1 Outline hematopoiesis, beginning with a pluripotent stem cell

See Figure 15-1.

Figure 15-1 Stem cell–based model of hematopoiesis.

(From Noble J: Textbook of Primary Care Medicine, 3rd ed. St. Louis, Mosby, 2001.)

2 What are the major primary and secondary organs that make up the human lymphoid system?

Primary lymphoid organs include bone marrow, thymus, and fetal liver and are the sites at which lymphocytes mature. Precursor B and T lymphocytes are produced in the bone marrow. In adults, B cells continue to mature in the bone marrow, but T cells leave the marrow to mature in the thymus. Maturation in these organs occurs in the absence of stimulating antigen and involves a stringent selection process that eliminates both poorly functioning and autoreactive lymphocytes. Secondary lymphoid organs include lymph nodes, spleen, and mucosa-associated lymphoid tissue (MALT). MALT ranges from poorly organized clusters of lymphoid cells to highly organized structures such as appendix, tonsils, and Peyer’s patches. These secondary lymphoid organs provide a site for mature lymphocytes to respond to antigen presented on the surface of dendritic cells and other professional antigen presenting cells (APCs).

3 What is the function of innate immune system?

The innate immune system forms the first line of defense against pathogens and toxic compounds. Cells of the innate immune system are activated non-specifically and contain various pattern recognition receptors (PRRs) that recognize a diverse group of pathogen-associated molecular patterns (PAMPs) shared by different classes of microbes (e.g., LPS and flagellin). The intensity of the innate immune response does not increase with repetitive exposure to identical antigens.

The first component of innate immunity includes factors that prevent microbes from entering the body. Examples of this are intact epithelium, respiratory cilia, alveolar macrophages, lysozyme, cationic peptides (defensins), RNase (ribonuclease), and normal flora. The second component includes factors that destroy or limit the growth of microbes that have already entered the body. Examples of this are phagocytes, natural killer (NK) cells, complement, interferon (IFN), iron-sequestering proteins (e.g., lactoferrin), fever and the inflammatory response (i.e., release of interleukin 1 [IL-1], IL-6, and tumor necrosis factor [TNF]). Neutrophils form the most abundant type of phagocyte and are first to reach the site of infection. Here, they engulf and destroy microbes. Pus is a viscous exudate that is composed of dead neutrophils at the infection site.

4 What is adaptive/acquired immunity?

Adaptive immunity involves the response of B and T cells following exposure to an antigen to which they are specific. It is characterized by memory, resulting in rapid, stronger, and more efficient elimination of the source of the offending antigen upon repeat exposure to said antigen (Table 15-1). It is very important to recognize that stimulation of the adaptive immune response depends on prior activation of the innate immune system. Dendritic cells are considered to form the bridge between the innate and adaptive immune systems, because they present antigen to naive T cells. Stimulation of dendritic cell PRRs results in antigen processing and presentation to naive T cells via major histocompatibility complex (MHC)-T-cell receptor (TCR) interactions.

Table 15-1 Characteristics of Innate and Acquired Immunity

Note: The primary and secondary immune responses refer to the activity of the adaptive immune system. The primary response is the activity of this system after first exposure to the pathogen, whereas the secondary response occurs after immunologic memory has been generated from a previous exposure, so the secondary response is more rapid and powerful.

5 What are the basic characteristics of cell-mediated and humoral immunity?

Cell-mediated immunity involves helper T (T_H) cells and is characterized by T_H cell–mediated defense against viruses, fungi, and mycobacteria. It is also responsible for type IV hypersensitivity reactions, tumor destruction, and graft rejection.

Non-specific activation of dendritic cells and macrophages leads to secretion of IL-12, which stimulate naive T helper (T_H0) cells to differentiate into T_H1 cells. This T-cell subset secretes IL-2, leading to propagation of the T_H cell response and activation/conversion of T_C cells to cytotoxic T lymphocytes (CTLs), which function to destroy tumors and virus-infected cells. Note that IL-2 secretion from T_H1 cells constitutes only part of the signal sequence necessary for CTL activation; CTLs also require TCR and CDB interaction with MHC class I on infected cells (see question 6, next). T_H1 cells also secrete interferon-γ (IFN-γ), which activates macrophages and stimulates intracellular microorganism destruction.

Humoral immunity is primarily mediated by B cell production of antibodies. Antibodies have a diverse set of roles, including neutralization of pathogens and toxins, opsonization of pathogens to facilitate phagocytosis, complement activation, stimulation of mast cell and basophil degranulation, and B cell activation. Antibodies are grouped into five different isotypes (IgM, IgD, IgA, IgE, IgG) according to their constant domains. Production of IgA, IgE, and IgG relies on B cell interaction with T cells in a process known as class switching (see question 9).

6 Describe the difference between class I and class II major histocompatibility complex molecules

T cells possess T-cell receptors (TCRs) that interact with MHC molecules. MHC class I molecules are found on the surface of all nucleated cells, thus excluding only mature erythrocytes, and interact with the TCR of CD8⁺ T cells (i.e., cytotoxic T lymphocytes). The CD8 molecule is necessary to complete the interaction of the TCR and MHC class I. MHC class II lies on the plasma membrane of antigen-presenting cells (dendritic cells, macrophages, and memory B cells). It interacts with the TCR of CD4⁺ T cells (i.e., T_H cells), with the CD4 molecule serving a necessary role for this interaction to occur. An easy way to remember which MHC molecule interacts with which T-cell type is to use the rule of 8:

For the purpose of boards, MHC class I displays nonself peptides that have been processed intracellularly (as might be seen in a cell infected with a virus), and MHC class II displays exogenous nonself peptides (obtained via phagocytosis or endocytosis). This is an important distinction to make. MHC class I, when displaying nonself peptides, marks the cell for destruction by the CTL with the TCR specific for that peptide. This destruction is mediated by Fas-Fas ligand binding (leading to apoptosis), granzymes (proteases), or perforins (creates a channel in the plasma membrane and mediates cytolysis), thus eliminating the infected cell. On the other hand, when MHC class II displays nonself peptides, it leads to activation of the T_H cell with the TCR specific for that peptide. This activation process upregulates T_H cytokine production, resulting in subsequent macrophage activation and proliferation of plasma cells with the appropriate specificity, thus catalyzing the elimination of the offending extracellular agent.

7 How do antibodies eliminate extracellular pathogens?

Extracellular pathogens (e.g., bacteria, free virions) commonly induce production of humoral antibodies. Extracellular pathogens that are coated with opsonizing antibodies (IgG) are efficiently phagocytized by macrophages and neutrophils. Following phagocytosis and processing of extracellular antigens, expression of antigenic peptides in association with MHC class II on the surface of antigen-presenting cells (APCs) stimulates T_H2 cells, which further enhances the humoral response. Keep in mind that IgG and IgM can activate complement that may lyse, neutralize, or opsonize extracellular pathogens.

8 By what process can antibodies catalyze the elimination of intracellular pathogens?

Antibody-dependent cellular cytotoxicity (ADCC) involves the binding of IgG (attached to antigens on the surface of the target cell) to Fcγ receptors. This leads to destruction of the cell by phagocytic cells or NK cells possessing these receptors.

9 What are the five classes (isotypes) of immunoglobulins? Describe their respective distributions in the body

See Table 15-2.

Table 15-2 Immunoglobulin Isotypes

Note: When B cells directly interact with CD4⁺ helper T cells, all classes of immunoglobulin can potentially be produced. The TCR interacts with MHC II on the B cell, and CD40L expressed on the activated T cell simultaneously binds to the constitutively expressed CD40 on the B cell. This interaction facilitates the production of various cytokines from the T cell that influence B cell class switching via VDJ recombination and production of IgG, IgE, and IgA. B cells capable of producing these antibodies are referred to as plasma cells.

In contrast, the T cell–independent response consists almost exclusively of IgM. The T cell–independent response generally occurs when the primary antigen is a polysaccharide (e.g., bacterial capsule, lipopolysaccharide on the gram-negative outer membrane), because these molecules are not processed by APCs in the same manner as for peptide antigen. Therefore, T_H cells are not activated and isotype switching cannot occur. This means that only IgM will be produced and no secondary (memory) immune response will occur. Vaccines containing polysaccharide capsules (e.g., meningococcal vaccine, pneumovax, Haemophilus influenzae type B vaccine) are thus commonly conjugated to proteins in order to promote T_H cell activation, class switching and the formation of immunologic memory.

10 Most humans can produce 10⁶ to 10⁹ unique immunoglobulin (Ig) molecules. However, the number of immunoglobulin genes is orders of magnitude less than this. How is this possible?

This is primarily due to gene rearrangement. Note that each Ig molecule consists of two identical light chains (classified as λ or κ by the constant region) and two identical heavy chains (classified as α, δ, ε, γ, and μ according to the isotype, as determined by the constant region). Diversity is gained from “mixing and matching” these chains. However, each of these four chains also contains a variable region, which is created by gene rearrangement. In this process, various unique gene segments in groups named V, D (heavy chains only), and J are excised from the strand, reordered to create a unique code for the variable region, and reconnected by RAG1 and RAG2 (recombination-activating genes) to each other and to the constant region gene segments. This type of rearrangement is also seen in the α and β chains of the TCR to achieve diversity in these molecules as well.

Step 1 Secret

For the purpose of the USMLE, it is not important to memorize the detailed mechanism by which V(D)J recombination occurs. Rather, focus on understanding the significance of recombination—that is, the diversity of antigen recognition sites results from the recombination process. It is, however, particularly high-yield to understand details regarding general antibody structure (Fig. 15-2).

Figure 15-2 The structure of immunoglobulin (Ig) molecules. Note: The 12 complement-determining regions (CDRs) are the areas that most determine to which antigen the antibody will bind.

(From Mason RJ, Murray JF, Broaddus VC, et al: Murray & Nadel’s Textbook of Respiratory Medicine, 4th ed. Philadelphia, WB Saunders, 2005.)

11 What are complement proteins and how do they function in an immune response?

Complement proteins comprise a network of soluble plasma proteins that become activated as a cascade by IgM and IgG (classical pathway) or by surface molecules of microorganisms (alternative and lectin pathways). The complement proteins have many important biologic activities. The membrane attack complex mediates cell lysis, whereas other components participate in opsonization, chemotaxis, neutralization of pathogens, and clearance of immune complexes (Fig. 15-3 and Table 15-3).

Figure 15-3 The complement cascade.

(From Mandell GL, Bennett JE, Dolin R: Principles and Practice of Infectious Disease, 6th ed. Philadelphia, Churchill Livingstone, 2005.)

Table 15-3 Complement Pathway Components and Activity

12 Describe the ramifications of the most common complement protein deficiencies

See Table 15-4.

Table 15-4 Complement Protein Deficiencies

Note: Since complement represents a set of proteins produced by the liver, generalized complement deficiencies can be seen in liver failure or in dietary deficiencies of certain essential amino acids.

13 Which complement components and cytokines are required for neutrophil chemotaxis?

Neutrophil migration is dependent on IL-8 C5a (complement component), and LTB₄ (a leukotriene). Leukotriene synthesis is dependent upon the enzyme lipoxygenase, which is inhibited by the drug zileuton.

14 As a review, list the effector functions of the major leukocyte classes

See Table 15-5.

Table 15-5 Leukocytes and their Effector Functions

15 List the functions of the major cytokines secreted by various classes of immune cells

Secreted by T cells:

IL-3: Hematopoietic stem cell differentiation into myeloid progenitors

Secreted by T_H1 cells:

IL-2: T-cell proliferation

IL-12: T_H1 cell differentiation

INF-γ: Activation of macrophages and T_H1 cells, suppression of T_H2 cells, killing of intracellular pathogens

Secreted by T_H2 cells:

IL-4: T_H2 cell differentiation, promotes IgE and IgG class switch

IL-5: Promotes eosinophil proliferation, aids in IgA class switch

IL-10: Anti-inflammatory; activates T_H2 cells and inhibits T_H1 cells

Secreted by macrophages:

TNF-α: Acute-phase cytokine that activates adhesion molecule expression on endothelium; promotes vascular leak; responsible for septic shock and cachexia

IL-1: Acute-phase cytokine; activates adhesion molecule expression on endothelium and causes fever

IL-6: Acute-phase cytokine; promotes fever

IL-8: Neutrophil chemotaxis

IL-12: T_H1 cell differentiation, NK cell activation

16 List the major cell surface markers used to identify various classes of immune cells

See Table 15-6.

Table 15-6 Cell Surface Markers Used to Identify Classes Of Immune Cells

1 What is the differential diagnosis for this presentation?

Asthma, anaphylactic shock, bronchiolitis (inflammation of the small airways usually following a viral infection), foreign body aspiration, and toxin or allergen ingestion must be considered.

2 What is the most likely diagnosis?

Acute systemic anaphylaxis (anaphylactic shock)—the combination of bronchospasm, urticaria, and hypotension makes this diagnosis much more likely than the others. It should be noted that anaphylaxis is the most serious result of drug hypersensitivity. However, the most common type of drug hypersensitivity involves cutaneous symptoms, such as generalized flushing, urticaria (hives), or mild angioedema. Penicillins are the most common cause of medication-induced anaphylaxis, but this is still a very rare phenomenon with this class of antibiotics.

3 Can free penicillin cause anaphylaxis?

No. Penicillin is only large enough to bind to one arm of an antibody (i.e., the variable regions of only one heavy chain and one light chain) and thus is referred to as univalent. Penicillin must be bound to a carrier protein to induce a response, and therefore it is a hapten. When penicillin is bound to its carrier protein, it can cross-link IgE (bind to one arm of two separate but identical antibodies and increase the propinquity of the corresponding Fc receptors on the mast cell surface), thus triggering the release of mediators.

4 What type of hypersensitivity is anaphylaxis? What is its immunopathogenesis?

Anaphylaxis is the systemic form of the immediate hypersensitivity response (type I). Upon first exposure to antigen, the antigen binds to a B cell, which presents the antigen on MHC class II to a T_H2 cell. The T cell provides cytokines to induce IgE class switching. IgE binds to Fc receptors on the surface of mast cells and basophils, where it persists until the second exposure to antigen. This antigen then cross-links the IgE bound to mast cells and basophils, triggering the release of pre-formed histamine (via cGMP, cAMP, and Ca²⁺) and the de novo synthesis and release of lipid mediators (e.g., leukotrienes, prostaglandins, platelet-activating factor [PAF]). In the immediate phase (occurs within minutes; caused by degranulation of pre-formed mediators), histamine induces a widespread increase in vascular permeability and smooth muscle contraction. In the late phase (>6 hours later, as these mediators were synthesized at the time of exposure), leukotrienes elicit prolonged bronchoconstriction, mucus secretion, and an increase in vascular permeability, while prostaglandin D₂ and PAF cause leukocyte migration and activation. Eosinophilia occurs along with the late phase reaction due to release of eosinophil chemotactic factor. Eosinophils release cationic granule proteins intended to destroy parasites, but in the absence of parasites, tissue damage and remodeling occur.

5 What is the pathophysiologic explanation for the wheezing and diarrhea that developed?

Release of mast cell mediators, such as histamine and leukotrienes, causes an increase in vascular permeability and contraction of certain smooth muscle types in multiple organ systems. In the airways, this leads to laryngeal edema, bronchoconstriction, and mucus hypersecretion, resulting in the clinical symptom of wheezing. In the gastrointestinal tract, the consequence of smooth muscle contraction and edema is nausea, vomiting, and diarrhea.

6 How does anaphylaxis result in the urticaria observed in this patient?

The widespread vasodilation and increased vascular permeability that develop in anaphylaxis allow fluid to accumulate in the superficial dermis, producing small wheals with a pale center encircled by a red flare. This same mechanism is responsible for causing edema in the deep dermis (angioedema) and the laryngeal edema that can develop in anaphylaxis.

7 Why did the child NOT have a reaction to amoxicillin when it was first administered for his previous ear infection?

He was not sensitized to the allergen at that time. Initial exposure to an allergen generates allergen-specific IgE antibodies after several days, which become bound to Fc receptors on the mast cell surface (sensitization). Re-exposure to the same allergen (challenge phase) cross-links the IgE-bound mast cells and stimulates degranulation (see question 4).

8 What clinical testing can be performed to confirm that this immediate hypersensitivity reaction was caused by amoxicillin?

A few days following the attack, a skin test for amoxicillin and other common drug allergens can be performed. If the child is allergic, intradermal injections of amoxicillin will cause degranulation of local mast cells at the site of injection. The resultant release of histamine and other mediators will cause a large wheal and flare to develop within 30 minutes. The wheal is a central raised area reflecting leakage of plasma from venules (edema), and the flare is a surrounding red area caused by vasodilation (erythema).

9 Why was the child immediately given epinephrine?

Epinephrine alleviates the symptoms of anaphylaxis through its positive adrenergic effects. By stimulating β₂-receptors, it relaxes bronchial smooth muscles to open the airways; by stimulating peripheral α₁-receptors, it constricts small blood vessels, thereby reducing vascular leakage and raising blood pressure.

10 Why was the child given diphenhydramine and methylprednisolone?

11 What was the motivation for developing the second-generation H₁ receptor antagonists such as fexofenadine (Allegra) and loratadine (Claritin)?

These agents do not cross the blood-brain barrier like the first-generation H₁ receptor antagonists and do not cause as much drowsiness. They are generally used for allergic rhinitis, another (milder) type I hypersensitivity response.

12 List the common classes of drugs that are used to treat type I hypersensitivity disorders and their general mode of action

See Table 15-7.

Table 15-7 Drugs Used to Treat Type I Hypersensitivity Disorders and their General Mode of Action

1 What is the differential diagnosis for this presentation?

Hemolytic anemia, chronic liver disease (jaundice is common, as is a macrocytic anemia), occult hemorrhage, drug toxicity, tumor obstructing biliary tract (anemia is common in cancer, and biliary obstruction would lead to jaundice), and Gilbert syndrome (jaundice can occur if stressor—anemia in this case—is severe enough to induce markedly reduced uridine diphosphate (UDP) glucuronyl transferase activity) are all considered.

2 What additional tests should be ordered to further analyze the anemia and jaundice?

All patients with anemia should receive a complete blood count (CBC) with erythrocyte indices (mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC], red blood cell (RBC) distribution width, and a reticulocyte count). The patient is also presenting with jaundice, necessitating liver function tests as well as direct (conjugated) and indirect (unconjugated) bilirubin levels.

3 What is the most likely diagnosis?

Drug-induced warm autoimmune hemolytic anemia (AIHA).

4 What is the significance of a positive direct Coombs’ test and how does it support the diagnosis considered in this patient?

A Coombs’ test is an antiglobulin assay that detects both immunoglobulin that is attached to the surface of a patient’s RBCs (direct test) and the presence of circulating immunoglobulin against RBCs (indirect test). A positive direct Coombs’ test supports, but does not prove, the presence of an immune-mediated hemolytic process.

Note: Distinguishing between direct and indirect Coombs’ assays can be tricky, but it is important to understand the differences between the methodologies and uses for the two tests. In a direct Coombs’ test, anti-Ig antibody is added to a patient’s RBCs. If the RBCs are already bound to immunoglobulin present in the patient’s own blood, the addition of anti-Ig antibody will cause agglutination. This test is used to detect the presence of immunoglobulins against a patient’s own RBCs (e.g., hemolytic disease of the newborn, drug-induced AIHA, transfusion reactions). In contrast, an indirect Coombs’ test requires addition of normal RBCs to a patient’s serum. If immunoglobulins against the RBCs are present in the serum, agglutination will occur. Thus, an indirect test measures for immunoglobulins against foreign blood products that are not bound to the patient’s own RBCs (e.g., screening for antibodies prior to blood transfusion, detecting Rh antibodies).

5 How do medications such as penicillin cause autoimmune hemolytic anemia?

Certain antibiotics (e.g., penicillin, cephalosporins, streptomycin, tetracycline) and other small molecules may nonspecifically adsorb to proteins on RBC surfaces, forming a complex similar to a hapten-carrier complex. These drugs are generally too small to elicit an immune response by themselves; however, they can become immunogenic when combined with larger molecules such as membrane-associated proteins. In such individuals, this complex can induce the formation of antibodies, which then bind to the adsorbed drug on RBCs. It is the presence of these autoantibodies that is detected in the direct Coombs’ test.

6 What type of hypersensitivity does autoimmune hemolytic anemia represent?

This is an example of a type II hypersensitivity reaction, in which IgM or IgG antibodies bind to cell surface antigens. Other examples of type II hypersensitivity include blood transfusion reactions, hyperacute rejection of organ transplants, and hemolytic disease of the newborn.

7 What is the mechanism by which hemolysis occurs in drug-induced autoimmune hemolytic anemia?

IgG damages RBCs by binding and activating effector cells carrying Fcγ receptors (e.g., neutrophils, macrophages, and NK cells). Note that both IgG and IgM can induce complement-mediated lysis of RBCs, but this mode of destruction is not common in this form of AIHA.

Note: In some individuals, the extended use of certain drugs, including methyldopa, levodopa, quinidine, and procainamide, can stimulate production of anti-RBC antibodies. The mechanism by which the autoantibodies are induced is unknown, and the antibodies do not cross-react with the drug that appears to elicit their production.