Autoimmunity and Autoimmune Disease

Immune disorders can be classified in several ways: by the source of the inducing, or reacting, antigen (heterologous, homologous, autologous); by the model hypersensitivity reactions types I-IV (IgE-, antibody-, immune complex-, or cell-mediated); and by disease expression characterized by clinical and immunological features, such as allergy/hypersensitivity, autoimmune disease, immune deficiency, and immunoproliferative disorder.

A central function of the immune system is to distinguish foreign antigens, such as infectious agents, from self components of body tissues. The immune system normally acquires self tolerance (unresponsiveness to self) by clonal deletion of autoreactive T cells in the thymus in the perinatal period and by functional suppression of autoreactive T and B cells at later stages of development. Nevertheless, sometimes there is a failure in the maintenance of self tolerance, a failure to discriminate between self and non-self antigens, and an autoimmune response, characterized by the activation and clonal expansion of autoreactive lymphocytes and the production of autoantibodies, is produced against autologous antigens of normal body tissues.

While there are many laboratory animal models of autoimmunity, there is currently no general unifying theory to explain how an autoimmune process gets started in the usual clinical setting. Suffice it to say that autoimmunity and autoimmune diseases are multifactorial in origin. Contributory factors include: genetic predisposition (as shown by HLA associations, family, and twin studies), host factors (such as weakness of immunoregulatory controls, defects in suppressor T cells, or polyclonal stimulation of B cells resistant to controls), environmental factors (such as certain microbial infections), and antigen-driven mechanisms (sequestered antigen, cross-reacting exogenous antigen, or molecular mimicry) which may bypass self tolerance in a nominally normal immune system. Current investigations of several autoimmune diseases are focused on the concept of molecular mimicry by which a viral, bacterial, or other foreign antigen bearing antigenic epitopes with aminoacid sequence homology to epitopes on a normal host protein elicits, in responsive individuals, the production of antibodies and activated T cells that react with the host protein as well as with the foreign protein.

Analogous to the manner in which hypersensitivity reactions to exogenous antigens initiate tissue injury and inflammation, so also autoantibody-, immune complex (IC)- or cell-mediated reactions to autologous antigens can lead to tissue injury and inflammation, resulting in an autoimmune disease. However, not all autoantibodies are pathogenic: some are, some are not, and some autoantibodies are the result, rather than the cause, of tissue injury. This is shown, for example, by the transient appearance of myocardial autoantibodies in some patients after myocardial infarction or after invasive heart surgery.

Autoimmune diseases, mainly those in which autoimmunity contributes to, or has an association with, the pathogenesis of the disease, can be classified into two broad, but overlapping, groups: organ-specific and nonorgan-specific (or systemic) autoimmune diseases. In the first mentioned, local injury, inflammation, or dysfunction are produced by autoantibody- or cell-mediated reactions against a specific target antigen located in a specialized cell, tissue, or organ. Clinical examples include: autoimmune hemolytic anemia (erythrocyte autoantibodies); Hashimoto's thyroiditis (thyroid autoantibodies and autoreactive T cells); myasthenia gravis (acetylcholine receptor autoantibodies); Grave's disease characterized by diffuse goiter and hyperthyroidism (thyrotropin receptor autoantibodies); Goodpasture's syndrome comprising anti-GBM nephritis and pulmonary intraalveolar hemorrhage (anti-GBM autoantibodies); and type I (insulin -dependent) diabetes (pancreatic beta-cell autoreactive T cells and autoantibodies).

In a systemic autoimmune disease, by contrast, tissue injury and inflammation occur in multiple sites in organs without relation to their antigenic makeup and are usually initiated by the vascular leakage and tissue deposition of circulating autologous immune complexes. These ICs are formed by autoantibody responses to ubiquitous soluble cellular antigens of nuclear, or less commonly cytoplasmic, origin. Systemic lupus erythematosus (SLE), which is characterized by the production of multiple autoantibodies, particularly antinuclear and anti-DNA antibodies, is the classical example of an IC-mediated systemic autoimmune disease.

Immune Complex Diseases

Serum sickness

Historically at the beginning of the 20th century, children with life-threatening bacterial infections, such as diphtheria, were sometimes treated with a large injection of horse serum antitoxin. After a latent period of 8-12 days, some of the children developed a self-limited syndrome called serum sickness and consisting of fever, urticaria, muscle and joint pains, lymphadenopathy, and proteinuria. While the clinical use of heterologous serum is rare today and essentially limited to horse antirabies serum, tetanus antitoxin, and antisnake venum, the contemporary relevance of serum sickness is that the elucidation of its pathogenesis as a hypersensitivity reaction to foreign serum protein and an IC-mediated disease has advanced understanding of human glomerular and connective-tissue diseases.

Rabbit model of serum sickness

A laboratory model of serum sickness can be produced in rabbits by a one-shot i.v. injection of a foreign serum protein, such as BSA (bovine serum albumin). Blood serum and tissue specimens are taken at various intervals to determine the time-course of immunopathological events.

729 Time-course of immunopathological events in rabbit model of serum sickness induced by one-shot i.v. injection of bovine serum albumin (BSA) at time zero. After an interval of a week or so, the immune phase of BSA antigen elimination begins as newly produced anti-BSA antibody forms circulating BSA-anti-BSA complexes in an antigen-excess environment. The resulting expression of immune complex-initiated tissue injury and inflammation, as shown for glomerulonephritis, first appears as the immune phase begins, reaches greatest incidence (and severity) about the time the immune phase is completed, and decreases following the elimination of all complexes and the appearance of free antibody. (Modified from: Germuth, F.G., The comparative histologic and immunologic study in rabbits of induced hypersensitivity of the serum sickness type. J. Exper. Med., 97: 257-282, 1953).

The injected BSA is removed from the circulation in three stages : equilibration; catabolism; and, after a latent period of a week or so, the immune phase of antigen elimination during which anti-BSA antibody is produced in an antigen excess environment, forms soluble BSA-antiBSA complexes, and the circulating ICs are rapidly removed by the mononuclear phagocytic system. Thereafter, free antibody appears in the circulation.

Manifestations of serum sickness, expressed as tissue injury and acute inflammation in multiple sites, develop during the immune phase of antigen elimination: glomerulonephritis (GN), vasculitis, synovitis, and endocarditis.

652 Valvular endocarditis in acute serum sickness of rabbit. This photomicrograph illustrates acute endocarditis of the mitral valve with foci of fibrinoid necrosis within the valve leaflet and on the surface, where fibrin deposition is also beginning to take place. H&E.

The development and progression of the lesions coincide with the presence of soluble ICs in the circulation, a decrease in serum C' activity, and the tissue deposition of ICs and C' in the involved sites. The lesions begin to regress and heal after all complexes are eliminated and free antibody appears in the circulation.

The inflammatory lesions in experimental serum sickness are initiated by (type III) IC-mediated mechanisms. The pathogenic complexes are those produced in moderate antigen excess, tend to form antibody doublets (Ag3Ab2), have molecular masses < 1,000,000, and when deposited at sites of vascular permeability activate the classical C'-, neutrophil-, and macrophage-mediated mechanisms of tissue injury and inflammation. Vascular permeability is increased by platelet-derived amines and by C3a and C5a. Neutrophils are attracted by C5a. The GBM and other small vessel walls are injured by collagenases, proteases, other enzymes, and inflammatory mediators released from recruited neutrophils and macrophages and by the C5b- 9 membrane attack complex (MAC), resulting in vasculitis, GN, synovitis, serositis, and endocarditis.

Table - Schematic illustration of the sequential phases in the induction of systemic type III (immune complex) hypersensitivity.



Richly vascular or "filtering" structures such as the renal glomeruli are major targets of IC-mediated injury. IC deposits in the glomeruli can be seen by immunofluorescence microscopy as granular or "lumpy-bumpy" deposits of Igs (IGG), C'-components (C1, C3, C5-9), and antigen located along the GBM of peripheral capillary loops and within the mesangium.

730 Localization of circulating immune complexes in glomeruli of animal injected i.v. with fluorescent doubly-labeled BSA-anti-BSA complexes. Note the typical granular deposits of immune complexes (white) along the glomerular basement membrane (adjacent to and beneath the endothelium) and within the mesangium. IF.

By electron microscopy, corresponding electron dense deposits are seen along the GBM mainly as subendothelial deposits.

The rabbit model of acute serum sickness can be transformed into chronic serum sickness if daily injections of BSA are made in order to maintain ICs in the circulation for an extended period. The expression of systemic IC disease then becomes chronic and progressive.

Other models of IC-mediated disease

Natural animal models of systemic IC disease include those induced by replicating foreign antigens, as with persistent viral infections, or by ever present autoantigens, as with autoimmune-disease prone strains of laboratory mice.

The Arthus reaction, the classical model of local IC disease, is produced experimentally by the intracutaneous injection of antigen in previously sensitized animals which already have precipitating antibodies to the antigen. As antigen diffuses into an antibody excess environment, large ICs are precipitated within the blood vessel walls, initiating C'- and neutrophil- mediated mechanisms of local injury and inflammation usually expressed as acute necrotizing vasculitis of the skin.

It can also be noted that the immune mechanisms of glomerular injury include not only type III hypersensitivity but also antibody-mediated (type II) hypersensitivity resulting from the in-situ formation of ICs of specific antibody with tissue localized antigen, in the absence of circulating ICs. The target antigen may be an intrinsic glomerular antigen, such as the GBM, as epitomized by anti-GBM nephritis in which a tissue-specific anti-GBM autoantibody binds to the GBM and produces a characteristic linear immunofluorescence pattern of Ig and C' deposits along the entire length of the GBM, contrasting with the granular pattern typical of deposits of circulating ICs.

731 Localization of anti-glomerular basement membrane (anti-GBM) antibodies in glomeruli of animal injected i.v. with fluorescent anti-GBM antibodies. Note the characteristic linear pattern of anti-GBM antibody deposition along the entire length of the GBM. IF. (Contrast with previous illustration).

Or, the target antigen for (type II) antibody-mediated injury may be a nonglomerular antigen of exogenous origin (such as an infectious agent) or endogenous derivation (such as DNA in individuals with SLE) which becomes bound or "planted" in the glomeruli because of affinity for components of the GBM. With a planted antigen, the IC deposits along the GBM produce an interrupted granular immunofluorescence pattern.

Human immune complex diseases

Immune complex diseases are those in which the tissue deposition of circulating ICs initiates tissue injury and inflammation in multiple sites. Serum sickness is a prototype. Many human diseases are associated with circulating ICs, and some of them show granular deposits of Igs and C' in the inflammatory lesions of GN, vasculitis, and so on. The evoking antigen may be exogenous or autologous or is unknown in the majority of cases. The main IC-related human diseases are: the diffuse connective tissue diseases exemplified by SLE, a prototype systemic autoimmune disease; GN and vasculitis resulting from immune responses induced by microbial, autologous, or unknown antigens.

Human Diseases Associated with Immune Complexes


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DIFFUSE CONNECTIVE TISSUE DISEASES (CTDs)

Autoimmune Diseases:

• Systemic Lupus Erythematosus (SLE)
• Rheumatoid Arthritis (RA)
• Sjogren's Syndrome (SS)
• Progressive Systemic Sclerosis (PSS)
• Mixed Connective Tissue Disease (MCTD)

Other:

• Polyarteritis Nodosa (PAN)

GLOMERULONEPHRITIS (GN)

• Exogenous antigens ( bacterial, viral, parasitic)
• Autologous antigens (nuclear, IgG, thyroglobulin, tumor)

INFECTIOUS DISEASES

• Bacterial: infective endocarditis, disseminated streptococcal, staphylococcal, meningococcal, gonococcal infections, Lyme disease (borreliosis), syphilis, leprosy

• Viral: hepatitis B, cytomegalovirus infection, infectious mononucleosis

• Parasitic: malaria, toxoplasmosis, trypanosomiasis

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(Modified from: Theofilopoulos, A. N., and Dixon, F.J., Immune complexes in human diseases. Am. J. Pathol., 100: 531-594, 1980.)

Diffuse Connective Tissue Diseases

This diverse group of diseases of unknown etiologies and overlapping clinical and laboratory features was originally brought together under the descriptive term "diffuse collagen diseases" or "collagen vascular diseases" and is now generally called the diffuse connective tissue diseases (CTDs) or sometimes the autoimmune CTDs.

The CTDs include:

• rheumatoid arthritis (RA), a chronic systemic inflammatory disease characterized by polyarthritis (simultaneous inflammation of several joints) which may lead to joint destruction and deformity;

• systemic lupus erythematosus (SLE), a multisystem inflammatory disease involving many organs (skin, joints, kidneys);

• scleroderma/ progressive systemic sclerosis (PSS) characterized by dermal fibrosis and skin thickening (scleroderma) (see 629) and progressive fibrosis (systemic sclerosis) of internal organs (GI tract, kidneys, heart, lungs) (see 657);

  629 Advanced stage of scleroderma. Note the typical symmetrical tightening and thickening of the skin of the fingers, imparting a shiny or waxy appearance, the tapering and claw-like deformity of the fingers, and the ulceration of finger tips.

  657 Systemic sclerosis with progressive fibrosis of lung resulting in diffuse pulmonary interstitial and alveolar fibrosis. The remaining alveolar spaces are dilated, contain fluid, and are separated by septa and broad bands of dense fibrous tissue ("honeycomb" lung). H&E.

• Sjogren's syndrome (SS) characterized by a syndrome of dry mouth/dry eyes (due to lymphocytic infiltration of salivary and lacrymal glands), polyarthritis, and either a primary disease or secondary to RA or SLE;

• polymyositis/ dermatomyositis, primary inflammatory disorders of striated muscle, also with involvement of skin (dermatomyositis) (see 622) and internal organs (heart, lungs);

  622 (Top). Skin involvement by dermatomyositis. Note the classical dusky purple (heliotrope) rash over the upper eyelids. (Bottom). This same patient with dermatomyositis also has features of scleroderma as shown by typical skin thickening and contracture deformities of fingers and wrists. Combinations of features of more than one connective tissue disease in the same patient are called overlap syndromes.

• mixed connective tissue disease (MCTD) with overlapping clinical features of SLE, scleroderma, and polymyositis; and

• polyarteritis nodosa (PAN), a noninfectious necrotizing vasculitis of small and medium sized arteries of almost any organ (kidneys, heart, intestine).

Rheumatic fever was once included in this group but is now established as a poststreptococcal hypersensitivity disease.

Several of the CTDs are associated with antinuclear antibodies (Table-4), anti-IgG antibodies called rheumatoid factors, and other autoantibodies. IC-mediated mechanisms are involved in the pathogenesis of SLE and, although less compellingly, RA and PAN.

Table-4

RHEUMATOID ARTHRITIS (RA)

RA is a systemic chronic inflammatory disease of unknown etiology and is characterized by polyarthritis which may be progressive and permanently deforming and by extra-articular manifestations, such as rheumatoid nodules, pericarditis, and arteritis. Adult RA is commonly associated with a peculiar group of anti-IgG autoantibodies called rheumatoid factors which mainly belong to IgM or IgG classes, bind to the Fc portion of other IgG molecules, and form IgG-anti-IgG complexes in the circulation or joint fluid. RFs are detected in serum in up to 80% of adult patients with RA and often at high titer and in repeated tests. Nevertheless, RFs are not specific for RA and occur in other CTDs, such as Sjogren's syndrome, in chronic infectious diseases, such as infective endocarditis, tuberculosis, and hepatitis B and, although usually at low titer, in up to 20% of overtly normal elderly individuals.

Pathologically, RA is characterized by chronic inflammatory and proliferative changes in the synovial membrane (interior of joint capsule lined with synoviocytes), accompanied by inflammatory effusion in the joint space and destructive erosion of joint cartilage and adjacent bone cortex. Ultimately, the joint space may become obliterated and the bone ends united by fibrous or bony union (ankylosis).

In advanced RA, the synovial membrane becomes densely infiltrated with lymphocytes, plasma cells, and macrophages (see 626) and extends over the articular surface as a membrane, called rheumatoid pannus (see 733), which erodes and replaces the underlying articular cartilage (see 625).

626 Rheumatoid synovitis. This is a photomicrograph of the tip of an enlarged and richly vascularized synovial villus. The surface is covered with proliferated (hyperplastic) synoviocytes. The core of the villus is densely infiltrated with mononuclear cells (lymphocytes, plasma cells in abundance in this field, and macrophages), which are seen in perivascular clusters as though emerging from the small blood vessels or engaged in intense immunological activity. H&E.

733 Rheumatoid synovitis with pannus formation. This photomicrograph shows a hyperplastic and chronically inflamed synovial villus which extends over the surface of the articular cartilage as a fibrous inflammatory membrane or pannus (from Latin: a cloth). At points of contact, the inflammatory membrane erodes and replaces the underlying articular cartilage. H&E.

625 Advanced stage of rheumatoid arthritis. Virtually all of the articular cartilage is replaced by rheumatoid inflammatory membrane. One nonviable remnant of eroded cartilage (9 o'clock) remains attached to the subchondral bone, and fragments of cartilage (3 o'clock) are embedded in the inflammatory membrane. H&E.

RA may also be accompanied by extra-articular lesions of which the rheumatoid nodule, a granuloma occurring in subcutaneous sites or elsewhere, is characteristic.

734 Rheumatoid nodules occur in subcutaneous tissue most often but are also seen in tendon (as here), lungs, and heart. The rheumatoid nodule is a characteristic granuloma and is histologically composed of three layers: a central zone of collagen necrosis and fibrinoid change, a middle zone of epithelioid cells (modified macrophages) often elongated and palisaded, and an outer zone of granulation tissue infiltrated by lymphocytes, plasma cells, and macrophages. Note the collagen necrosis and fibrinoid change in the confluent rheumatoid granulomas which occupy the center of this tendon. H&E.

RFs in RA are produced not only in spleen and lymph nodes but also locally, and significantly, by plasma cells in the rheumatoid synovial membrane.

748 Rheumatoid synovitis with plasma cells forming rheumatoid factor (RF). A frozen section was "stained" with a labeled reactant (fluorescent aggregated human IgG) for RF. Two hypertrophic synovial villi are shown at low power (vertically oriented), and their stalks are stuffed with RF-forming cells (white), which were further identifiable by morphological features at higher power and with counterstains as typical mature and immature plasma cells. IF.

RF complexes formed with IgG locally, in synovial fluid, or in the circulation may activate C'-, neutrophil-, and macrophage-mediated mechanisms of joint and extra-articular injury and inflammation.

Cell-mediated immune mechanisms and proinflammatory cytokines are now a focus of interest in the study of the pathogenesis of RA. Briefly, T-lymphocytes, mainly T-helper/inducer cells and many of them activated, are the most abundant cells in the rheumatoid synovial membrane. Many activated macrophages, macrophage-like synoviocytes, and interdigitating reticular cells (strongly expressing HLA-DR antigen) are intermixed with the recruited T-cells and B-cells. Among the several inflammatory cytokines released by the activated macrophages are TNF-alpha and IL-1 which are considered to be major mediators of joint inflammation in RA. Recent clinical approaches in the treatment of active RA include the use of new classes of immunomodulatory drugs that inhibit the action of these inflammatory cytokines, for example, that bind to and block TNF-alpha and its interaction with its cell surface receptor.

POLYARTERITIS NODOSA (PAN)

PAN, or simply polyarteritis, is a classical model of noninfectious systemic necrotizing vasculitis (a generic term) and involves small and medium-sized arteries of almost any organ (kidney, heart, intestine, skin) with the possible exception of lung. Pathologically, polyarteritis is characterized by segmental fibrinoid necrosis and polymorphonuclear leukocyte infiltration of all three layers of the vessel wall.

736 Polyarteritis nodosa. The entire wall of this small artery (of the kidney) is the site of acute fibrinoid necrosis which is shown by intense smudgy eosinophilia and loss of histological structure and is accompanied by an infiltration of neutrophils, some of which are also necrotic, in the vessel wall and surrounding tissue. Many of the lining endothelial cells are detached and displaced into the lumen, setting the stage for possible occlusive thrombosis and infarction of regions supplied by the artery. H&E.

Involvement of the intima results in thrombotic occlusion and infarction of regions supplied by the affected artery. Destruction of the elastic lamina results in aneurysmal dilatations (nodosa) in some cases.

Significantly, up to 20-30% of patients with polyarteritis have hepatitis B virus antigenemia. HB surface antigen (HBsAg)- anti-HBsAg complexes are present in the circulation and deposited along with C' in the vascular lesions of such patients.

SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)

SLE is a multisystem chronic inflammatory disease of unknown etiology which has a strong predilection for young women in the reproductive years, affects many organs, and is characterized by the production of multiple autoantibodies, typically antinuclear and anti-DNA antibodies. In some patients, autoantibodies are also produced against platelets, lymphocytes, and other cellular antigens.

The female: male ratio of SLE is about 9 to 1, and its prevalence in women is reported to be as high as 1:700. SLE is about a tenth as common as RA.

The clinical expression of SLE is extremely varied, but the most common findings include: fever, erythematous rash, arthritis, serositis (pleuritis, pericarditis), and nephritis. Skin photosensitivity, oral ulcers, hematologic disorders, and neurological abnormalities are also seen (see Table-5). SLE is a multisystem disease and mimics many other disorders.

Table-5: Revised Criteria for the Classification of Systemic Lupus Erythematosus

Criterion:	Definition:
1. Malar rash	Fixed erythema, flat or raised, over the malar eminences, tending to spare the nasolabial folds
2. Discoid rash	Erythematous raised patches with adherent keratotic scaling and follicular plugging; atrophic scarring may occur in older lesions
3. Photosensitivity	Skin rash as a result of unusual reaction to sunlight, by patient history or physician observation
4. Oral ulcers	Oral or nasopharyngeal ulceration, usually painless, observed by a physician
5. Arthritis	Nonerosive arthritis involving two or more peripheral joints, characterized by tenderness, swelling, or effusion
6. Serositis	Pleuritis - convincing history or pleuritic pain or rub heard by a physician or evidence of pleural effusion, or Pericarditis - documented by ECG or rub or evidence of pericardial effusion
7. Renal disorder	Persistent proteinuria greater than 0.5 grams per day or greater than 3+ if quantitation not performed, or Cellular casts - may be red cell, hemoglobin, granular, tubular, or mixed
8. Neurologic disorder	Seizures - in the absence of offending drugs or known metabolic derangements, e.g., uremia, ketoacidosis, or electrolyte imbalance, or Psychosis - in the absence of offending drugs or known metabolic derangements, e.g., uremia, ketoacidosis, or electrolyte imbalance
9. Hematologic disorder	Hemolytic anemia - with reticulocytosis, or Leukopenia - less than 4,000/mm^3 (4.0x10^9/L) on two or more occasions, or Lymphopenia - less than 1,500/mm^3 (1.5x10^9/L) on two or more occasions, or Thrombocytopenia - less than 100,000/mm^3 (100x10^9/L) in the absence of offending drugs
10. Immunologic disorder	Positive LE cell preparation, or Anti-DNA: antibody to native DNA in abnormal titer, or Anti-Sm: presence of antibody to Sm nuclear antigen, or False positive serologic test for syphilis known to be positive for at least 6 months and confirmed by Treponema pallidum immobilization or fluorescent treponemal antibody absorption test
11. Antinuclear antibody	An abnormal titer of antinuclear antibody by immunofluorescence or an equivalent assay at any point in time and in the absence of drugs known to be associated with "drug-induced lupus" syndrome
The proposed classification is based on 11 criteria. For the purpose of identifying patients in clinical studies, a person shall be said to have systemic lupus erythematosus if any 4 or more of the 11 criteria are present, serially or simultaneously, during any interval of observation. In 1997, anti-phospholipid antibody was added to the list of criteria for the classification of SLE (Hochberg, M.C., Arthritis Rheum 40: 1725, 1997). ( From: Tan, E.M., Cohen, A.S., Fries, J.F., et al. The 1982 revised criteria for the classification of systemic lupus erythematosus (SLE). Arthritis Rheum. 25: 1271-1277, 1982)

Etiology and Pathogenesis. The cause of SLE is unknown. The presence of multiple autoantibodies and other immunologic abnormalities point to basic defects in immunoregulatory controls that normally maintain immune tolerance to self antigens.

SLE is characterized by B- cell activation and production of antinuclear autoantibodies against DNA and histone proteins of cell nuclei (of nucleosomes, unit particles of chromatin). Intracellular molecular components are released from tissue cells during the natural process of programmed cell death (apoptosis).

B-cell activation generally requires T-cell help. Obligatory and enhanced T-cell help for B-cells is shown in SLE lymphocytes by prolonged expression and costimulatory interaction of the helper T-cell surface ligand CD40L with the B-cell receptor CD40 (Desai-Mehta, A., et al., J Clin Invest 97:2063-73, 1996; Koshy, M., et al., J Clin Invest 98: 826-37, 1996).

Contributory factors may be genetic, hormonal, or environmental in nature. Genetic influence is suggested by family clusters of SLE, significant (approx. 25%) concordance of SLE in homozygous twins, and associations with HLA (DR2, DR3) and with inherited C' deficiencies. (Genetic studies of animal models indicate that multiple genes are involved in SLE-like disease expression). Hormonal influence is suggested by the 9:1 female:male ratio of SLE, the predilection for females during the reproductive years, and the frequency of onset in the early postpartum period. (In laboratory animal models, estrogens increase and androgens decrease the incidence of SLE-like disease). Environmental factors are indicated by drug-induced (iatrogenic) lupus syndromes caused by procainamide, hydralazine, and other drugs, and by skin sensitivity to sunlight exposure which may provoke the onset of SLE. The possible etiologic role of a specific infectious agent is speculative at this time.

Immunological Aspects of SLE. The discovery in 1948 of the "LE cell" was a major advance in diagnosis and led to the recognition that SLE and related CTDs are associated with the presence of antinuclear autoantibodies (ANAs) (see Table-6). ANAs are a generic group of serum, mainly IgG, antibodies which react by indirect immunoflurescence test with acetone-fixed cell nuclei from a wide range of tissues and animal species (thus ANAs lack tissue and species specificity).

Table-6: Connective Tissue Diseases Associated with Antinuclear Antibodies

ANAs also react specifically with isolated and characterized nuclear antigens, such as deoxyribonucleoprotein, DNA in double stranded (ds) and single (ss) stranded configuration, and histones. Some ANAs also react with non-histone proteins, such as Sm antigen, various nuclear ribonucleoproteins (RNPs), nucleolar RNPs, and others. None of these reactivities has absolute specificity for any disease entity. The most specific for active SLE are anti-dsDNA antibodies which are rarely found at significant levels in other diseases.

Anti-DNA antibodies are involved in the pathogenesis of (type III) IC-initiated tissue injury and inflammation in SLE expressed as nephritis, vasculitis, and serositis. Autoantibodies are also produced in some lupus patients against platelets, erythrocytes, and T-cells, and these may cause (type II) antibody-mediated cell injury resulting in thrombocytopenia, hemolytic anemia, and lymphopenia.

Diagnosis of SLE. The diagnosis of SLE is based upon the appropriate clinical features and is confirmed by serological, pathological, and other laboratory findings. The fluorescent antinuclear antibody (FANA/ANA) test is the most widely used diagnostic screening test for SLE.

623 Positive antinuclear antibody (ANA) test given by SLE serum. In this illustration, the target antigen is acetone-fixed cultured cells attached to glass. The test serum is diluted, incubated with the target antigen, then washed off and followed by fluorescent anti-human Igs which react with ANAs bound to the antigen. The rim pattern of nuclear fluorescence given by the predominant ANA in this SLE serum is typical of anti-dsDNA antibodies and a characteristic of SLE. Further evidence (not illustrated) for anti-DNA antibody specificity is obtained by a parallel test using DNAse treated target cells from which all DNA is removed: the test serum no longer reacts with nuclei devoid of DNA. IF. Cell cytoplasm is counterstained with a conventional red dye to enhance color contrast.

This test is positive, often in high titer, in >95% of patients with active untreated SLE, but positive reactions also occur in other CTDs, as previously noted, other conditions (see Table-7), and rarely in overtly healthy subjects, particularly family members of patients with SLE. While the ANA test has high sensitivity, it has low specificity for SLE. It is a useful diagnostic screening test in the evaluation of patients suspected of having SLE or a strongly ANA-related CTD or in ruling out these diseases.

Table-7: Some Diseases (Other than CTDs) Associated with Antinuclear Antibodies

Other Autoimmune Diseases

• Hashimoto's thyroiditis
• Myasthenia gravis

Infectious diseases

• Tuberculosis
• Histoplasmosis
• Infectious mononucleosis
• Chronic active hepatitis

Miscellaneous

• Pernicious anemia
• Ulcerative colitis
• Alcoholic cirrhosis of the liver
• Lymphoproliferative disorders

The pattern of nuclear fluorescence observed in the ANA test suggests the predominant type of antibody in the test serum and the nature of the reactive antigen (see Table-8). Anti-ds DNA antibodies typically give a rim pattern of nuclear fluorescence and are associated with SLE. Anti-histone antibodies typically give a homogeneous pattern and are associated with drug-induced LE and also SLE. Antibodies to nonhistone proteins and nuclear RNPs give a speckled pattern and are associated with SLE, Sjogren's syndrome, MCTD, and scleroderma. Antibodies to nucleolar RNPs are associated with scleroderma.

Table-8: ANA Fluorescence Patterns and CTD Association

Nuclear Fluorescence Pattern	Disease Association	Nature of Reactive Antigen
Rim (peripheral)	SLE	dsDNA
Homogeneous (diffuse)	Drug-induced LE, SLE	Histones, deoxyribonucleopr.
Speckled	SLE, Sjogren's, MCTD, scleroderma	Nonhistone proteins, nuclear RNPs, etc.
Nucleolar	Scleroderma	Nucleolar RNPs

When the clinical evaluation warrants, a special antibody assay may be obtained to supplement the results of an ANA screening test (see Table-9).

Table-9: Special Antibody Assays in the Connective Tissue Diseases

anti-ds DNA	SLE	anti-La	Sjogren's, SLE
anti-Sm	SLE	anti-Scl 70	Scleroderma
anti-histone	Drug induced LE, SLE	anti-nucleolar	Scleroderma
anti-U1 RNP	MCTD, SLE	anti-centromere	CREST*
anti-Ro	SLE, Sjogren's	anti-Jo-1	Polymyositis
*Acronym for syndrome of scleroderma with calcinosis (C), Raynaud's phenomenon (paroxysmal blanching and numbness of fingers) (R), esophageal dysmotility (E), sclerodactyly (S), and telangiectasia (T).

While none of these antibody reactivities has absolute specificity for any disease entity, a positive result for anti-ds DNA antibodies has high specifity for SLE. The majority (~80%) of patients with active untreated SLE give positive assays for anti-ds DNA, whereas these antibodies are rarely found in significant levels in other diseases. Anti-Sm antibodies also have high specificity for SLE but occur in only 30% of patients.

The LE cell test is no longer done in most laboratories, having been superseded by the more sensitive fluorescent ANA test or its equivalent. The LE cell test is also positive in ~80% of patients with active untreated SLE, and positive results sometimes occur in RA and other CTDs. The basis of this in-vitro test is that when serum containing antibodies with anti-DNA histone specificity reacts with damaged (antibody permeable) cell nuclei, the nuclei swell and form basophilic round bodies (LE bodies) which are phagocytized by PMNs in the reaction mixture, thus forming typical inclusion body-containing LE cells.

737 Positive LE cell test in SLE. This test is performed by adding glass beads to a test tube of the patient's anticoagulated blood, shaking repeatedly to damage some of the cells and to release nuclei and nuclear fragments, incubating the preparation at 37 degrees Celsius for a suitable time, and then preparing smears and staining them with Wright's stain. ANAs with anti-DNA histone specificity bind to the nuclear fragments and form homogeneous round bodies (LE bodies). The LE bodies are phagocytized by viable neutrophils (or macrophages) in the reaction mixture, thus forming cytoplasmic inclusion body-containing LE cells. The LE cell (center of field) is typically recognized as a neutrophil containing a large round, homogeneous, slightly basophilic (violet stained) cytoplasmic inclusion body which displaces the basophilic (dark blue-stained) bilobate nucleus to one side. Wright-Giemsa.

Comparable LE bodies formed in vivo by a similar reaction are occasionally found in pleural or synovial fluid or in tissue sections (bone marrow, kidney, lymph node) and called hematoxylin bodies (see 620) which are diagnostic of SLE although present in only a minority (<10%) of cases.

620 Hematoxylin bodies in SLE. This photomicrograph shows hematoxylin bodies (at 3 and 9 o'clock) in a section of bone marrow from a patient with SLE. Hematoxylin bodies, pathognomonic of SLE, are analogous to LE bodies and are basophilic hematoxylin-stained (black), extracellular bodies formed in tissues by in-vivo combination of nuclear fragments with ANAs of anti-DNA histone specificity. (Hematoxylin bodies must not be confused with senescent megakaryocytes.) H&E.

Other serological abnormalities of significance in SLE include: decreased C' activity; polyclonal hypergammaglobulinemia; cryoglobulinemia (cold precipitable globulins); and biological false positive STS, which is related to the presence of anti-phospholipid antibodies and associated with a syndrome of thrombosis and recurrent abortion.

Pathology of SLE. It is important to bear in mind that SLE is characterized by the total picture of clinical, immunological, and pathological features and that many of the pathological changes are nonspecific and sometimes may appear disproportionally small in comparison with the extent of clinical manifestations.

The principle pathological changes are seen in the skin, joints (nonerosive synovitis), kidneys (lupus nephritis), serous membranes (pleuritis, pericarditis), and heart (endocarditis, pericarditis).

The most typical histopathological lesions, aside from the presence of pathognomonic hematoxylin bodies noted above, occur in blood vessels, kidneys, and skin.

In active SLE, vasculitis with subendothelial fibrinoid deposits may involve small arteries, arterioles, and capillaries of affected organs (kidneys, spleen, heart, lungs) and, rarely in the severest cases, acute necrotizing vasculitis may involve the entire vessel wall. Granular deposits of Igs and C' are seen in the acute vascular lesions by fluorescence microscopy.

In later stages, perivascular fibrosis occurs. The small penicillar arteries of splenic pulp typically develop concentric perivascular laminations of fibrous tissue to produce the typical "onion skin" lesion of spleen (see 618) which is characteristic of, but no longer regarded as specific for, SLE.

618 Periarterial fibrosis of spleen in SLE. Note the typical concentric perivascular laminations of fibrous tissue forming around small arteries ("onion skin" lesion). H&E.

The renal glomeruli are major targets of immune injury in SLE. Some 50% of SLE patients have glomerular disease (lupus nephritis) shown by urinalysis (hematuria, proteinuria, casts) or expressed clinically. About 70% of patients have glomerular disease indicated by light microscopy, and nearly all have glomerular abnormalities shown by immunofluorescence and electron microscopy.

There are several histopathological patterns of glomerular disease in lupus nephritis: mesangial, focal proliferative, diffuse proliferative (see 742) (see 650), and membranous lupus nephritis (see 654).

1014 Fulminant fatal renal glomerular disease in SLE (diffuse proliferative lupus nephritis). This postmortem specimen shows the cortical and sectioned surfaces of one of two similarly involved kidneys. Note the fine punctate hemorrhages dotting the cortical surface, the larger areas of hemorrhage, and the mottled and irregular pitted appearance of the cortex. The dot-like cortical hemorrhages are due, as can be shown by histologic study, to inflammatory damage and rupture of the glomerular capillaries.

A similar gross appearance of punctate cortical hemorrhages may be seen in the malignant phase of hypertension (malignant nephrosclerosis) and in some forms of primary glomerulonephritis.

650 Diffuse proliferative lupus nephritis. In general, the basic histopathological changes in glomerular diseases include one or more of the following: cellular proliferations (proliferative change); glomerular basement membrane (GBM) thickening (membranous change); leukocyte exudation; hyalinization and sclerosis. These changes may involve virtually all glomeruli (diffuse), a minority of glomeruli (focal), a part of each glomerulus (segmental), or predominately the mesangium (mesangial).

This photomicrograph of diffuse proliferative lupus nephritis shows an increase in the size of the glomeruli and in the number of cells, reflecting the proliferation of endothelial, mesangial, and epithelial cells, including "cresentic" proliferation of parietal epithelial cells which are encroaching upon the urinary space. Additionally, GBM thickening is seen in peripheral capillary loops in a segment of the glomerulus at 9 o'clock. H&E.

654 Diffuse membranous lupus nephritis. This photomicrograph of a percutaneous renal biopsy shows a glomerulus with pronounced GBM thickening of peripheral capillary loops and without an increase in cell number. Note the extreme degree of GBM thickening and hyaline change (homogeneous eosinophilia) in the capillary loops near 1 o'clock. H&E.

All of them show immunofluorescent granular deposits of Igs (see 738) and C' (see 739) within the glomeruli.

738 Immunofluorescence micrograph of glomerulus from diffuse proliferative lupus nephritis stained for human IgG. Note the fine and coarsely granular deposits of IgG along the GBM of peripheral capillary loops. IF.

739 Immunofluorescence micrograph of glomerulus from diffuse proliferative lupus nephritis stained for human C'3. Fine granular deposits of C'3 are located along the GBM of capillary loops and in the mesangium (6 o'clock). IF.

These presumed immune deposits are fine or coarsely granular and are located in the mesangium, or along the GBM, or throughout the peripheral capillary loops in a more massive "lumpy-bumpy" pattern.

740 Immunofluorescence micrograph of glomerulus from diffuse membranous lupus nephritis. Note the granular and massive "lumpy-bumpy" deposits of IGs along the GBM of peripheral capillary loops. IF.

The deposits contain IgG and protein components of the classical C' pathway, but IgM, IgA, proteins of the alternative C' pathway, and fibrin are sometimes present as well.

Electron microscopy shows the presence of electron dense deposits in corresponding locations in the mesangium and along the GBM beneath the vascular endothelium (subendothelial) or beneath the visceral epithelium (subepithelial). Subendothelial deposits when abundant increase the thickness and apparent rigidity of the GBM of peripheral capillary loops and produce the highly refractile "wire loop" lesion seen by light microccopy and characteristic of SLE.

741 "Wire loop" lesion in diffuse proliferative lupus nephritis. This photomicrograph shows an enlarged glomerulus with increased cellularity in the left half of the glomerulus and characteristic GBM thickening and bright red staining of the GBM of peripheral capillary loops ("wire loop" lesion) on the right half of the glomerulus (3 o'clock). H&E.

There is strong evidence that C' activating DNA-anti-DNA complexes are involved in the pathogenesis of lupus nephritis, vasculitis, and serositis. Briefly summarized: circulating ICs, including DNA-anti-DNA complexes, are present in patients with SLE; high or changing levels of anti-dsDNA antibodies and decreased levels of serum C' correlate with disease activity; Igs, C', and, when studied, DNA antigen are deposited in the glomerular lesions; and Igs eluted from the glomeruli in vitro show a greatly increased concentration of anti-dsDNA (and anti-ssDNA) antibodies compared to serum. That is not to say that DNA-anti-DNA complexes are the only cause of IC-mediated mechanisms, because the antigen-antibody components of circulating ICs in SLE patients are not fully characterized.

DNA-anti-DNA complexes are also formed in situ in the glomeruli in SLE. DNA, presumably derived from the normal turnover of cells, is sometimes present in the plasma of patients with active SLE, and this circulating DNA has affinity for the GBM (apparently for type IV collagen). Circulating anti-DNA antibodies combine with this "planted" DNA to form finely granular deposits of Igs and C' along the GBM.

Skin lesions involving the face, trunk, and extremities occur frequently in SLE and take many forms: facial erythema, such as the classical "butterfly" rash distributed over the cheeks and base of the nose, urticaria, maculopapular lesions, ulceration, and alopecia. The presence of liquefaction degeneration of the basal layer of the epidermis and fibrinoid change at the epidermal-dermal junction is a microscopic characteristic of acute SLE. Immunofluorescence microscopy shows granular deposits of Igs and C' along the epidermal-dermal junction of acute lesions and also of uninvolved "normal" skin (lupus band test). The presence of these deposits in both involved and normal skin distinguishes SLE from other CTDs, such as scleroderma and dermatomyositis, and chronic discoid lupus (cutaneous LE) in which deposits occur only in the skin lesions. It is postulated that the deposits are ICs precipitated in situ as nuclear DNA released from damaged or senescent basal epidermal cells reacts with anti-DNA antibodies diffusing from dermal capillaries.

The joint involvement in SLE consists of nonerosive synovitis with an exudation of neutrophils and some lymphocytes (see 630) but with the absence of joint destruction and deformity, such as seen in RA.

630 Nonerosive synovitis in SLE. Note the inflammatory exudate of occasional neutrophils and numerous lymphocytes immediately beneath the surface layer of synoviocytes. H&E.

Fibrinous pleuritis and pericarditis occur in SLE and may result in fibrous thickening and partial or complete obliteration of serosal cavities.

In addition to pericarditis, a nonbacterial verrucous endocarditis (Libman-Sacks endocarditis) distinguishable from that of rheumatic endocarditis and involving mitral and tricuspid valves is characteristic of SLE.

Hematologic abnormalities occur in SLE. In some patients, autoantibodies are produced against specific cell surface antigens of platelets, erythrocytes, and lymphocytes. These antibodies may cause (type II) antibody-mediated cell injury resulting in thrombocytopenia, hemolytic anemia, and lymphopenia.

Clinical Course. The course of SLE is chronic and variable, with flares of disease activity followed by complete or near-complete remission. Some laboratory tests, in addition to traditional CBC, ESR, urinalysis, serum creatinine, VDRL, that may be useful in evaluating disease activity include: presence and titers of ANA, anti-dsDNA antibody and other ANA subtypes, serum levels and classes of Igs and CÕ components, anticardiolipin antibody, and cryoglobulins.

Treatment is nonspecific , supportive, and includes nonsteroidal antiinflammatory drugs (NSAID), corticosteroids (sometimes in high doses), and immunosuppressive drugs. The five-year survival figures now approach 95%.

Causes of death include renal failure due to lupus nephritis, malignant hypertension secondary to lupus nephritis, vascular lesions affecting the CNS or other vital organs, and complicating secondary infections.

Animal Models of SLE. Laboratory models serve as a basis for comparison with human SLE. New Zealand mice of the F1 hybrid cross NZBxNZW develop most of the autoimmune abnormalities seen in human SLE and succomb from SLE-like IC-mediated GN which runs a fulminant fatal course in females. This IC disease is mediated not only by DNA-anti-DNA complexes as in human SLE but also by endogenous retroviral envelope glycoprotein gp70 -anti-gp70 complexes.

619 Immunofluorescence micrograph of glomerulus from NZBxNZW mouse with murine SLE-like nephritis. (A) Granular deposits of endogenous retroviral envelope glycoprotein gp70 are seen along the GBM of peripheral capillary loops and in the mesangium. (B) Granular and heavy deposits of host IgG are present in similar locations in a serial section of the same glomerulus. IF.

Genetic studies show that multiple genes and immunoregulatory defects are involved in the autoimmune manifestations of New Zealand mice. In this model, the expressed endogenous retroviral antigens also participate like autoantigens.

Studies of two other murine models of autoimmune disease show that loss-of-function mutations of either of two genes, the Fas gene or the Fas ligand gene, respectively, underlie the failure in Fas-mediated apoptosis of lymphocyres, the loss of self tolerance, and the autoimmune and immunoproliferative manifestations that characterize these mouse strains (Suda, T., and Nagata, S., J. Allergy Clin. Immunol., 100, S97-S101, 1997).

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