because of the inflammatory environment of the joints the osteoclasts become overly activated in the inflammatory and activation of osteoclasts.54 environment of the joints. TNF-α, in conjunction with other Some patients with RA develop extra-articular manifesta- cytokines and a molecule called RANKL (receptor activator of tions, which occur outside of the joints.54 These patients are nuclear factor kappa-B ligand), induces the differentiation of most likely to have had a history of smoking, early disease 4466_Ch15_233-262 30/08/16 5:06 PM Page 247 onset, and test positive for anti-CCP or RF. Extra-articular effective in treating RA, patients must be monitored closely manifestations include the formation of subcutaneous nod- because they are at greater risk for infection. ules, pericarditis, lymphadenopathy, splenomegaly, intersti- tial lung disease, or vasculitis. Some patients have small Diagnosis of RA is based on a combination of clinical manifes- masses of tissue called nodules over the bones. Nodules can tations, radiographic findings, and laboratory testing. RF is the also be found in the myocardium, pericardium, heart valves, antibody that is most often tested to aid in making the initial pleura, lungs, spleen, and larynx. About 10% of patients diagnosis. The importance of testing for the presence of RF is with RA develop secondary Sjögren’s syndrome, an autoim- also reflected in the fact that it is one of the classification crite- mune disorder characterized by the presence of dry eyes and ria for RA.64,65 Recall that RF is an autoantibody, usually of the dry mouth in addition to connective tissue disease. A small IgM class, that reacts with the Fc portion of IgG. Approxi- percent develop Felty’s syndrome, a combination of chronic, mately 70% to 90% of patients with RA test positive for RF.54,59 nodular RA coupled with neutropenia and splenomegaly. Thus, a negative result does not rule out the presence of RA. The most common cause of death in RA is cardiovascular Conversely, a positive test result is not specific for RA because disease, presumably because of the acceleration of arte- RF is also present in about 5% of healthy individuals and in riosclerosis by proinflammatory cytokines released during 10% to 25% of those over the age of 65.59 In addition, RF can th