Lecture 4 - Autoimmunity Pt2

Ability of immune system to not mount immune response to self antigens

By establishing self tolerance so the immune system does not respond to self antigens

Central Tolerance and Peripheral Tolerance

Bone Marrow and Thymus

Peripheral Tissues

Immune response to self antigens of a specific organ

Damage to organ structure and function

Systemic–auto Ag found on any type of cell throughout body (DNA)

Widespread attack of self antigens

Damage to numerous structures

Autoimmune activity is widely spread throughout the body

They affect multiple organs and tissues and include two important groups; Systemic autoimmune rheumatic diseases (SARDs) and Anti-neutrophil cytoplasmic antibodies (ANCA).

Diseases that involve inflammation of the joints and their associated structures

Associated vasculitides, which are characterized by inflammation of the blood vessels

A chronic inflammatory disease, relapsing, flares correlates with disease activity

A multisystem autoimmune disease

Women more than men (9:1 ratio), with 90% of cases being women

African Americans more than Whites

Between ages 15 and 45 years

Environmental factors, genetic susceptibility, and abnormalities within the immune system

Some drugs (procainamide, isoniazid, hydralazine), UV exposure, infections, hormones (estrogen-containing contraceptives)

>60 genetic loci associated with lupus in humans have been discovered including HLA-A1, B8, and DR3

Defects in CRs on phagocytic cells, defects in FcRs, and deficiencies in C1q, C2, or C4; polymorphisms in cytokine genes, etc

Over 100 autoantibodies (dsDNA, histones, nucleoli, lymphocytes, erythrocytes, platelets, phospholipids, ribosomal components, and endothelium)

Found in >70% of patients and highly specific

Impaired immune complex clearance (IC deposit on tissues) and autoantibodies against RBCs, PLTs, and endothelial cells

Anti-phospholipid antibodies (associated with increased miscarriage, stillbirth, and preterm delivery in pregnant women with lupus) and lupus anticoagulant

Butterfly rash; discoid lupus of head and neck with scarring and disfigurement; photosensitivity with UV-induced DNA damage; oral ulcers; arthritis; serositis; neurologic; hematologic; renal involvement; autoantibodies; cardiopulmonary

Renal involvement is present in about half of lupus patients; nephritis is a major cause of illness and death

CBC, platelet count, creatinine levels, and urinalysis to assess renal function; it may show leukopenia, possible thrombocytopenia and anemia; ESR may also be high, but CRP may be low or normal

Further tests should be done to differentiate the type of ANA

Antibodies to dsDNA and antibodies to the Sm nuclear antigen are highly specific for SLE

Indirect Immunofluorescence (IIF)

Fluorescent anti-nuclear antibody (FANA) testing has been the most widely used and accepted test because it is highly sensitive, detects a wide range of antibodies, and is inexpensive and easy to perform

A commercially prepared slide with fixed HEp2 cell line; incubation with patient’s serum (serial dilutions, starting with 1:80 or 1:160), ANA will bind to HEp2 nucleus; wash to remove unbound Ab molecules; add anti-human Ig labeled with a fluorescent dye and incubate; wash again to remove unbound Ab; put a cover slip, mount the slide and view it under microscope (400× magnification); titers above 160 are clinically significant

Homogeneous—This pattern is characterized by uniform staining of the entire nucleus in interphase (nondividing) cells and of the condensed chromosomal region in metaphase cells. It is associated with antibodies to dsDNA, histones, and nucleosomes. The homogeneous pattern is found in patients with SLE, drug-induced lupus, chronic autoimmune hepatitis (AIH), and Juvenile idiopathic arthritis.

Speckled—These patterns are characterized by discrete, fluorescent specks throughout the nuclei of interphase cells. The speckled group is divided into three subgroups depending on whether the specks are dense fine, tiny/fine, or large/coarse. The fine and coarse speckled patterns are associated with antibodies to ENAs and can be found in patients with SLE, Sjögren’s syndrome, systemic sclerosis (SSc), and other SARDs.

Nucleolar—Prominent staining of the nucleoli within the nuclei of interphase cells is seen in this pattern. The size, shape, and number of the nucleoli per cell are variable, and staining can be smooth, clumpy, or speckled, depending on the type of antibody present. Staining may or may not be present in the dividing cells. The nucleolar patterns are primarily caused by antibodies to RNA and RNP and are seen mainly in patients with SSc but can also be present in patients with other SARDs.

Polystyrene microtiter plates, nitrocellulose strips, or synthetic beads coated with auto-Ags; incubation with patient’s serum.

Detection by enzyme/fluo/chemilum tagged AHG.

Antibodies to a range of Ags can be identified: dsDNA, Sm, U1-RNP, SS-A/Ro, SS-B/La, Jo-1, Scl-70, PM/Scl, Mi-2, CENP-B, and histones.

Advantage: automation, ease of performance, and yielding objective results. Disadvantage: sensitivity lower than IIF, false positive results.

Immunofluorescence Using Crithidia luciliae. This IIF assay is used to detect antibodies to dsDNA.

A hemoflagellate organism called Crithidia luciliae as the substrate.

This trypanosome has a circular organelle called a kinetoplast that is composed mainly of dsDNA.

In this test, patient serum is incubated on a microscope slide coated with C. luciliae organisms, and binding is detected with a fluorescent-labeled anti-Ig conjugate; washing of the slide is performed after each step to remove unbound antibody.

Can be used to determine the immunologic specificity of a positive FANA test, particularly when a speckled pattern is observed.

It detects antibody to several ENAs including antibodies to Sm, RNP, SS-A/Ro, SS-B/La, Scl-70 (DNA topoisomerase I), and Jo-1.

If the antigens are identical, they will react with the same antibody, and the precipitate line forms a continuous arc.

If the antigens share no identical determinants, they will react with different antibodies, and two crossed lines are formed.

Partial identity. Antibodies react with two similar antigens, forming two lines and a spur that points to the simpler antigen (the antigen that reacts least with the antibody).

For example, suppose antigen 3 has an epitope in common with antigen 1, and there are two antibodies in the center well (anti-1 and anti-3). One of the antibodies reacts with the epitope common to both antigens. The other antibody reacts with a different epitope on antigen 1 (that is absent on antigen 3). Two precipitin lines form, with a spur pointing toward antigen 3.

Association with >100 genetic regions; the strongest associations with specific HLA-DRB1 alleles or PTPN22 gene polymorphisms. These patients are positive for rheumatoid factor (RF) or antibodies to cyclic citrullinated peptides (CCP).

Epigenetic factors, such as altered DNA methylation affecting the transcription of critical genes, are also believed to play a role in the pathogenesis of RA.

The strongest environmental risk factor for RA is believed to be cigarette smoking, which doubles the risk of developing the disease.

Caused by an inflammatory process that results in the destruction of bone and cartilage in the joints.

Infiltration of the inflamed synovium with T and B lymphocytes, plasma cells, dendritic cells, mast cells, and granulocytes is evidence of immunologic activity within the joint.

The balance between proinflammatory and anti-inflammatory cytokines in RA appears to be tipped toward continual inflammation: IL-1, IL-6, IL-17 and TNF-α (TNF-α plays the key role).

These cytokines trigger the release of matrix metalloproteinases from fibroblasts and macrophages; these enzymes degrade important structural proteins in the cartilage.

Bone lesions due to hyperactivation of osteoclasts.

Rheumatoid factor (RF), an IgM against FC portion of IgG, enhances activity of macrophages and Ag presentation to APCs; Anti-cyclic citrullinated peptide antibody [anti-CCP or ACPA].

Citrullinated proteins released from dead granulocytes or macrophages; overexpression of these antigens may provoke an immune response in individuals with certain HLA-DRB1 alleles.

Initial test is RF, positive in 70–90%; negative RF does not exclude RA; positive RF is not specific to RA; RF is also positive in about 5% of healthy individuals and in 10% to 25% of those over the age of 65.

RF can be found in patients with other SARDs, such as SLE, Sjögren’s syndrome, scleroderma, as well as in people with some chronic infections.

RF is done using latex agglutination on a slide, detects only IgM isotype.

ELISA and nephelometry are more sensitive and precise, they detect IgG isotype (more specific).

Anti-CCP is done using ELISA, great test for early diagnosis where RF may be negative; a large number of RF-negative RA patients are anti-CCP positive.

Chronic inflammation of the exocrine glands (eyes, saliva); most individuals with Sjögren syndrome present with sicca symptoms, such as xerophthalmia (dry eyes), xerostomia (dry mouth), and parotid gland enlargement.

Primary Sjögren’s syndrome occurs in the absence of other diseases.

Secondary Sjögren’s syndrome occurs in patients who also have other diseases, most commonly SLE or RA.

The majority of patients with the primary form of the disease test positive for RF and ANAs, exhibiting a fine speckled pattern of fluorescence that is indicative of anti–SS-A/Ro and/or anti–SS-B/La.

SSc, also known as scleroderma, is a rare SARD: excessive fibrosis and vascular abnormalities that affect the skin and joints and progress over time to involve internal organs.

Symptoms: hardening of the skin, musculoskeletal pain, Raynaud’s phenomenon, and heartburn.

The combination of calcinosis cutis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia has been referred to as the CREST syndrome.

Complications involving the internal organs often result in functional impairment, leading to death.

At least 10 different types of ANAs have been identified in patients with SSc. Three of these are useful to determine the diagnosis, subgrouping, and prognosis.

Anti-centromere antibodies are found only in patients with limited cutaneous SSc and correlate with a better survival rate.

Antibodies to topoisomerase I (also known as Scl-70) are associated with diffuse cutaneous SSc and a poor prognosis.

Antibodies to RNA polymerase III are associated with the diffuse form of the disease and rapid thickening of the skin but have a better prognosis than those with anti-Scl-70.

High titer of autoantibodies to U1-RNP.

Anti JO-1.

A common autoimmune disease, affecting about 8 out of every 1,000 individuals.

The disease is most often seen in middle-aged women; in addition, women are 5 to 10 times more likely to develop the disease than men.

Patients develop an enlarged thyroid called a goiter, which is irregular and rubbery.

Patients also produce thyroid-specific autoantibodies and cytotoxic T cells leading to destruction of the thyroid gland, which results in hypothyroidism.

Dry skin, decreased sweating, puffy face with edematous eyelids, pallor with a yellow tinge, weight gain, fatigue, and dry and brittle hair.

Antibodies to Tg and TPO.

Graves’ disease is characterized by hyperthyroidism, a state of excessive thyroid function.

Graves’ disease is, in fact, one of the most frequently occurring autoimmune diseases and the most common cause of hyperthyroidism.

Women exhibit greater susceptibility.

Thyrotoxicosis: an excess of thyroid hormones, with a diffusely enlarged goiter that is firm instead of rubbery

Nervousness, insomnia, depression, weight loss, heat intolerance, sweating, rapid heartbeat, palpitations, breathlessness, fatigue, cardiac dysrhythmias, and restlessness.

Hypertrophy of the eye muscles and increased connective tissue in the orbit cause the eyeball to bulge out so that the patient has a large-eyed, staring expression.

The main antibodies involved in the pathogenesis of Graves’ disease are the TSH receptor antibodies (TRAbs).

The type responsible for the hyperthyroidism seen in Graves’ disease are called thyroid-stimulating immunoglobulins (TSIs). When TSIs bind to the TSH receptor, they mimic the action of TSH, resulting in uncontrolled receptor stimulation with excessive release of thyroid hormones.

For patients with Hashimoto’s thyroiditis, antibodies to TPO are the best indicator of the disease because they are found in up to 95% of patients but in only 10% to 15% of the general population.

TRAbs, on the other hand, are highly indicative of Graves’ disease because they are present in 98% to 100% of patients; TRAbs.

About 5% to 10% of patients with DM (a complete or nearly complete deficiency in insulin). Of these patients, 90% have an immune-mediated form of the disease known as type 1A diabetes.

T1D is a chronic autoimmune disease that involves selective destruction of the beta cells of the pancreas.

Insufficient insulin production, hyperglycemia, and toxic effects on the body.

Long-term effects include cardiovascular complications, renal disease, nerve damage, blindness, and infections of the lower extremities, which can lead to amputation.

Patients require lifelong insulin injections to control glucose levels and lower the risk of these complications.

Family history (role of genetics, multiple genes involved).

Progressive inflammation of the islets of Langerhans in the pancreas leads to fibrosis and destruction of most beta cells.

Accumulation of CD8+ lymphocytes, plasma cells and macrophages.

Autoantibody production precedes the development of clinically evident T1D by several years.

According to the ADA, a person is considered to have diabetes if he or she meets one of four criteria:
1. A fasting glucose greater than 126 mg/dL on more than one occasion (normal value is lower than 100 mg/dL)
2. A random plasma glucose level of 200 mg/dL or more with classic symptoms of diabetes
3. An oral glucose tolerance test of 200 mg/dL or more in a 2-hour sample with a 75 g glucose load
4. Or a hemoglobin A1c value (HbA1c) greater than 6.5%.

T1D needs to be differentiated from type 2 DM by serologic tests: ICA detection in serum.

If T1D suspected: tests for detection of antibodies to GAD and IA-2A confirm diagnosis.

An autoimmune disorder involving inflammation and destruction of the CNS.

It affects more than 900,000 Americans and millions of individuals worldwide.

Common between the ages of 20 and 50 and is twice as common in women as in men.

Multiple genes may contribute to the development of MS, but the disease is most closely associated with inheritance of the HLA allele, DRB1*1501.

Exposure to sunlight, vitamin D deficiency, cigarette smoking, and infection with EBV after early childhood.

Formation of plaques in the white matter of the brain and spinal cord.

T cells and macrophages predominate within the plaques and orchestrate progressive destruction of the myelin sheath of axons (demyelination).

Antibody binds to the myelin membrane and may stimulate specialized phagocytes called microglial cells.

Clinical picture and detection of lesions in the brain using MRI.

Oligoclonal bands in CSF electrophoresis.

A calculated ratio of cerebral spinal fluid (CSF) IgG/albumin ÷ serum IgG/albumin.

It will be increased in MS.

Detection of autoantibody against myelin basic protein (positive in majority of patients)